Bio

Bio


Professor Carolyn Bertozzi's research interests span the disciplines of chemistry and biology with an emphasis on studies of cell surface sugars important to human health and disease. Her research group profiles changes in cell surface glycosylation associated with cancer, inflammation and bacterial infection, and uses this information to develop new diagnostic and therapeutic approaches, most recently in the area of immuno-oncology.

Dr. Bertozzi completed her undergraduate degree in Chemistry at Harvard University and her Ph.D. at UC Berkeley, focusing on the chemical synthesis of oligosaccharide analogs. During postdoctoral work at UC San Francisco, she studied the activity of endothelial oligosaccharides in promoting cell adhesion at sites of inflammation. She joined the UC Berkeley faculty in 1996. A Howard Hughes Medical Institute Investigator since 2000, she came to Stanford University in June 2015, among the first faculty to join the interdisciplinary institute ChEM-H (Chemistry, Engineering & Medicine for Human Health). Named a MacArthur Fellow in 1999, Dr. Bertozzi has received many awards for her dedication to chemistry, and to training a new generation of scientists fluent in both chemistry and biology. She has been elected to the Institute of Medicine, National Academy of Sciences, and American Academy of Arts and Sciences; and received the Lemelson-MIT Prize, the Heinrich Wieland Prize, and the ACS Award in Pure Chemistry, among many others. Her efforts in undergraduate education have earned the UC Berkeley Distinguished Teaching Award and the Donald Sterling Noyce Prize for Excellence in Undergraduate Teaching.

Today, the Bertozzi Group at Stanford studies the glycobiology underlying diseases such as cancer, inflammatory disorders such as arthritis, and infectious diseases such as tuberculosis. The work has advanced understanding of cell surface oligosaccharides involved in cell recognition and inter-cellular communication.

Dr. Bertozzi's lab also develops new methods to perform controlled chemical reactions within living systems. The group has developed new tools for studying glycans in living systems, and more recently nanotechnologies for probing biological systems. Such "bioorthogonal" chemistries enable manipulation of biomolecules in their living environment.

Several of the technologies developed in the Bertozzi lab have been adapted for commercial use. Actively engaged with several biotechnology start-ups, Dr. Bertozzi founded Redwood Bioscience of Emeryville, California, and has served on the research advisory board of GlaxoSmithKline.

Academic Appointments


Administrative Appointments


  • Investigator, Howard Hughes Medical Institute (2000 - Present)

Honors & Awards


  • Arthur C. Cope Award, American Chemical Society (2017)
  • National Academy of Sciences Award in the Chemical Sciences, National Academy of Sciences (2016)
  • Ernest Orlando Lawrence Award, U.S. Department of Energy (2015)
  • Heinrich Wieland Prize, Heinrich Wieland Prize (2012)
  • Lemelson-MIT Prize, Massachusetts Institute of Technology (2010)
  • Ernst Schering Prize, Ernst Schering Research Foundation (2007)
  • Distinguished Teaching Award, UC Berkeley College of Chemistry (2001)
  • Award in Pure Chemistry, American Chemical Society (2001)
  • MacArthur Foundation ?Genius? Award, MacArthur Foundation (1999)
  • Arthur C. Cope Scholar Award, American Chemical Society (1999)
  • Honorary Degree, Freie University Berlin (2014)
  • Honorary Doctorate Degree, Duke University (2014)
  • Hans Bloemendal Award, Radboud Univ. Nijmegen (2013)
  • Honorary Doctorate Degree, Brown University (2012)
  • Tetrahedron Young Investigator Award, Executive Board of Editors and the Publisher of Tetrahedron Publications (2011)
  • Albert Hofmann Medal, U. Zurich (2009)
  • Harrison Howe Award, Rochester Section, American Chemical Society (2009)
  • W. H. Nichols Award, New York Section, American Chemical Society (2009)
  • Li Ka Shing Women in Science Award, Li Ka Shing Foundation Women in Science Program (2008)
  • Roy L. Whistler International Award in Carbohydrate Chemistry, International Carbohydrate Organization (2008)
  • Willard Gibbs Medal, Chicago Section, American Chemical Society (2008)
  • T.Z. and Irmgard Chu Distinguished Professorship in Chemistry, UC Berkeley (2005-14)
  • Havinga Medal, U. Leiden (2005)
  • Agnes Fay Morgan Research Award, Iota Sigma Pi (2004)
  • Fellow, American Association for the Advancement of Science (2002)
  • Irving Sigal Young Investigator Award, Protein Society (2002)
  • Donald Sterling Noyce Prize for Excellence in Undergraduate Teaching, UC Berkeley College of Chemistry (2001)
  • Department of Chemistry Teaching Award, UC Berkeley (2000)
  • Merck Academic Development Program Award, Merck (2000)
  • Presidential Early Career Award in Science and Engineering (PECASE), The U.S. White House (2000)
  • Camille Dreyfus Teacher-Scholar Award, Camille and Henry Dreyfus Foundation (1999)
  • Beckman Young Investigator Award, Arnold and Mabel Beckman Foundation (1998)
  • Glaxo Wellcome Scholar, Glaxo Wellcome (1998)
  • Prytanean Faculty Award, Prytanean Women's Honor Society, UC Berkeley (1998)
  • Research Innovation Award, Research Corporation (1998)
  • Young Investigator Award, Office of Naval Research (1998)
  • Horace S. Isbell Award in Carbohydrate Chemistry, American Chemical Society (1997)
  • New Investigator Award in Pharmacology, Burroughs Wellcome (1997)
  • Sloan Research Fellow, Alfred P. Sloan Foundation (1997)
  • Pew Scholars Award in the Biomedical Sciences, Pew Charitable Trusts (1996)
  • Young Investigator Award, Exxon Education Fund (1996)
  • Dreyfus New Faculty Award, Camille and Henry Dreyfus Foundation (1995)

Boards, Advisory Committees, Professional Organizations


  • Member, National Academy of Inventors (2013 - Present)
  • Member, Institute of Medicine (2011 - Present)
  • Member, German Academy of Sciences Leopoldina (2008 - Present)
  • Member, National Academy of Sciences (2005 - Present)
  • Member, American Academy of Arts and Sciences (2003 - Present)
  • Chair, Scientific Advisory Board, Redwood Bioscience
  • Board Member, Board of Scientific Counslors, Broad Institutue
  • Board Member, Catalent Biologics Board
  • Board Member, Research Advisory Baord, Glaxo Smithkline

Professional Education


  • Postdoc, UC San Francisco, Immunology
  • PhD, UC Berkeley, Chemistry (1993)
  • AB, Harvard University, Chemistry (1988)

Teaching

2019-20 Courses


Stanford Advisees


Publications

All Publications


  • DNA-PKcs has KU-dependent function in rRNA processing and haematopoiesis. Nature Shao, Z., Flynn, R. A., Crowe, J. L., Zhu, Y., Liang, J., Jiang, W., Aryan, F., Aoude, P., Bertozzi, C. R., Estes, V. M., Lee, B. J., Bhagat, G., Zha, S., Calo, E. 2020

    Abstract

    The DNA-dependent protein kinase (DNA-PK), which comprises the KU heterodimer and a catalytic subunit (DNA-PKcs), is a classical non-homologous end-joining (cNHEJ) factor1. KU binds to DNA ends, initiates cNHEJ, and recruits and activates DNA-PKcs. KU also binds to RNA, but the relevance of this interaction in mammals is unclear. Here we use mouse models to show that DNA-PK has an unexpected role in the biogenesis of ribosomal RNA (rRNA) and in haematopoiesis. The expression of kinase-dead DNA-PKcs abrogates cNHEJ2. However, most mice that both expressed kinase-dead DNA-PKcs and lacked the tumour suppressor TP53 developed myeloid disease, whereas all other previously characterized mice deficient in both cNHEJ and TP53 expression succumbed to pro-B cell lymphoma3. DNA-PK autophosphorylates DNA-PKcs, which is its best characterized substrate. Blocking the phosphorylation of DNA-PKcs at the T2609 cluster, but not the S2056 cluster, led to KU-dependent defects in 18S rRNA processing, compromised global protein synthesis in haematopoietic cells and caused bone marrow failure in mice. KU drives the assembly of DNA-PKcs on a wide range of cellular RNAs, including the U3 small nucleolar RNA, which is essential for processing of 18S rRNA4. U3 activates purified DNA-PK and triggers phosphorylation of DNA-PKcs at T2609. DNA-PK, but not other cNHEJ factors, resides in nucleoli in an rRNA-dependent manner and is co-purified with the small subunit processome. Together our data show that DNA-PK has RNA-dependent, cNHEJ-independent functions during ribosome biogenesis that require the kinase activity of DNA-PKcs and its phosphorylation at the T2609 cluster.

    View details for DOI 10.1038/s41586-020-2041-2

    View details for PubMedID 32103174

  • Author Correction: Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nature neuroscience Marschallinger, J., Iram, T., Zardeneta, M., Lee, S. E., Lehallier, B., Haney, M. S., Pluvinage, J. V., Mathur, V., Hahn, O., Morgens, D. W., Kim, J., Tevini, J., Felder, T. K., Wolinski, H., Bertozzi, C. R., Bassik, M. C., Aigner, L., Wyss-Coray, T. 2020

    Abstract

    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

    View details for DOI 10.1038/s41593-020-0595-9

    View details for PubMedID 32005940

  • Lipid-droplet-accumulating microglia represent a dysfunctional and proinflammatory state in the aging brain. Nature neuroscience Marschallinger, J., Iram, T., Zardeneta, M., Lee, S. E., Lehallier, B., Haney, M. S., Pluvinage, J. V., Mathur, V., Hahn, O., Morgens, D. W., Kim, J., Tevini, J., Felder, T. K., Wolinski, H., Bertozzi, C. R., Bassik, M. C., Aigner, L., Wyss-Coray, T. 2020

    Abstract

    Microglia become progressively activated and seemingly dysfunctional with age, and genetic studies have linked these cells to the pathogenesis of a growing number of neurodegenerative diseases. Here we report a striking buildup of lipid droplets in microglia with aging in mouse and human brains. These cells, which we call 'lipid-droplet-accumulating microglia' (LDAM), are defective in phagocytosis, produce high levels of reactive oxygen species and secrete proinflammatory cytokines. RNA-sequencing analysis of LDAM revealed a transcriptional profile driven by innate inflammation that is distinct from previously reported microglial states. An unbiased CRISPR-Cas9 screen identified genetic modifiers of lipid droplet formation; surprisingly, variants of several of these genes, including progranulin (GRN), are causes of autosomal-dominant forms of human neurodegenerative diseases. We therefore propose that LDAM contribute to age-related and genetic forms of neurodegeneration.

    View details for DOI 10.1038/s41593-019-0566-1

    View details for PubMedID 31959936

  • Updates to the Symbol Nomenclature for Glycans guidelines GLYCOBIOLOGY Neelamegham, S., Aoki-Kinoshita, K., Bolton, E., Frank, M., Lisacek, F., Luetteke, T., O'Boyle, N., Packer, N. H., Stanley, P., Toukach, P., Varki, A., Woods, R. J., Darvill, A., Dell, A., Henrissat, B., Bertozzi, C., Hart, G., Narimatsu, H., Freeze, H., Yamada, I., Paulson, J., Prestegard, J., Marth, J., Vliegenthart, J. G., Etzler, M., Aebi, M., Kanehisa, M., Taniguchi, N., Edwards, N., Rudd, P., Seeberger, P., Mazumder, R., Ranzinger, R., Cummings, R., Schnaar, R., Perez, S., Kornfeld, S., Kinoshita, T., York, W., Knirel, Y., SNFG Discussion Grp 2019; 29 (9): 620?24

    Abstract

    The Symbol Nomenclature for Glycans (SNFG) is a community-curated standard for the depiction of monosaccharides and complex glycans using various colored-coded, geometric shapes, along with defined text additions. It is hosted by the National Center for Biotechnology Information (NCBI) at the NCBI-Glycans Page (www.ncbi.nlm.nih.gov/glycans/snfg.html). Several changes have been made to the SNFG page in the past year to update the rules for depicting glycans using the SNFG, to include more examples of use, particularly for non-mammalian organisms, and to provide guidelines for the depiction of ambiguous glycan structures. This Glycoforum article summarizes these recent changes.

    View details for DOI 10.1093/glycob/cwz045

    View details for Web of Science ID 000493194700001

    View details for PubMedID 31184695

  • Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Choi, J., Wagner, L. S., Timmermans, S. E., Malaker, S. A., Schumann, B., Gray, M. A., Debets, M. F., Takashima, M., Gehring, J., Bertozzi, C. R. 2019; 141 (34): 13442?53

    Abstract

    O-Linked ?-N-acetylgalactosamine (O-GalNAc) glycans constitute a major part of the human glycome. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). Despite proven disease relevance, correlating the activity of individual GalNAc-Ts with biological function remains challenging due to a lack of tools to probe their substrate specificity in a complex biological environment. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. The approach was applicable to multiple GalNAc-T isoenzymes, including GalNAc-T1 and -T2 that prefer nonglycosylated peptide substrates and GalNAcT-10 that prefers a preglycosylated peptide substrate. A detailed investigation of enzyme kinetics and specificities revealed the robustness of the approach to faithfully report on GalNAc-T activity and paves the way for studying substrate specificities in living systems.

    View details for DOI 10.1021/jacs.9b04695

    View details for Web of Science ID 000484082700023

    View details for PubMedID 31373799

  • CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity. Nature chemical biology Tsui, C. K., Barfield, R. M., Fischer, C. R., Morgens, D. W., Li, A., Smith, B. A., Gray, M. A., Bertozzi, C. R., Rabuka, D., Bassik, M. C. 2019

    Abstract

    Antibody-drug conjugates (ADCs) selectively deliver chemotherapeutic agents to target cells and are important cancer therapeutics. However, the mechanisms by which ADCs are internalized and activated remain unclear. Using CRISPR-Cas9 screens, we uncover many known and novel endolysosomal regulators as modulators of ADC toxicity. We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of screens with ADCs bearing different linkers, we show that a subset of late endolysosomal regulators selectively influence toxicity of noncleavable linker ADCs. Surprisingly, we find cleavable valine-citrulline linkers can be processed rapidly after internalization without lysosomal delivery. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal new regulators of endolysosomal trafficking, provide important insights for ADC design and identify candidate combination therapy targets.

    View details for DOI 10.1038/s41589-019-0342-2

    View details for PubMedID 31451760

  • An RNA-centric dissection of host complexes controlling flavivirus infection. Nature microbiology Ooi, Y. S., Majzoub, K., Flynn, R. A., Mata, M. A., Diep, J., Li, J. K., van Buuren, N., Rumachik, N., Johnson, A. G., Puschnik, A. S., Marceau, C. D., Mlera, L., Grabowski, J. M., Kirkegaard, K., Bloom, M. E., Sarnow, P., Bertozzi, C. R., Carette, J. E. 2019

    Abstract

    Flaviviruses, including dengue virus (DENV) and Zika virus (ZIKV), cause severe human disease. Co-opting cellular factors for viral translation and viral genome replication at the endoplasmic reticulum is a shared replication strategy, despite different clinical outcomes. Although the protein products of these viruses have been studied in depth, how the RNA genomes operate inside human cells is poorly understood. Using comprehensive identification of RNA-binding proteins by mass spectrometry (ChIRP-MS), we took an RNA-centric viewpoint of flaviviral infection and identified several hundred proteins associated with both DENV and ZIKV genomic RNA in human cells. Genome-scale knockout screens assigned putative functional relevance to the RNA-protein interactions observed by ChIRP-MS. The endoplasmic-reticulum-localized RNA-binding proteins vigilin and ribosome-binding protein 1 directly bound viral RNA and each acted at distinct stages in the life cycle of flaviviruses. Thus, this versatile strategy can elucidate features of human biology that control the pathogenesis of clinically relevant viruses.

    View details for DOI 10.1038/s41564-019-0518-2

    View details for PubMedID 31384002

  • A novel therapeutic modality of inhibiting the glyco-immune checkpoint axis to treat cancer Cao, L., Petrone, A., Gatlin, W., Che, J., Das, A., LeBlanc, R., Siddiquee, Z., Nerle, S., Stanczak, M., Mayo, M., Xu, L., Normington, K., Brown, J., Yao, W., Bertozzi, C., Broderick, J., Laubli, H., Peng, L. AMER ASSOC CANCER RESEARCH. 2019
  • Physical Principles of Membrane Shape Regulation by the Glycocalyx CELL Shurer, C. R., Kuo, J., Roberts, L., Gandhi, J. G., Colville, M. J., Enoki, T. A., Pan, H., Su, J., Noble, J. M., Hollander, M. J., O'Donnell, J. P., Yin, R., Pedram, K., Mockl, L., Kourkoutis, L. F., Moerner, W. E., Bertozzi, C. R., Feigenson, G. W., Reesink, H. L., Paszek, M. J. 2019; 177 (7): 1757-+
  • A Sugar Cloak of Invisibility BIOCHEMISTRY Waldman, A. J., Bertozzi, C. R. 2019; 58 (19): 2385?86
  • A Sugar Cloak of Invisibility. Biochemistry Waldman, A. J., Bertozzi, C. R. 2019

    View details for PubMedID 31041861

  • Physical Principles of Membrane Shape Regulation by the Glycocalyx. Cell Shurer, C. R., Kuo, J. C., Roberts, L. M., Gandhi, J. G., Colville, M. J., Enoki, T. A., Pan, H., Su, J., Noble, J. M., Hollander, M. J., O'Donnell, J. P., Yin, R., Pedram, K., Mockl, L., Kourkoutis, L. F., Moerner, W. E., Bertozzi, C. R., Feigenson, G. W., Reesink, H. L., Paszek, M. J. 2019

    Abstract

    Cells bend their plasma membranes into highly curved forms to interact with the local environment, but how shape generation is regulated is not fully resolved. Here, we report a synergy between shape-generating processes in the cell interior and the external organization and composition of the cell-surface glycocalyx. Mucin biopolymers and long-chain polysaccharides within the glycocalyx can generate entropic forces that favor or disfavor the projection of spherical and finger-like extensions from the cell surface. A polymer brush model of the glycocalyx successfully predicts the effects of polymer size and cell-surface density on membrane morphologies. Specific glycocalyx compositions can also induce plasma membrane instabilities to generate more exotic undulating and pearled membrane structures and drive secretion of extracellular vesicles. Together, our results suggest a fundamental role for the glycocalyx in regulating curved membrane features that serve in communication between cells and with the extracellular matrix.

    View details for PubMedID 31056282

  • CD22 blockade restores homeostatic microglial phagocytosis in ageing brains NATURE Pluvinage, J. V., Haney, M. S., Smith, B. H., Sun, J., Iram, T., Bonanno, L., Li, L., Lee, D. P., Morgens, D. W., Yang, A. C., Shuken, S. R., Gate, D., Scott, M., Khatri, P., Luo, J., Bertozzi, C. R., Bassik, M. C., Wyss-Coray, T. 2019; 568 (7751): 187-+
  • The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Malaker, S. A., Pedram, K., Ferracane, M. J., Bensing, B. A., Krishnan, V., Pett, C., Yu, J., Woods, E. C., Kramer, J. R., Westerlind, U., Dorigo, O., Bertozzi, C. R. 2019; 116 (15): 7278?87
  • CD22 blockade restores homeostatic microglial phagocytosis in ageing brains. Nature Pluvinage, J. V., Haney, M. S., Smith, B. A., Sun, J., Iram, T., Bonanno, L., Li, L., Lee, D. P., Morgens, D. W., Yang, A. C., Shuken, S. R., Gate, D., Scott, M., Khatri, P., Luo, J., Bertozzi, C. R., Bassik, M. C., Wyss-Coray, T. 2019

    Abstract

    Microglia maintain homeostasis in the central nervous system through phagocytic clearance of protein aggregates and cellular debris. This function deteriorates during ageing and neurodegenerative disease, concomitant with cognitive decline. However, the mechanisms of impaired microglial homeostatic function and the cognitive effects of restoring this function remain unknown. We combined CRISPR-Cas9 knockout screens with RNAsequencing analysis to discover age-related genetic modifiers of microglial phagocytosis. These screens identified CD22, a canonical Bcell receptor, as a negative regulator of phagocytosis that is upregulated on aged microglia. CD22 mediates the anti-phagocytic effect of alpha2,6-linked sialic acid, and inhibition of CD22 promotes the clearance of myelin debris, amyloid-beta oligomers and alpha-synuclein fibrils in vivo. Long-term central nervous system delivery of an antibody that blocks CD22 function reprograms microglia towards a homeostatic transcriptional state and improves cognitive function in aged mice. These findings elucidate a mechanism of age-related microglial impairment and a strategy to restore homeostasis in the ageing brain.

    View details for PubMedID 30944478

  • Towards Mycobacterium tuberculosis detection at the point-of-care: solvatochromic probes permits the detection of mycobacteria within minutes Kamariza, M., Bertozzi, C. AMER CHEMICAL SOC. 2019
  • Orthogonal enzyme/substrate engineering to profile biological substrates of glycosyltransferases Bertozzi, C. AMER CHEMICAL SOC. 2019
  • Glyco-immune modulation in the tumor microenvironment Bertozzi, C. AMER CHEMICAL SOC. 2019
  • Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O-2 activation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Appel, M. J., Meier, K. K., Lafrance-Vanasse, J., Lim, H., Tsai, C., Hedman, B., Hodgson, K. O., Tainer, J. A., Solomon, E. I., Bertozzi, C. R. 2019; 116 (12): 5370?75
  • Formylglycine-generating enzyme binds substrate directly at a mononuclear Cu(I) center to initiate O2 activation. Proceedings of the National Academy of Sciences of the United States of America Appel, M. J., Meier, K. K., Lafrance-Vanasse, J., Lim, H., Tsai, C., Hedman, B., Hodgson, K. O., Tainer, J. A., Solomon, E. I., Bertozzi, C. R. 2019

    Abstract

    The formylglycine-generating enzyme (FGE) is required for the posttranslational activation of type I sulfatases by oxidation of an active-site cysteine to Calpha-formylglycine. FGE has emerged as an enabling biotechnology tool due to the robust utility of the aldehyde product as a bioconjugation handle in recombinant proteins. Here, we show that Cu(I)-FGE is functional in O2 activation and reveal a high-resolution X-ray crystal structure of FGE in complex with its catalytic copper cofactor. We establish that the copper atom is coordinated by two active-site cysteine residues in a nearly linear geometry, supporting and extending prior biochemical and structural data. The active cuprous FGE complex was interrogated directly by X-ray absorption spectroscopy. These data unambiguously establish the configuration of the resting enzyme metal center and, importantly, reveal the formation of a three-coordinate tris(thiolate) trigonal planar complex upon substrate binding as furthermore supported by density functional theory (DFT) calculations. Critically, inner-sphere substrate coordination turns on O2 activation at the copper center. These collective results provide a detailed mechanistic framework for understanding why nature chose this structurally unique monocopper active site to catalyze oxidase chemistry for sulfatase activation.

    View details for PubMedID 30824597

  • Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum NATURE CHEMICAL BIOLOGY Zhou, X., Rodriguez-Rivera, F. P., Lim, H., Bell, J. C., Bernhardt, T. G., Bertozzi, C. R., Theriot, J. A. 2019; 15 (3): 221-+
  • Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum. Nature chemical biology Zhou, X., Rodriguez-Rivera, F. P., Lim, H. C., Bell, J. C., Bernhardt, T. G., Bertozzi, C. R., Theriot, J. A. 2019

    Abstract

    Members of the Corynebacterineae, including Corynebacterium and Mycobacterium, have an atypical cell envelope characterized by an additional mycomembrane outside of the peptidoglycan layer. How this multilayered cell envelope is assembled remains unclear. Here, we tracked the assembly dynamics of different envelope layers in Corynebacterium glutamicum and Mycobacterium smegmatis by using metabolic labeling and found that the septal cell envelope is assembled sequentially in both species. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. This diffusion barrier is resolved through perforations in the peripheral peptidoglycan, thus leading to the confluency of the mycomembrane before daughter cell separation (V snapping). Furthermore, the same junctional peptidoglycan also serves as a mechanical link holding the daughter cells together and undergoes mechanical fracture during V snapping. Finally, we show that normal V snapping in C. glutamicum depends on complete assembly of the septal cell envelope.

    View details for PubMedID 30664686

  • Inverting family GH156 sialidases define an unusual catalytic motif for glycosidase action. Nature communications Bule, P., Chuzel, L., Blagova, E., Wu, L., Gray, M. A., Henrissat, B., Rapp, E., Bertozzi, C. R., Taron, C. H., Davies, G. J. 2019; 10 (1): 4816

    Abstract

    Sialic acids are a family of related sugars that play essential roles in many biological events intimately linked to cellular recognition in both health and disease. Sialidases are therefore orchestrators of cellular biology and important therapeutic targets for viral infection. Here, we sought to define if uncharacterized sialidases would provide distinct paradigms in sialic acid biochemistry. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. Consistent with an inverting mechanism, EnvSia156 reveals a His/Asp active center in which the His acts as a Brønsted acid and Asp as a Brønsted base in a single-displacement mechanism. A predominantly hydrophobic aglycone site facilitates accommodation of a variety of 2-linked sialosides; a versatility that offers the potential for glycan hydrolysis across a range of biological and technological platforms.

    View details for DOI 10.1038/s41467-019-12684-7

    View details for PubMedID 31645552

  • Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae. PLoS genetics Lim, H. C., Sher, J. W., Rodriguez-Rivera, F. P., Fumeaux, C., Bertozzi, C. R., Bernhardt, T. G. 2019; 15 (8): e1008284

    Abstract

    Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. These bacteria are surrounded by a multilayered cell envelope composed of a cytoplasmic membrane, a peptidoglycan (PG) cell wall, a second polysaccharide layer called the arabinogalactan (AG), and finally an outer membrane-like layer made of mycolic acids. Several anti-tuberculosis drugs target the biogenesis of this complex envelope, but their efficacy is declining due to resistance. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. To this end, we generated the first high-density library of transposon insertion mutants in the model organism C. glutamicum. Transposon-sequencing was then used to define its essential gene set and identify loci that, when inactivated, confer hypersensitivity to ethambutol (EMB), a drug that targets AG biogenesis. Among the EMBs loci were genes encoding RipC and the FtsEX complex, a PG cleaving enzyme required for proper cell division and its predicted regulator, respectively. Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. A combination of quantitative microscopy, mutational analysis, and interaction studies indicate that SteA and SteB form a complex that localizes to the cytokinetic ring to promote cell separation by RipC-FtsEX and may coordinate its PG remodeling activity with the biogenesis of other envelope layers during cell division.

    View details for DOI 10.1371/journal.pgen.1008284

    View details for PubMedID 31437147

  • The mucin-selective protease StcE enables molecular and functional analysis of human cancer-associated mucins. Proceedings of the National Academy of Sciences of the United States of America Malaker, S. A., Pedram, K., Ferracane, M. J., Bensing, B. A., Krishnan, V., Pett, C., Yu, J., Woods, E. C., Kramer, J. R., Westerlind, U., Dorigo, O., Bertozzi, C. R. 2019

    Abstract

    Mucin domains are densely O-glycosylated modular protein domains that are found in a wide variety of cell surface and secreted proteins. Mucin-domain glycoproteins are known to be key players in a host of human diseases, especially cancer, wherein mucin expression and glycosylation patterns are altered. Mucin biology has been difficult to study at the molecular level, in part, because methods to manipulate and structurally characterize mucin domains are lacking. Here, we demonstrate that secreted protease of C1 esterase inhibitor (StcE), a bacterial protease from Escherichia coli, cleaves mucin domains by recognizing a discrete peptide- and glycan-based motif. We exploited StcE's unique properties to improve sequence coverage, glycosite mapping, and glycoform analysis of recombinant human mucins by mass spectrometry. We also found that StcE digests cancer-associated mucins from cultured cells and from ascites fluid derived from patients with ovarian cancer. Finally, using StcE, we discovered that sialic acid-binding Ig-type lectin-7 (Siglec-7), a glycoimmune checkpoint receptor, selectively binds sialomucins as biological ligands, whereas the related receptor Siglec-9 does not. Mucin-selective proteolysis, as exemplified by StcE, is therefore a powerful tool for the study of mucin domain structure and function.

    View details for PubMedID 30910957

  • Quantitative Super-Resolution Microscopy of the Mammalian Glycocalyx. Developmental cell Möckl, L., Pedram, K., Roy, A. R., Krishnan, V., Gustavsson, A. K., Dorigo, O., Bertozzi, C. R., Moerner, W. E. 2019

    Abstract

    The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Despite its relevance in both health and disease, studies of the glycocalyx remain hampered by a paucity of methods to spatially classify its components. We combine metabolic labeling, bioorthogonal chemistry, and super-resolution localization microscopy to image two constituents of cell-surface glycans, N-acetylgalactosamine (GalNAc) and sialic acid, with 10-20 nm precision in 2D and 3D. This approach enables two measurements: glycocalyx height and the distribution of individual sugars distal from the membrane. These measurements show that the glycocalyx exhibits nanoscale organization on both cell lines and primary human tumor cells. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. In the latter case, we trace increased height to an effector gene, GALNT7. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology.

    View details for DOI 10.1016/j.devcel.2019.04.035

    View details for PubMedID 31105009

  • The Phosphoinositide Kinase PIKfyve Promotes Cathepsin-S-Mediated Major Histocompatibility Complex Class II Antigen Presentation. iScience Baranov, M. V., Bianchi, F., Schirmacher, A., van Aart, M. A., Maassen, S., Muntjewerff, E. M., Dingjan, I., Ter Beest, M., Verdoes, M., Keyser, S. G., Bertozzi, C. R., Diederichsen, U., van den Bogaart, G. 2018; 11: 160?77

    Abstract

    Antigen presentation to Tcells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. Maturation is driven by the phosphoinositide kinase PIKfyve. Blocking PIKfyve activity by small molecule inhibitors caused a delay in the conversion of phagosomes into lysosomes and in phagosomal acidification, whereas production of reactive oxygen species (ROS) increased. Elevated ROS resulted in reduced activity of cathepsin S and B, but not X, causing a proteolytic defect of MHC class II chaperone invariant chain Ii processing. We developed a novel universal MHC class II presentation assay based on a bio-orthogonal "clickable" antigen and showed that MHC class II presentation was disrupted by the inhibition of PIKfyve, which in turn resulted in reduced activation of CD4+ Tcells. Our results demonstrate a key role of PIKfyve in the processing and presentation of antigens, which should be taken into consideration when targeting PIKfyve in autoimmune disease and cancer.

    View details for PubMedID 30612035

  • Making Glycoproteomics via Mass Spectrometry More Accessible to the greater Scientific Community Driessen, M. D., Going, C. C., Woo, C. M., Pitteri, S. J., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2018: 1013
  • New therapeutic avenues for NGLY1 deficiency Gerling-Driessen, U. M., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2018: 1041
  • A TENSION-MEDIATED GLYCOCALYX FEEDBACK LOOP PROMOTES A MESENCHYMAL, STEM-LIKE PHENOTYPE IN GLIOBLASTOMA Kaushik, S., Barnes, J., Bainer, R. O., Sa, J. K., Woods, E. C., Kai, F., Przybyla, L., Lakins, J. N., Phillips, J. J., Nam, D., Bertozzi, C. R., Weaver, V. M. OXFORD UNIV PRESS INC. 2018: 265?66
  • Is the Nobel Prize in Chemistry Having a Boston Red Sox Moment? ACS CENTRAL SCIENCE Bertozzi, C. R. 2018; 4 (10): 1291
  • A novel germline variant in CSF3R reduces N-glycosylation and exerts potent oncogenic effects in leukemia. Cancer research Spiciarich, D. R., Oh, S. T., Foley, A., Hughes, S. B., Mauro, M. J., Abdel-Wahab, O., Press, R. D., Viner, R., Thompson, S. L., Chen, Q., Azadi, P., Bertozzi, C. R., Maxson, J. E. 2018

    Abstract

    Mutations in the colony stimulating factor 3 receptor (CSF3R) have been identified in the vast majority of patients with chronic neutrophilic leukemia and are present in other kinds of leukemia, such as AML. Here we studied the function of novel germline variants in CSF3R at amino acid N610. These N610 substitutions were potently oncogenic and activated the receptor independently of its ligand GCSF. These mutations activated the JAK-STAT signaling pathway and conferred sensitivity to JAK inhibitors. Mass spectrometry revealed that the N610 residue is part of a consensus N-linked glycosylation motif in the receptor, usually linked to complex glycans. N610 was also the primary site of sialylation of the receptor. Membrane-proximal N-linked glycosylation was critical for maintaining the ligand dependence of the receptor. Mutation of the N610 site prevented membrane-proximal N-glycosylation of CSF3R, which then drove ligand-independent cellular expansion. Kinase inhibitors blocked growth of cells with an N610 mutation. This study expands the repertoire of oncogenic mutations in CSF3R that are therapeutically targetable and provides insight into the function of glycans in receptor regulation.

    View details for PubMedID 30348809

  • Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling CELL HOST & MICROBE Walton, E. M., Cronan, M. R., Cambier, C. J., Rossi, A., Marass, M., Foglia, M. D., Brewer, W., Poss, K. D., Stainier, D. R., Bertozzi, C. R., Tobin, D. M. 2018; 24 (4): 514-+

    Abstract

    Mycobacterial infection leads to the formation of characteristic immune aggregates called granulomas, a process accompanied by dramatic remodeling of the host vasculature. As granuloma angiogenesis favors the infecting mycobacteria, it may be actively promoted by bacterial determinants during infection. Using Mycobacterium marinum-infected zebrafish as a model, we identify the enzyme proximal cyclopropane synthase of alpha-mycolates (PcaA) as an important bacterial determinant of granuloma-associated angiogenesis. cis-Cyclopropanation of mycobacterial mycolic acids by pcaA drives the activation of host Vegf signaling within granuloma macrophages. Cyclopropanation of the mycobacterial cell wall glycolipid trehalose dimycolate is both required and sufficient to induce robust host angiogenesis. Inducible genetic inhibition of angiogenesis and Vegf signaling during granuloma formation results in bacterial growth deficits. Together, these data reveal a mechanism by which PcaA-mediated cis-cyclopropanation of mycolic acids promotes bacterial growth and dissemination in vivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.

    View details for PubMedID 30308157

  • A tension-mediated glycocalyx-integrin feedback loop promotes mesenchymal-like glioblastoma NATURE CELL BIOLOGY Barnes, J., Kaushik, S., Bainer, R. O., Sa, J. K., Woods, E. C., Kai, F., Przybyla, L., Lee, M., Lee, H., Tung, J. C., Maller, O., Barrett, A. S., Lu, K. V., Lakins, J. N., Hansen, K. C., Obernier, K., Alvarez-Buylla, A., Bergers, G., Phillips, J. J., Nam, D., Bertozzi, C. R., Weaver, V. M. 2018; 20 (10): 1203-+

    Abstract

    Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. Recurrent GBMs often exhibit mesenchymal, stem-like phenotypes that could explain their resistance to therapy. Analyses revealed that recurrent GBMs have increased tension and express high levels of glycoproteins that increase the bulkiness of the glycocalyx. Studies showed that a bulky glycocalyx potentiates integrin mechanosignalling and tissue tension and promotes a mesenchymal, stem-like phenotype in GBMs. Gain- and loss-of-function studies implicated integrin mechanosignalling as an inducer of GBM growth, survival, invasion and treatment resistance, and a mesenchymal, stem-like phenotype. Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Strategies to ameliorate GBM tissue tension offer a therapeutic approach to reduce mortality due to GBM.

    View details for PubMedID 30202050

  • Glycopolymers for immune modulation in the tumor microenvironment Bertozzi, C. AMER CHEMICAL SOC. 2018
  • Quantitative super-resolution microscopy reveals the architecture of the mammalian glycocalyx and its changes during cancer progression Moeckl, L., Pedram, K., Roy, A., Gustavsson, A., Bertozzi, C., Moerner, W. AMER CHEMICAL SOC. 2018
  • Glycosyltransferase bump-hole engineering to dissect mucin-type O-glycosylation in the living cell Schumann, B., Debets, M., Wisnovsky, S., Agbay, A., Wagner, L., Choi, J., Gray, M., Bertozzi, C. AMER CHEMICAL SOC. 2018
  • Computation-Guided Rational Design of a Peptide Motif That Reacts with Cyanobenzothiazoles via Internal Cysteine-Lysine Relay JOURNAL OF ORGANIC CHEMISTRY Keyser, S. L., Utz, A., Bertozzi, C. R. 2018; 83 (14): 7467?79

    Abstract

    Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. Our data show that the cysteine residue reversibly reacts with the nitrile group on the CBT moiety to form an intermediate thioimidate, which undergoes irreversible SN transfer to the lysine residue, yielding an amidine-linked product. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments.

    View details for DOI 10.1021/acs.joc.8b00625

    View details for Web of Science ID 000439761100020

    View details for PubMedID 29771122

  • Multiple Click-Selective tRNA Synthetases Expand Mammalian Cell-Specific Proteomics JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yang, A. C., Du Bois, H., Olsson, N., Gate, D., Lehallier, B., Berdnik, D., Brewer, K. D., Bertozzi, C. R., Elias, J. E., Wyss-Coray, T. 2018; 140 (23): 7046?51

    Abstract

    Bioorthogonal tools enable cell-type-specific proteomics, a prerequisite to understanding biological processes in multicellular organisms. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Azide-labeled proteins are chemoselectively tagged via azide-alkyne cycloadditions with fluorophores for imaging or affinity resins for mass spectrometric characterization. Both mutant synthetases label human, hamster, and mouse cell line proteins and selectively activate their azido-bearing amino acids over 10-fold above the canonical. ScTyrY43G and MmPheT413G label overlapping but distinct proteomes in human cell lines, with broader proteome coverage upon their coexpression. In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals.

    View details for DOI 10.1021/jacs.8b03074

    View details for Web of Science ID 000435525500001

    View details for PubMedID 29775058

  • IL-1R and MyD88 Contribute to the Absence of a Bacterial Microbiome on the Healthy Murine Cornea FRONTIERS IN MICROBIOLOGY Wan, S. J., Sullivan, A. B., Shieh, P., Metruccio, M. E., Evans, D. J., Bertozzi, C. R., Fleiszig, S. J. 2018; 9
  • Acute Modulation of Mycobacterial Cell Envelope Biogenesis by Front-Line Tuberculosis Drugs ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Rodriguez-Rivera, F. P., Zhou, X., Theriot, J. A., Bertozzi, C. R. 2018; 57 (19): 5267?72

    Abstract

    Front-line tuberculosis (TB) drugs have been characterized extensively in?vitro and in?vivo with respect to gene expression and cell viability. However, little work has been devoted to understanding their effects on the physiology of the cell envelope, one of the main targets of this clinical regimen. Herein, we use metabolic labeling methods to visualize the effects of TB drugs on cell envelope dynamics in mycobacterial species. We developed a new fluorophore-trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super-resolution microscopy. We also probed the relationship between mycomembrane and peptidoglycan dynamics using a dual metabolic labeling strategy. Finally, we found that metabolic labeling of both cell envelope structures reports on drug effects on cell physiology in two hours, far faster than a genetic sensor of cell envelope stress. Our work provides insight into acute drug effects on cell envelope biogenesis in live mycobacteria.

    View details for PubMedID 29392891

    View details for PubMedCentralID PMC5924460

  • Isotype-specific agglutination-PCR (ISAP): Asensitive and multiplex method for measuring allergen-specific IgE. The Journal of allergy and clinical immunology Tsai, C., Mukai, K., Robinson, P. V., Gray, M. A., Waschmann, M. B., Lyu, S., Tsai, M., Chinthrajah, R. S., Nadeau, K. C., Bertozzi, C. R., Galli, S. J. 2018; 141 (5): 1901

    View details for PubMedID 29248495

  • Site-specific incorporation of quadricyclane into a protein and photocleavage of the quadricyclane ligation adduct. Bioorganic & medicinal chemistry Tomlin, F. M., Gordon, C. G., Han, Y., Wu, T. S., Sletten, E. M., Bertozzi, C. R. 2018

    Abstract

    The quadricyclane (QC) ligation is a bioorthogonal reaction between a quadricyclane moiety and a nickel bis(dithiolene) derivative. Here we show that a QC amino acid can be incorporated into a protein site-specifically using the pyrrolysine-based genetic code expansion platform, and subsequently used for ligation chemistry. Additionally, we exploited the photolability of the QC ligation product to render the adduct cleavable with a handheld UV lamp. We further developed a protein purification method that involves QC ligation of biotin to a protein of interest, capture on streptavidin resin, and finally release using only UV light. The QC ligation thus brings novel chemical manipulations to the realm of bioorthogonal chemistry.

    View details for PubMedID 29754834

  • Targeting glyco-immune checkpoints for cancer immune therapy Bertozzi, C. AMER CHEMICAL SOC. 2018
  • F Rapid detection of Mycobacterium tuberculosis in sputum with a solvatochromic trehalose probe SCIENCE TRANSLATIONAL MEDICINE Kamariza, M., Shieh, P., Ealand, C. S., Peters, J. S., Chu, B., Rodriguez-Rivera, F. P., Sait, M., Treuren, W. V., Martinson, N., Kalscheuer, R., Kana, B. D., Bertozzi, C. R. 2018; 10 (430)

    Abstract

    Tuberculosis (TB) is the leading cause of death from an infectious bacterial disease. Poor diagnostic tools to detect active disease plague TB control programs and affect patient care. Accurate detection of live Mycobacterium tuberculosis (Mtb), the causative agent of TB, could improve TB diagnosis and patient treatment. We report that mycobacteria and other corynebacteria can be specifically detected with a fluorogenic trehalose analog. We designed a 4-N,N-dimethylamino-1,8-naphthalimide-conjugated trehalose (DMN-Tre) probe that undergoes >700-fold increase in fluorescence intensity when transitioned from aqueous to hydrophobic environments. This enhancement occurs upon metabolic conversion of DMN-Tre to trehalose monomycolate and incorporation into the mycomembrane of Actinobacteria. DMN-Tre labeling enabled the rapid, no-wash visualization of mycobacterial and corynebacterial species without nonspecific labeling of Gram-positive or Gram-negative bacteria. DMN-Tre labeling was detected within minutes and was inhibited by heat killing of mycobacteria. Furthermore, DMN-Tre labeling was reduced by treatment with TB drugs, unlike the clinically used auramine stain. Lastly, DMN-Tre labeled Mtb in TB-positive human sputum samples comparably to auramine staining, suggesting that this operationally simple method may be deployable for TB diagnosis.

    View details for PubMedID 29491187

    View details for PubMedCentralID PMC5985656

  • Antibody detection by agglutination-PCR (ADAP) enables early diagnosis of HIV infection by oral fluid analysis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Tsai, C., Robinson, P. V., Cortez, F., Elma, M. B., Seftel, D., Pourmandi, N., Pandori, M. W., Bertozzi, C. R. 2018; 115 (6): 1250?55

    Abstract

    Oral fluid (OF) is a highly effective substrate for population-based HIV screening efforts, as it is noninfectious and significantly easier to collect than blood. However, anti-HIV antibodies are found at far lower concentrations in OF compared with blood, leading to poor sensitivity and a longer period of time from infection to detection threshold. Thus, despite its inherent advantages in sample collection, OF is not widely used for population screening. Here we report the development of an HIV OF assay based on Antibody Detection by Agglutination-PCR (ADAP) technology. This assay is 1,000-10,000 times more analytically sensitive than clinical enzyme-linked immunoassays (EIAs), displaying both 100% clinical sensitivity and 100% specificity for detecting HIV antibodies within OF samples. We show that the enhanced analytical sensitivity enables this assay to correctly identify HIV-infected individuals otherwise missed by current OF assays. We envision that the attributes of this improved HIV OF assay can increase testing rates of at-risk individuals while enabling diagnosis and treatment at an earlier time point.

    View details for PubMedID 29358368

  • Quantitative Super-Resolution Imaging Reveals Mammalian Glycocalyx Dynamics Moeckl, L., Pedram, K., Roy, A., Bertozzi, C., Moerner, W. CELL PRESS. 2018: 537A?538A
  • Imaging Mycobacterial Trehalose Glycolipids. Methods in enzymology Kamariza, M., Shieh, P., Bertozzi, C. R. 2018; 598: 355?69

    Abstract

    Cell surface trehalose mycolates are important modulators of mycobacterial pathogenesis and host immune response. We discuss the use of fluorescent and fluorogenic trehalose probes for the detection of the mycobacterial trehalose glycolipids. These probes enable real-time imaging of trehalose mycolate biosynthesis and mycomembrane dynamics in the laboratory as well as in clinical settings for the detection of mycobacteria in patient samples.

    View details for PubMedID 29306442

  • Is the Nobel Prize in Chemistry Having a Boston Red Sox Moment? ACS central science Bertozzi, C. R. 2018; 4 (10): 1291

    View details for PubMedID 30410963

    View details for PubMedCentralID PMC6202648

  • Mapping and quantification of over 2,000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (IsoTaG). Molecular & cellular proteomics : MCP Woo, C. M., Lund, P. J., Huang, A. C., Davis, M. M., Bertozzi, C. R., Pitteri, S. 2018

    Abstract

    Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example.  Mounting evidence indicates that the modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc), the only mammalian glycan found on nuclear and cytoplasmic proteins, helps regulate T cell activation.  Yet, a mechanistic understanding of how O-GlcNAc functions in T cell activation remains elusive, partly because of the difficulties in mapping and quantifying O-GlcNAc sites.  Thus, to advance insight into the role of O-GlcNAc in T cell activation, we performed glycosite mapping studies via direct glycopeptide measurement on resting and activated primary human T cells with a technique termed Isotope Targeted Glycoproteomics.  This approach led to the identification of 2,219 intact O-linked glycopeptides across 1,045 glycoproteins.  A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk.  Consistent with other studies, we find that O-GlcNAc sites in T cells lack a strict consensus sequence.  To validate our results, we employed gel shift assays based on conjugating mass tags to O-GlcNAc groups.  Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells.  Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation.

    View details for PubMedID 29351928

  • N-Carboxyanhydride Polymerization of Glycopolypeptides That Activate Antigen-Presenting Cells through Dectin-1 and Dectin-2. Angewandte Chemie (International ed. in English) Zhou, M. N., Delaveris, C. S., Kramer, J. R., Kenkel, J. A., Engleman, E. G., Bertozzi, C. R. 2018; 57 (12): 3137?42

    Abstract

    The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. These receptors are promising targets for vaccine development and cancer immunotherapy. However, currently available agonists are heterogeneous glycoconjugates and polysaccharides from natural sources. Herein, we designed and synthesized the first chemically defined ligands for dectin-1 and dectin-2. They comprised glycopolypeptides bearing mono-, di-, and trisaccharides and were built through polymerization of glycosylated N-carboxyanhydrides. Through this approach, we achieved glycopolypeptides with high molecular weights and low dispersities. We identified structures that elicit a pro-inflammatory response through dectin-1 or dectin-2 in antigen-presenting cells. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications.

    View details for PubMedID 29370452

    View details for PubMedCentralID PMC5842139

  • How many human proteoforms are there? Nature chemical biology Aebersold, R., Agar, J. N., Amster, I. J., Baker, M. S., Bertozzi, C. R., Boja, E. S., Costello, C. E., Cravatt, B. F., Fenselau, C., Garcia, B. A., Ge, Y., Gunawardena, J., Hendrickson, R. C., Hergenrother, P. J., Huber, C. G., Ivanov, A. R., Jensen, O. N., Jewett, M. C., Kelleher, N. L., Kiessling, L. L., Krogan, N. J., Larsen, M. R., Loo, J. A., Ogorzalek Loo, R. R., Lundberg, E., MacCoss, M. J., Mallick, P., Mootha, V. K., Mrksich, M., Muir, T. W., Patrie, S. M., Pesavento, J. J., Pitteri, S. J., Rodriguez, H., Saghatelian, A., Sandoval, W., Schlüter, H., Sechi, S., Slavoff, S. A., Smith, L. M., Snyder, M. P., Thomas, P. M., Uhlén, M., Van Eyk, J. E., Vidal, M., Walt, D. R., White, F. M., Williams, E. R., Wohlschlager, T., Wysocki, V. H., Yates, N. A., Young, N. L., Zhang, B. 2018; 14 (3): 206?14

    Abstract

    Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Here, we outline what we know from current databases and measurement strategies including mass spectrometry-based proteomics. In doing so, we examine prevailing notions about the number of modifications displayed on human proteins and how they combine to generate the protein diversity underlying health and disease. We frame central issues regarding determination of protein-level variation and PTMs, including some paradoxes present in the field today. We use this framework to assess existing data and to ask the question, "How many distinct primary structures of proteins (proteoforms) are created from the 20,300 human genes?" We also explore prospects for improving measurements to better regularize protein-level biology and efficiently associate PTMs to function and phenotype.

    View details for PubMedID 29443976

  • A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression ELIFE Woods, E. C., Kai, F., Barnes, J., Pedram, K., Pickup, M. W., Hollander, M. J., Weaver, V. M., Bertozzi, C. R. 2017; 6

    Abstract

    Metastasis depends upon cancer cell growth and survival within the metastatic niche. Tumors which remodel their glycocalyces, by overexpressing bulky glycoproteins like mucins, exhibit a higher predisposition to metastasize, but the role of mucins in oncogenesis remains poorly understood. Here we report that a bulky glycocalyx promotes the expansion of disseminated tumor cells in vivo by fostering integrin adhesion assembly to permit G1 cell cycle progression. We engineered tumor cells to display glycocalyces of various thicknesses by coating them with synthetic mucin-mimetic glycopolymers. Cells adorned with longer glycopolymers showed increased metastatic potential, enhanced cell cycle progression, and greater levels of integrin-FAK mechanosignaling and Akt signaling in a syngeneic mouse model of metastasis. These effects were mirrored by expression of the ectodomain of cancer-associated mucin MUC1. These findings functionally link mucinous proteins with tumor aggression, and offer a new view of the cancer glycocalyx as a major driver of disease progression.

    View details for PubMedID 29266001

  • Exploring the role of Nrf1 in NGly1 deficiency Gerling-Driessen, U. M., Tomlin, F. M., Cambier, C. J., Liu, Y., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2017: 1226?27
  • Making Glycoproteomics via Mass Spectrometry More Accessible to the greater Scientific Community Driessen, M. D., Going, C. C., Woo, C. M., Pitteri, S. J., Bertozzi, C. R. OXFORD UNIV PRESS INC. 2017: 1212
  • Atoms out of Blobs: CryoEM Takes the Nobel Prize in Chemistry ACS CENTRAL SCIENCE Bertozzi, C. 2017; 3 (10): 1056

    View details for PubMedID 29104918

    View details for PubMedCentralID PMC5658776

  • Human and Baculovirus-Insect Manufacturing Platforms Generate Chemically and Functionally Distinct AAV Vectors with Sexually Dimorphic Liver Transduction. Paulk, N. K., Rumachik, N., Malaker, S., Adams, C., Leib, R., Bertozzi, C. R., Kay, M. WILEY. 2017: 373A
  • Antibody-enzyme conjugates for targeted glycocalyx editing Bertozzi, C. AMER CHEMICAL SOC. 2017
  • Polymerization of glycosylated NCAs for preparation of biomedical materials and synthetic glycoproteins Kramer, J., Zhou, M., Delaveris, C., Bertozzi, C. AMER CHEMICAL SOC. 2017
  • Synthesis of solvatochromic probes to label the mycobacterial cell wall and their use in studies of host-pathogen interactions Keyser, S., Utz, A., Kamariza, M., Bertozzi, C. AMER CHEMICAL SOC. 2017
  • Award Address (Arthur C. Cope Award sponsored by the Arthur C. Cope Fund). Wild world of bioorthogonal chemistry Bertozzi, C. AMER CHEMICAL SOC. 2017
  • Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Spiciarich, D. R., Nolley, R., Maund, S. L., Purcell, S. C., Herschel, J., Iavarone, A. T., Peehl, D. M., Bertozzi, C. R. 2017; 56 (31): 8992?97

    Abstract

    Sialylated glycans are found at elevated levels in many types of cancer and have been implicated in disease progression. However, the specific glycoproteins that contribute to the cancer cell-surface sialylation are not well characterized, specifically in bona fide human disease tissue. Metabolic and bioorthogonal labeling methods have previously enabled the enrichment and identification of sialoglycoproteins from cultured cells and model organisms. Herein, we report the first application of this glycoproteomic platform to human tissues cultured ex?vivo. Both normal and cancerous prostate tissues were sliced and cultured in the presence of the azide-functionalized sialic acid biosynthetic precursor Ac4 ManNAz. The compound was metabolized to the azidosialic acid and incorporated into cell surface and secreted sialoglycoproteins. Chemical biotinylation followed by enrichment and mass spectrometry led to the identification of glycoproteins that were found at elevated levels or uniquely in cancerous prostate tissue. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance.

    View details for PubMedID 28649697

    View details for PubMedCentralID PMC5675001

  • Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding FASEB JOURNAL Jolly, A. L., Agarwal, P., Metruccio, M. M., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. 2017; 31 (6): 2393-2404

    Abstract

    Cell surface glycosylation is thought to be involved in barrier function against microbes at mucosal surfaces. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N-azidoacetylgalactosamine (Ac4GalNAz). Ac4GalNAz treatment labeled the surface of healthy mouse corneas, leaving most cells viable, and bacteria preferentially associated with GalNAz-labeled regions. Surprisingly, corneas from MyD88-/- mice displayed similar GalNAz labeling to wild-type corneas, but labeling was reduced and patchy on IL-1 receptor (IL-1R)-knockout mouse corneas (P < 0.05, ANOVA). Tissue paper blotting removed GalNAz-labeled surface cells, causing DAPI labeling (permeabilization) of underlying cells. MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. They also suggest increased P. aeruginosa adhesion to MyD88-/- and blotted corneas is not due to reduction in total surface glycosylation, and for tissue paper blotting is likely due to cell permeabilization.-Jolly, A. L., Agarwal, P., Metruccio, M. M. E., Spiciarich, D. R., Evans, D. J., Bertozzi, C. R., Fleiszig, S. M. J. Corneal surface glycosylation is modulated by IL-1R and Pseudomonas aeruginosa challenge but is insufficient for inhibiting bacterial binding.

    View details for DOI 10.1096/fj.201601198R

    View details for Web of Science ID 000401553400015

    View details for PubMedCentralID PMC5434651

  • Visualization of mycobacterial membrane dynamics in live cells. Journal of the American Chemical Society Rodriguez-Rivera, F. P., Zhou, X., Theriot, J. A., Bertozzi, C. R. 2017; 139 (9): 3488-3495

    Abstract

    Mycobacteria are endowed with a highly impermeable mycomembrane that confers intrinsic resistance to many antibiotics. Several unique mycomembrane glycolipids have been isolated and structurally characterized, but the underlying organization and dynamics of glycolipids within the cell envelope remain poorly understood. We report here a study of mycomembrane dynamics that was enabled by trehalose-fluorophore conjugates capable of labeling trehalose glycolipids in live actinomycetes. We identified fluorescein-trehalose analogues that are metabolically incorporated into the trehalose mycolates of representative Mycobacterium, Corynebacterium, Nocardia, and Rhodococcus species. Using these probes, we studied the mobilities of labeled glycolipids by time-lapse microscopy and fluorescence recovery after photobleaching experiments and found that mycomembrane fluidity varies widely across species and correlates with mycolic acid structure. Finally, we discovered that treatment of mycobacteria with ethambutol, a front-line tuberculosis (TB) drug, significantly increases mycomembrane fluidity. These findings enhance our understanding of mycobacterial cell envelope structure and dynamics and have implications for development of TB drug cocktails.

    View details for DOI 10.1021/jacs.6b12541

    View details for PubMedID 28075574

    View details for PubMedCentralID PMC5345120

  • Subcellular Partitioning and Intramacrophage Selectivity of Antimicrobial Compounds against Mycobacterium tuberculosis. Antimicrobial agents and chemotherapy Schump, M. D., Fox, D. M., Bertozzi, C. R., Riley, L. W. 2017; 61 (3)

    Abstract

    The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. However, inside infected macrophages, bacteria encounter an environment which differs substantially from broth culture and are subject to important host-dependent pharmacokinetic phenomena which modulate drug activity. Here, we describe how pH-dependent partitioning drives asymmetric antimicrobial drug distribution in M. tuberculosis-infected macrophages. Specifically, weak bases with moderate activity against M. tuberculosis (fluoxetine, sertraline, and dibucaine) were shown to accumulate intracellularly due to differential permeability and relative abundance of their ionized and nonionized forms. Nonprotonatable analogs of the test compounds did not show this effect. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. We further demonstrate that the efficacy of a clinically used compound, clofazimine, is augmented by pH-based partitioning in a macrophage infection model. Because the parameters which govern this effect are well understood and are amenable to chemical modification, this knowledge may enable the rational development of more effective antibiotics against tuberculosis.

    View details for DOI 10.1128/AAC.01639-16

    View details for PubMedID 28052847

    View details for PubMedCentralID PMC5328571

  • Development of IsoTaG, a Chemical Glycoproteomics Technique for Profiling Intact N- and O-Glycopeptides from Whole Cell Proteomes. Journal of proteome research Woo, C. M., Felix, A., Byrd, W. E., Zuegel, D. K., Ishihara, M., Azadi, P., Iavarone, A. T., Pitteri, S. J., Bertozzi, C. R. 2017

    Abstract

    Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This same structural diversity has imposed major challenges on the development of methods to study the intact glycoproteome. We recently introduced a method termed isotope-targeted glycoproteomics (IsoTaG), which utilizes isotope recoding to characterize azidosugar-labeled glycopeptides bearing fully intact glycans. Here, we describe the broad application of the method to analyze glycoproteomes from a collection of tissue-diverse cell lines. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. Application of the IsoTaG platform to 15 cell lines metabolically labeled with Ac4GalNAz or Ac4ManNAz revealed 1375 N- and 2159 O-glycopeptides, variously modified with 74 discrete glycan structures. Glycopeptide-bound glycans observed by IsoTaG were found to be comparable to released N-glycans identified by permethylation analysis. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome.

    View details for DOI 10.1021/acs.jproteome.6b01053

    View details for PubMedID 28244757

  • Click-Chemistry Based High Throughput Screening Platform for Modulators of Ras Palmitoylation SCIENTIFIC REPORTS Ganesan, L., Shieh, P., Bertozzi, C. R., Levental, I. 2017; 7

    Abstract

    Palmitoylation is a widespread, reversible lipid modification that has been implicated in regulating a variety of cellular processes. Approximately one thousand proteins are annotated as being palmitoylated, and for some of these, including several oncogenes of the Ras and Src families, palmitoylation is indispensable for protein function. Despite this wealth of disease-relevant targets, there are currently few effective pharmacological tools to interfere with protein palmitoylation. One reason for this lack of development is the dearth of assays to efficiently screen for small molecular inhibitors of palmitoylation. To address this shortcoming, we have developed a robust, high-throughput compatible, click chemistry-based approach to identify small molecules that interfere with the palmitoylation of Ras, a high value therapeutic target that is mutated in up to a third of human cancers. This assay design shows excellent performance in 384-well format and is sensitive to known, non-specific palmitoylation inhibitors. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. The screening approach described here provides an integrated platform to identify specific modulators of palmitoylated proteins, demonstrated here for Ras and Fyn, but potentially applicable to pharmaceutical targets involved in a variety of human diseases.

    View details for DOI 10.1038/srep41147

    View details for Web of Science ID 000392525600001

    View details for PubMedID 28112226

    View details for PubMedCentralID PMC5255568

  • Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars ANALYTICAL AND BIOANALYTICAL CHEMISTRY Woo, C. M., Felix, A., Zhang, L., Elias, J. E., Bertozzi, C. R. 2017; 409 (2): 579-588

    Abstract

    Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

    View details for DOI 10.1007/s00216-016-9934-9

    View details for Web of Science ID 000391364200021

  • Inhibition of NGLY1 Inactivates the Transcription Factor Nrf1 and Potentiates Proteasome Inhibitor Cytotoxicity. ACS central science Tomlin, F. M., Gerling-Driessen, U. I., Liu, Y. C., Flynn, R. A., Vangala, J. R., Lentz, C. S., Clauder-Muenster, S., Jakob, P., Mueller, W. F., Ordoñez-Rueda, D., Paulsen, M., Matsui, N., Foley, D., Rafalko, A., Suzuki, T., Bogyo, M., Steinmetz, L. M., Radhakrishnan, S. K., Bertozzi, C. R. 2017; 3 (11): 1143?55

    Abstract

    Proteasome inhibitors are used to treat blood cancers such as multiple myeloma (MM) and mantle cell lymphoma. The efficacy of these drugs is frequently undermined by acquired resistance. One mechanism of proteasome inhibitor resistance may involve the transcription factor Nuclear Factor, Erythroid 2 Like 1 (NFE2L1, also referred to as Nrf1), which responds to proteasome insufficiency or pharmacological inhibition by upregulating proteasome subunit gene expression. This "bounce-back" response is achieved through a unique mechanism. Nrf1 is constitutively translocated into the ER lumen, N-glycosylated, and then targeted for proteasomal degradation via the ER-associated degradation (ERAD) pathway. Proteasome inhibition leads to accumulation of cytosolic Nrf1, which is then processed to form the active transcription factor. Here we show that the cytosolic enzyme N-glycanase 1 (NGLY1, the human PNGase) is essential for Nrf1 activation in response to proteasome inhibition. Chemical or genetic disruption of NGLY1 activity results in the accumulation of misprocessed Nrf1 that is largely excluded from the nucleus. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Through a small molecule screen, we identified a cell-active NGLY1 inhibitor that disrupts the processing and function of Nrf1. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis.

    View details for PubMedID 29202016

  • Miraculous Chemistry on the Han River. ACS central science Bertozzi, C. 2017; 3 (5): 360?61

    View details for PubMedID 28573193

    View details for PubMedCentralID PMC5445543

  • A Model for Accelerating Patient-to-Bench Research. ACS central science Bertozzi, C. 2017; 3 (11): 1129?30

    View details for PubMedID 29202011

    View details for PubMedCentralID PMC5704282

  • Chemical Modulation of Protein O-GlcNAcylation via OGT Inhibition Promotes Human Neural Cell Differentiation. ACS chemical biology Andres, L. M., Blong, I. W., Evans, A. C., Rumachik, N. G., Yamaguchi, T., Pham, N. D., Thompson, P., Kohler, J. J., Bertozzi, C. R. 2017; 12 (8): 2030?39

    Abstract

    The enzymes that determine protein O-GlcNAcylation, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), act on key transcriptional and epigenetic regulators, and both are abundantly expressed in the brain. However, little is known about how alterations in O-GlcNAc cycling affect human embryonic stem cell (hESC) neural differentiation. Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac4-5SGlcNAc). Treatment of hESCs with Ac4-5SGlcNAc during induction limited protein O-GlcNAcylation and also caused a dramatic decrease in global levels of UDP-GlcNAc. Concomitantly, a subpopulation of neural progenitor cells (NPCs) acquired an immature neuronal morphology and expressed early neuronal markers such as ?-III tubulin (TUJ1) and microtubule associated protein 2 (MAP2), phenotypes that took longer to manifest in the absence of OGT inhibition. These data suggest that chemical inhibition of OGT and perturbation of protein O-GlcNAcylation accelerate the differentiation of hESCs along the neuronal lineage, thus providing further insight into the dynamic molecular mechanisms involved in neuronal development.

    View details for PubMedID 28541657

    View details for PubMedCentralID PMC5850955

  • Chemical Glycoproteomics CHEMICAL REVIEWS Palaniappan, K. K., Bertozzi, C. R. 2016; 116 (23): 14277-14306

    Abstract

    Chemical tools have accelerated progress in glycoscience, reducing experimental barriers to studying protein glycosylation, the most widespread and complex form of posttranslational modification. For example, chemical glycoproteomics technologies have enabled the identification of specific glycosylation sites and glycan structures that modulate protein function in a number of biological processes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. In this review, we describe the important role that chemical glycoproteomics methods are playing in such efforts. We summarize developments in four key areas: enrichment of glycoproteins and glycopeptides from complex mixtures, emphasizing methods that exploit unique chemical properties of glycans or introduce unnatural functional groups through metabolic labeling and chemoenzymatic tagging; identification of sites of protein glycosylation; targeted glycoproteomics; and functional glycoproteomics, with a focus on probing interactions between glycoproteins and glycan-binding proteins. Our goal with this survey is to provide a foundation on which continued technological advancements can be made to promote further explorations of protein glycosylation.

    View details for DOI 10.1021/acs.chemrev.6b00023

    View details for Web of Science ID 000389962700001

    View details for PubMedID 27960262

    View details for PubMedCentralID PMC5327817

  • Biosynthesis and Regulation of Sulfomenaquinone, a Metabolite Associated with Virulence in Mycobacterium tuberculosis ACS INFECTIOUS DISEASES Sogi, K. M., Holsclaw, C. M., Fragiadakis, G. K., Nomura, D. K., Leary, J. A., Bertozzi, C. R. 2016; 2 (11): 800-806

    Abstract

    Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. Here, we report the identification of the SMK biosynthetic operon that, in addition to a previously identified sulfotransferase stf3, includes a putative cytochrome P450 gene (cyp128) and a gene of unknown function, rv2269c. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. Loss of Stf3 expression, but not that of Cyp128, is correlated with elevated levels of menaquinone-9, an essential component in the electron-transport chain in M. tuberculosis. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis.

    View details for DOI 10.1021/acsinfecdis.6b00106

    View details for Web of Science ID 000388161300007

    View details for PubMedID 27933784

  • Piperidine-based glycodendrons as protein N-glycan prosthetics BIOORGANIC & MEDICINAL CHEMISTRY Hudak, J. E., Belardi, B., Appel, M. J., Solania, A., Robinson, P. V., Bertozzi, C. R. 2016; 24 (20): 4791-4800

    Abstract

    The generation of homogeneously glycosylated proteins is essential for defining glycoform-specific activity and improving protein-based therapeutics. We present a novel glycodendron prosthetic which can be site-selectively appended to recombinant proteins to create 'N-glycosylated' glycoprotein mimics. Using computational modeling, we designed the dendrimer scaffold and protein attachment point to resemble the native N-glycan architecture. Three piperidine-melamine glycodendrimers were synthesized via a chemoenzymatic route and attached to human growth hormone and the Fc region of human IgG. These products represent a new class of engineered biosimilars bearing novel glycodendrimer structures.

    View details for DOI 10.1016/j.bmc.2016.05.050

    View details for Web of Science ID 000385905800005

    View details for PubMedID 27283789

    View details for PubMedCentralID PMC5052108

  • Isotope-targeted glycoproteomics (IsoTaG) analysis of sialylated N- and O-glycopeptides on an Orbitrap Fusion Tribrid using azido and alkynyl sugars. Analytical and bioanalytical chemistry Woo, C. M., Felix, A., Zhang, L., Elias, J. E., Bertozzi, C. R. 2016: -?

    Abstract

    Protein glycosylation is a post-translational modification (PTM) responsible for many aspects of proteomic diversity and biological regulation. Assignment of intact glycan structures to specific protein attachment sites is a critical step towards elucidating the function encoded in the glycome. Previously, we developed isotope-targeted glycoproteomics (IsoTaG) as a mass-independent mass spectrometry method to characterize azide-labeled intact glycopeptides from complex proteomes. Here, we extend the IsoTaG approach with the use of alkynyl sugars as metabolic labels and employ new probes in analysis of the sialylated glycoproteome from PC-3 cells. Using an Orbitrap Fusion Tribrid mass spectrometer, we identified 699 intact glycopeptides from 192 glycoproteins. These intact glycopeptides represent a total of eight sialylated glycan structures across 126 N- and 576 O-glycopeptides. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome.

    View details for PubMedID 27695962

  • Precision glycocalyx editing as a strategy for cancer immunotherapy. Proceedings of the National Academy of Sciences of the United States of America Xiao, H., Woods, E. C., Vukojicic, P., Bertozzi, C. R. 2016; 113 (37): 10304-10309

    Abstract

    Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody-sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody-enzyme conjugates is therefore a promising avenue for cancer immune therapy.

    View details for DOI 10.1073/pnas.1608069113

    View details for PubMedID 27551071

  • Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells. Data in brief Sheta, R., Roux-Dalvai, F., Woo, C. M., Fournier, F., Bourassa, S., Bertozzi, C. R., Droit, A., Bachvarov, D. 2016; 8: 342-349

    Abstract

    This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

    View details for DOI 10.1016/j.dib.2016.05.060

    View details for PubMedID 27331112

    View details for PubMedCentralID PMC4908283

  • Glyco-seek: Ultrasensitive Detection of Protein-Specific Glycosylation by Proximity Ligation Polymerase Chain Reaction. Journal of the American Chemical Society Robinson, P. V., Tsai, C., de Groot, A. E., McKechnie, J. L., Bertozzi, C. R. 2016; 138 (34): 10722-10725

    Abstract

    We report a non-destructive biochemical technique, termed "Glyco-seek", for analysis of O-GlcNAcylated proteins. Glyco-seek combines chemoenzymatic labeling, proximity ligation, and quantitative polymerase chain reaction to detect O-GlcNAcylated proteins with ultrahigh sensitivity. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. We show that Glyco-seek detects attomoles of glycoproteins of interest from cell lysates, with sensitivity several orders of magnitude higher than that of current techniques. We used the method to directly assay the O-GlcNAcylation status of a low-abundance transcription factor from cell lysates without need for isolation or enrichment.

    View details for DOI 10.1021/jacs.6b03861

    View details for PubMedID 27454154

    View details for PubMedCentralID PMC5327792

  • Nuclear repartitioning of galectin-1 by an extracellular glycan switch regulates mammary morphogenesis. Proceedings of the National Academy of Sciences of the United States of America Bhat, R., Belardi, B., Mori, H., Kuo, P., Tam, A., Hines, W. C., Le, Q., Bertozzi, C. R., Bissell, M. J. 2016; 113 (33): E4820-7

    Abstract

    Branching morphogenesis in the mammary gland is achieved by the migration of epithelial cells through a microenvironment consisting of stromal cells and extracellular matrix (ECM). Here we show that galectin-1 (Gal-1), an endogenous lectin that recognizes glycans bearing N-acetyllactosamine (LacNAc) epitopes, induces branching migration of mammary epithelia in vivo, ex vivo, and in 3D organotypic cultures. Surprisingly, Gal-1's effects on mammary patterning were independent of its glycan-binding ability and instead required localization within the nuclei of mammary epithelia. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Specifically, ?2,6-sialylation of terminal LacNAc residues in the end buds masked Gal-1 ligands, thereby liberating the protein for nuclear translocation. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. Conversely, unsialylated LacNAc glycans, enriched in the epithelial ducts, sequestered Gal-1 in the extracellular environment, ultimately attenuating invasive potential. We also found that malignant breast cells possess higher levels of nuclear Gal-1 and ?2,6-SA and lower levels of LacNAc than nonmalignant cells in culture and in vivo and that nuclear localization of Gal-1 promotes a transformed phenotype. Our findings suggest that differential glycosylation at the level of tissue microanatomy regulates the nuclear function of Gal-1 in the context of mammary gland morphogenesis and in cancer progression.

    View details for DOI 10.1073/pnas.1609135113

    View details for PubMedID 27496330

    View details for PubMedCentralID PMC4995945

  • Nuclear repartitioning of galectin-1 by an extracellular glycan switch regulates mammary morphogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Bhat, R., Belardi, B., Mori, H., Kuo, P., Tam, A., Hines, W. C., Quynh-Thu Le, Q. T., Bertozzi, C. R., Bissell, M. J. 2016; 113 (33): E4820-E4827
  • A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells. Journal of proteomics Sheta, R., Woo, C. M., Roux-Dalvai, F., Fournier, F., Bourassa, S., Droit, A., Bertozzi, C. R., Bachvarov, D. 2016; 145: 91-102

    Abstract

    Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets.Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts - GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

    View details for DOI 10.1016/j.jprot.2016.04.009

    View details for PubMedID 27095597

  • Exercise Your Brain. ACS central science Bertozzi, C. R. 2016; 2 (7): 430-431

    View details for DOI 10.1021/acscentsci.6b00194

    View details for PubMedID 27504487

    View details for PubMedCentralID PMC4965850

  • Postdoc Labor Love. ACS central science Bertozzi, C. 2016; 2 (6): 359-360

    View details for DOI 10.1021/acscentsci.6b00167

    View details for PubMedID 27413776

    View details for PubMedCentralID PMC4919776

  • Click-EM for imaging metabolically tagged nonprotein biomolecules NATURE CHEMICAL BIOLOGY Ngo, J. T., Adams, S. R., Deerinck, T. J., Boassa, D., Rodriguez-Rivera, F., Palida, S. F., Bertozzi, C. R., Ellisman, M. H., Tsien, R. Y. 2016; 12 (6): 459-U128

    Abstract

    EM has long been the main technique for imaging cell structures with nanometer resolution but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal 'click chemistry' ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of click-EM in imaging metabolically tagged DNA, RNA and lipids in cultured cells and neurons and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes.

    View details for DOI 10.1038/NCHEMBIO.2076

    View details for Web of Science ID 000376160600017

    View details for PubMedID 27110681

    View details for PubMedCentralID PMC4871776

  • Achieving Gender Balance in the Chemistry Professoriate Is Not Rocket Science. ACS central science Bertozzi, C. R. 2016; 2 (4): 181-182

    View details for DOI 10.1021/acscentsci.6b00102

    View details for PubMedID 27163045

    View details for PubMedCentralID PMC4850509

  • Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology. PLoS pathogens Lantos, A. B., Carlevaro, G., Araoz, B., Ruiz Diaz, P., Camara, M. d., Buscaglia, C. A., Bossi, M., Yu, H., Chen, X., Bertozzi, C. R., Mucci, J., Campetella, O. 2016; 12 (4)

    Abstract

    Trypanosoma cruzi, the flagellate protozoan agent of Chagas disease or American trypanosomiasis, is unable to synthesize sialic acids de novo. Mucins and trans-sialidase (TS) are substrate and enzyme, respectively, of the glycobiological system that scavenges sialic acid from the host in a crucial interplay for T. cruzi life cycle. The acquisition of the sialyl residue allows the parasite to avoid lysis by serum factors and to interact with the host cell. A major drawback to studying the sialylation kinetics and turnover of the trypomastigote glycoconjugates is the difficulty to identify and follow the recently acquired sialyl residues. To tackle this issue, we followed an unnatural sugar approach as bioorthogonal chemical reporters, where the use of azidosialyl residues allowed identifying the acquired sugar. Advanced microscopy techniques, together with biochemical methods, were used to study the trypomastigote membrane from its glycobiological perspective. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Together with determining their shedding and turnover rates, we also report that several membrane proteins, including TS and its substrates, both glycosylphosphatidylinositol-anchored proteins, are separately distributed on parasite surface and contained in different and highly stable membrane microdomains. Notably, labeling for ?(1,3)Galactosyl residues only partially colocalize with sialylated mucins, indicating that two species of glycosylated mucins do exist, which are segregated at the parasite surface. Moreover, sialylated mucins were included in lipid-raft-domains, whereas TS molecules are not. The location of the surface-anchored TS resulted too far off as to be capable to sialylate mucins, a role played by the shed TS instead. Phosphatidylinositol-phospholipase-C activity is actually not present in trypomastigotes. Therefore, shedding of TS occurs via microvesicles instead of as a fully soluble form.

    View details for DOI 10.1371/journal.ppat.1005559

    View details for PubMedID 27058585

    View details for PubMedCentralID PMC4825991

  • Ultrasensitive Antibody Detection by Agglutination-PCR (ADAP). ACS central science Tsai, C., Robinson, P. V., Spencer, C. A., Bertozzi, C. R. 2016; 2 (3): 139-147

    Abstract

    Antibodies are widely used biomarkers for the diagnosis of many diseases. Assays based on solid-phase immobilization of antigens comprise the majority of clinical platforms for antibody detection, but can be undermined by antigen denaturation and epitope masking. These technological hurdles are especially troublesome in detecting antibodies that bind nonlinear or conformational epitopes, such as anti-insulin antibodies in type 1 diabetes patients and anti-thyroglobulin antibodies associated with thyroid cancers. Radioimmunoassay remains the gold standard for these challenging antibody biomarkers, but the limited multiplexability and reliance on hazardous radioactive reagents have prevented their use outside specialized testing facilities. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). Antibodies bind to and agglutinate synthetic antigen-DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. ADAP detects zepto- to attomoles of antibodies in 2 ?L of sample with a dynamic range spanning 5-6 orders of magnitude. Using ADAP, we detected anti-thyroglobulin autoantibodies from human patient plasma with a 1000-fold increased sensitivity over an FDA-approved radioimmunoassay. Finally, we demonstrate the multiplexability of ADAP by simultaneously detecting multiple antibodies in one experiment. ADAP's combination of simplicity, sensitivity, broad dynamic range, multiplexability, and use of standard PCR protocols creates new opportunities for the discovery and detection of antibody biomarkers.

    View details for PubMedID 27064772

  • Happy Birthday ACS Central Science! ACS central science Bertozzi, C. R. 2016; 2 (3): 117-118

    View details for DOI 10.1021/acscentsci.6b00070

    View details for PubMedID 27163035

    View details for PubMedCentralID PMC4827488

  • Voices of biotech. Nature biotechnology Amit, I., Baker, D., Barker, R., Berger, B., Bertozzi, C., Bhatia, S., Biffi, A., Demichelis, F., Doudna, J., Dowdy, S. F., Endy, D., Helmstaedter, M., Junca, H., June, C., Kamb, S., Khvorova, A., Kim, D., Kim, J., Krishnan, Y., Lakadamyali, M., Lappalainen, T., Lewin, S., Liao, J., Loman, N., Lundberg, E., Lynd, L., Martin, C., Mellman, I., Miyawaki, A., Mummery, C., Nelson, K., Paz, J., Peralta-Yahya, P., Picotti, P., Polyak, K., Prather, K., Qin, J., Quake, S., Regev, A., Rogers, J. A., Shetty, R., Sommer, M., Stevens, M., Stolovitzky, G., Takahashi, M., Tang, F., Teichmann, S., Torres-Padilla, M., Tripathi, L., Vemula, P., Verdine, G., Vollmer, F., Wang, J., Ying, J. Y., Zhang, F., Zhang, T. 2016; 34 (3): 270-275

    View details for DOI 10.1038/nbt.3502

    View details for PubMedID 26963549

  • Training the next generation of biomedical investigators in glycosciences JOURNAL OF CLINICAL INVESTIGATION Agre, P., Bertozzi, C., Bissell, M., Campbell, K. P., Cummings, R. D., Desai, U. R., Estes, M., Flotte, T., Fogleman, G., Gage, F., Ginsburg, D., Gordon, J. I., Hart, G., Hascall, V., Kiessling, L., Kornfeld, S., Lowe, J., Magnani, J., Mahal, L. K., Medzhitov, R., Roberts, R. J., Sackstein, R., Sarkar, R., Schnaar, R., Schwartz, N., Varki, A., Walt, D., Weissman, I. 2016; 126 (2): 405-408

    Abstract

    This position statement originated from a working group meeting convened on April 15, 2015, by the NHLBI and incorporates follow-up contributions by the participants as well as other thought leaders subsequently consulted, who together represent research fields relevant to all branches of the NIH. The group was deliberately composed not only of individuals with a current research emphasis in the glycosciences, but also of many experts from other fields, who evinced a strong interest in being involved in the discussions. The original goal was to discuss the value of creating centers of excellence for training the next generation of biomedical investigators in the glycosciences. A broader theme that emerged was the urgent need to bring the glycosciences back into the mainstream of biology by integrating relevant education into the curricula of medical, graduate, and postgraduate training programs, thus generating a critical sustainable workforce that can advance the much-needed translation of glycosciences into a more complete understanding of biology and the enhanced practice of medicine.

    View details for DOI 10.1172/JCI85905

    View details for Web of Science ID 000370677300001

    View details for PubMedID 26829621

    View details for PubMedCentralID PMC4731185

  • Grand Challenges in Chemistry for 2016 and Beyond. ACS central science Bertozzi, C. R., Chang, C. J., Davis, B. G., Olvera de la Cruz, M., Tirrell, D. A., Zhao, D. 2016; 2 (1): 1-3

    View details for DOI 10.1021/acscentsci.6b00010

    View details for PubMedID 27163017

    View details for PubMedCentralID PMC4827666

  • Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis. Cell Freeman, S. A., Goyette, J., Furuya, W., Woods, E. C., Bertozzi, C. R., Bergmeier, W., Hinz, B., van der Merwe, P. A., Das, R., Grinstein, S. 2016; 164 (1-2): 128-140

    Abstract

    Phagocytosis is initiated by lateral clustering of receptors, which in turn activates Src-family kinases (SFKs). Activation of SFKs requires depletion of tyrosine phosphatases from the area of particle engagement. We investigated how the major phosphatase CD45 is excluded from contact sites, using single-molecule tracking. The mobility of CD45 increased markedly upon engagement of Fc? receptors. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. By micropatterning IgG, the ligand of Fc? receptors, we found that the barrier extended well beyond the perimeter of the receptor-ligand engagement zone. Second messengers generated by Fc? receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. The expanding integrin wave facilitates the zippering of Fc? receptors onto the target and integrates the information from sparse receptor-ligand complexes, coordinating the progression and ultimate closure of the phagocytic cup.

    View details for DOI 10.1016/j.cell.2015.11.048

    View details for PubMedID 26771488

    View details for PubMedCentralID PMC4715264

  • Isotope Targeted Glycoproteomics (IsoTaG) to Characterize Intact, Metabolically Labeled Glycopeptides from Complex Proteomes. Current protocols in chemical biology Woo, C. M., Bertozzi, C. R. 2016; 8 (1): 59-82

    Abstract

    Protein glycosylation plays many critical roles in biological function and creates the most diversity of all post-translational modifications (PTMs). Glycan structural diversity is directly correlated with difficulty in characterizing the intact glycoproteome by mass spectrometry (MS). In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). To use IsoTaG, cell culture samples are metabolically labeled with an azido- or alkynyl-sugar. Metabolically labeled glycoproteins are then tagged using Click chemistry and enriched with an isotopic recoding biotin probe. Intact glycopeptides are recovered by cleavage of the probe, analyzed with directed MS, and assigned by targeted mass-independent data analysis. The outlined procedure is well defined in cell culture and has been executed with over 15 cell lines. © 2016 by John Wiley & Sons, Inc.

    View details for DOI 10.1002/9780470559277.ch150185

    View details for PubMedID 26995354

    View details for PubMedCentralID PMC4809415

  • Precision glycocalyx editing as a strategy for cancer immunotherapy. Proceedings of the National Academy of Sciences of the United States of America Xiao, H., Woods, E. C., Vukojicic, P., Bertozzi, C. R. 2016

    Abstract

    Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Here, we report the development of antibody-sialidase conjugates that enhance tumor cell susceptibility to antibody-dependent cell-mediated cytotoxicity (ADCC) by selective desialylation of the tumor cell glycocalyx. We chemically fused a recombinant sialidase to the human epidermal growth factor receptor 2 (HER2)-specific antibody trastuzumab through a C-terminal aldehyde tag. The antibody-sialidase conjugate desialylated tumor cells in a HER2-dependent manner, reduced binding by natural killer (NK) cell inhibitory sialic acid-binding Ig-like lectin (Siglec) receptors, and enhanced binding to the NK-activating receptor natural killer group 2D (NKG2D). Sialidase conjugation to trastuzumab enhanced ADCC against tumor cells expressing moderate levels of HER2, suggesting a therapeutic strategy for cancer patients with lower HER2 levels or inherent trastuzumab resistance. Precision glycocalyx editing with antibody-enzyme conjugates is therefore a promising avenue for cancer immune therapy.

    View details for PubMedID 27551071

  • Back to the Lecture. ACS central science Bertozzi, C. R. 2016; 2 (8): 483?85

    View details for PubMedID 27610405

    View details for PubMedCentralID PMC4999963

  • Ingredients for a Positive Safety Culture. ACS central science Bertozzi, C. R. 2016; 2 (11): 764?66

    View details for PubMedID 27924302

    View details for PubMedCentralID PMC5126708

  • A Season of Giving to Science. ACS central science Bertozzi, C. R., Paley, M. A. 2016; 2 (12): 872?73

    View details for PubMedID 28058273

    View details for PubMedCentralID PMC5200921

  • A fluorogenic screening platform enables directed evolution of an alkyne biosynthetic tool CHEMICAL COMMUNICATIONS Zhu, X., Shieh, P., Su, M., Bertozzi, C. R., Zhang, W. 2016; 52 (75): 11239-11242

    Abstract

    Directed evolution was used to improve the activity of JamB, a membrane-bound bifunctional desaturase/acetylenase. To quickly assess the protein engineering outcomes, we developed a new platform for quantifying extracellular alkyne-tagged metabolites through a fluorogenic click reaction. Random mutagenesis yielded the best JamB variant with ?20-fold increased activity in E. coli.

    View details for DOI 10.1039/c6cc05990b

    View details for Web of Science ID 000384202600014

    View details for PubMedID 27561030

    View details for PubMedCentralID PMC5023497

  • Advice for "Hunting Season". ACS central science Bertozzi, C. R. 2015; 1 (9): 464-465

    View details for DOI 10.1021/acscentsci.5b00386

    View details for PubMedID 27163007

    View details for PubMedCentralID PMC4827657

  • A Bioorthogonal Reaction of N-Oxide and Boron Reagents ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Kim, J., Bertozzi, C. R. 2015; 54 (52): 15777-15781

    Abstract

    The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. In this report, we seek to expand the functional repertoire of such transformations by introducing a new bond-cleaving reaction between N-oxide and boron reagents. The reaction features a large dynamic range of reactivity, showcasing second-order rate constants as high as 2.3×10(3) ?M(-1) ?s(-1) using diboron reaction partners. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and effectively reduce N-oxides inside mammalian cells. This new bioorthogonal process based on miniscule components is thus well-suited for activating molecules within cells under chemical control. Furthermore, we demonstrate that the metabolic diversity of nature enables the use of naturally occurring functional groups that display inherent biocompatibility alongside abiotic components for organism-specific applications.

    View details for DOI 10.1002/anie.201508861

    View details for Web of Science ID 000368061800024

    View details for PubMedCentralID PMC4715665

  • Glycocalyx Engineering with a Recycling Glycopolymer that Increases Cell Survival In Vivo ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Woods, E. C., Yee, N. A., Shen, J., Bertozzi, C. R. 2015; 54 (52): 15782-15788
  • Chemistry Is Central to Repairing Genes and Global Health: Reflections on the 2015 Nobel Prizes in Chemistry and Physiology or Medicine. ACS central science Bertozzi, C. R. 2015; 1 (7): 343-344

    View details for DOI 10.1021/acscentsci.5b00336

    View details for PubMedID 27162990

    View details for PubMedCentralID PMC4827536

  • Chemically tunable mucin chimeras assembled on living cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kramer, J. R., Onoa, B., Bustamante, C., Bertozzi, C. R. 2015; 112 (41): 12574-12579

    View details for DOI 10.1073/pnas.1516127112

    View details for PubMedID 26420872

  • It's about the Students. ACS central science Bertozzi, C. R. 2015; 1 (6): 279-280

    View details for DOI 10.1021/acscentsci.5b00301

    View details for PubMedID 27162982

    View details for PubMedCentralID PMC4827521

  • Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry. Angewandte Chemie (International ed. in English) Agarwal, P., Beahm, B. J., Shieh, P., Bertozzi, C. R. 2015; 54 (39): 11504-11510

    Abstract

    Vertebrate glycans constitute a large, important, and dynamic set of post-translational modifications that are notoriously difficult to manipulate and image. Although the chemical reporter strategy has been used in conjunction with bioorthogonal chemistry to image the external glycosylation state of live zebrafish and detect tumor-associated glycans in mice, the ability to image glycans systemically within a live organism has remained elusive. Here, we report a method that combines the metabolic incorporation of a cyclooctyne-functionalized sialic acid derivative with a ligation reaction of a fluorogenic tetrazine, allowing for the imaging of sialylated glycoconjugates within live zebrafish embryos.

    View details for DOI 10.1002/anie.201504249

    View details for PubMedID 26230529

  • Live-Cell Labeling of Specific Protein Glycoforms by Proximity-Enhanced Bioorthogonal Ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Robinson, P. V., de Almeida-Escobedo, G., de Groot, A. E., McKechnie, J. L., Bertozzi, C. R. 2015; 137 (33): 10452-10455
  • On Being a Closer. ACS central science Bertozzi, C. R., Paley, M. A. 2015; 1 (5): 217-218

    View details for DOI 10.1021/acscentsci.5b00275

    View details for PubMedID 27162973

    View details for PubMedCentralID PMC4827550

  • Chemical Lectinology: Tools for Probing the Ligands and Dynamics of Mammalian Lectins In Vivo. Chemistry & biology Belardi, B., Bertozzi, C. R. 2015; 22 (8): 983-993

    Abstract

    The importance and complexity associated with the totality of glycan structures, i.e. the glycome, has garnered significant attention from chemists and biologists alike. However, what is lacking from this biochemical picture is how cells, tissues, and organisms interpret glycan patterns and translate this information into appropriate responses. Lectins, glycan-binding proteins, are thought to bridge this gap by decoding the glycome and dictating cell fate based on the underlying chemical identities and properties of the glycome. Yet, our understanding of the in vivo ligands and function for most lectins is still incomplete. This review focuses on recent advances in chemical tools to study the specificity and dynamics of mammalian lectins in live cells. A picture emerges of lectin function that is highly sensitive to its organization, which in turn drastically shapes immunity and cancer progression. We hope this review will inspire biologists to make use of these new techniques and stimulate chemists to continue developing innovative approaches to probe lectin biology in vivo.

    View details for DOI 10.1016/j.chembiol.2015.07.009

    View details for PubMedID 26256477

    View details for PubMedCentralID PMC4567249

  • On the Move. ACS central science Bertozzi, C. R. 2015; 1 (4): 157-158

    View details for DOI 10.1021/acscentsci.5b00245

    View details for PubMedID 27162965

    View details for PubMedCentralID PMC4827477

  • CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shieh, P., Dien, V. T., Beahm, B. J., Castellano, J. M., Wyss-Coray, T., Bertozzi, C. R. 2015; 137 (22): 7145-7151

    Abstract

    Fluorescent bioorthogonal smart probes across the visible spectrum will enable sensitive visualization of metabolically labeled molecules in biological systems. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Termed the CalFluors, these probes possess emission maxima that range from green to far red wavelengths, and enable sensitive biomolecule detection under no-wash conditions. We used the CalFluor probes to image various alkyne-labeled biomolecules (glycans, DNA, RNA, and proteins) in cells, developing zebrafish, and mouse brain tissue slices.

    View details for DOI 10.1021/jacs.5b02383

    View details for Web of Science ID 000356322300038

    View details for PubMedID 25902190

    View details for PubMedCentralID PMC4487548

  • Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis NATURE METHODS Woo, C. M., Iavarone, A. T., Spiciarich, D. R., Palaniappan, K. K., Bertozzi, C. R. 2015; 12 (6): 561-?

    Abstract

    Protein glycosylation is a heterogeneous post-translational modification (PTM) that plays an essential role in biological regulation. However, the diversity found in glycoproteins has undermined efforts to describe the intact glycoproteome via mass spectrometry (MS). We present IsoTaG, a mass-independent chemical glycoproteomics platform for characterization of intact, metabolically labeled glycopeptides at the whole-proteome scale. In IsoTaG, metabolic labeling of the glycoproteome is combined with (i) chemical enrichment and isotopic recoding of glycopeptides to select peptides for targeted glycoproteomics using directed MS and (ii) mass-independent assignment of intact glycopeptides. We structurally assigned 32 N-glycopeptides and over 500 intact and fully elaborated O-glycopeptides from 250 proteins across three human cancer cell lines and also discovered unexpected peptide sequence polymorphisms (pSPs). The IsoTaG platform is broadly applicable to the discovery of PTM sites that are amenable to chemical labeling, as well as previously unknown protein isoforms including pSPs.

    View details for DOI 10.1038/nmeth.3366

    View details for Web of Science ID 000355248100027

    View details for PubMedID 25894945

  • A Meeting Full of Firsts. ACS central science Bertozzi, C. R. 2015; 1 (2): 57-?

    View details for DOI 10.1021/acscentsci.5b00185

    View details for PubMedID 27162947

    View details for PubMedCentralID PMC4827500

  • Glycocalyx Engineering with a Recycling Glycopolymer that Increases Cell Survival In Vivo. Angewandte Chemie (International ed. in English) Woods, E. C., Yee, N. A., Shen, J., Bertozzi, C. R. 2015; 54 (52): 15782?88

    Abstract

    Synthetic glycopolymers that emulate cell-surface mucins have been used to elucidate the role of mucin overexpression in cancer. However, because they are internalized within hours, these glycopolymers could not be employed to probe processes that occur on longer time scales. In this work, we tested a panel of glycopolymers bearing a variety of lipids to identify those that persist on cell membranes. Strikingly, we found that cholesterylamine (CholA) anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least ten days, even while the cells are dividing. As with native mucins, the cell-surface display of CholA-anchored glycopolymers influenced the focal adhesion distribution. Furthermore, we show that these mimetics enhance the survival of nonmalignant cells in a zebrafish model of metastasis. CholA-anchored glycopolymers therefore expand the application of glycocalyx engineering in glycobiology.

    View details for PubMedID 26647316

    View details for PubMedCentralID PMC4736730

  • Symbol Nomenclature for Graphical Representations of Glycans. Glycobiology Varki, A., Cummings, R. D., Aebi, M., Packer, N. H., Seeberger, P. H., Esko, J. D., Stanley, P., Hart, G., Darvill, A., Kinoshita, T., Prestegard, J. J., Schnaar, R. L., Freeze, H. H., Marth, J. D., Bertozzi, C. R., Etzler, M. E., Frank, M., Vliegenthart, J. F., Lütteke, T., Perez, S., Bolton, E., Rudd, P., Paulson, J., Kanehisa, M., Toukach, P., Aoki-Kinoshita, K. F., Dell, A., Narimatsu, H., York, W., Taniguchi, N., Kornfeld, S. 2015; 25 (12): 1323?24

    View details for PubMedID 26543186

    View details for PubMedCentralID PMC4643639

  • A Bioorthogonal Reaction of N-Oxide and Boron Reagents. Angewandte Chemie (International ed. in English) Kim, J., Bertozzi, C. R. 2015; 54 (52): 15777?81

    Abstract

    The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. In this report, we seek to expand the functional repertoire of such transformations by introducing a new bond-cleaving reaction between N-oxide and boron reagents. The reaction features a large dynamic range of reactivity, showcasing second-order rate constants as high as 2.3×10(3) ?M(-1) ?s(-1) using diboron reaction partners. Diboron reagents display minimal cell toxicity at millimolar concentrations, penetrate cell membranes, and effectively reduce N-oxides inside mammalian cells. This new bioorthogonal process based on miniscule components is thus well-suited for activating molecules within cells under chemical control. Furthermore, we demonstrate that the metabolic diversity of nature enables the use of naturally occurring functional groups that display inherent biocompatibility alongside abiotic components for organism-specific applications.

    View details for PubMedID 26568479

    View details for PubMedCentralID PMC4715665

  • The cancer glycocalyx mechanically primes integrin-mediated growth and survival NATURE Paszek, M. J., DuFort, C. C., Rossier, O., Bainer, R., Mouw, J. K., Godula, K., Hudak, J. E., Lakins, J. N., Wijekoon, A. C., Cassereau, L., Rubashkin, M. G., Magbanua, M. J., Thorn, K. S., Davidson, M. W., Rugo, H. S., Park, J. W., Hammer, D. A., Giannone, G., Bertozzi, C. R., Weaver, V. M. 2014; 511 (7509): 319-?

    Abstract

    Malignancy is associated with altered expression of glycans and glycoproteins that contribute to the cellular glycocalyx. We constructed a glycoprotein expression signature, which revealed that metastatic tumours upregulate expression of bulky glycoproteins. A computational model predicted that these glycoproteins would influence transmembrane receptor spatial organization and function. We tested this prediction by investigating whether bulky glycoproteins in the glycocalyx promote a tumour phenotype in human cells by increasing integrin adhesion and signalling. Our data revealed that a bulky glycocalyx facilitates integrin clustering by funnelling active integrins into adhesions and altering integrin state by applying tension to matrix-bound integrins, independent of actomyosin contractility. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. Clinical studies revealed that large glycoproteins are abundantly expressed on circulating tumour cells from patients with advanced disease. Thus, a bulky glycocalyx is a feature of tumour cells that could foster metastasis by mechanically enhancing cell-surface receptor function.

    View details for DOI 10.1038/nature13535

    View details for Web of Science ID 000338992200029

    View details for PubMedID 25030168

  • Imaging bacterial peptidoglycan with near-infrared fluorogenic azide probes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shieh, P., Siegrist, M. S., Cullen, A. J., Bertozzi, C. R. 2014; 111 (15): 5456-5461

    Abstract

    Fluorescent probes designed for activation by bioorthogonal chemistry have enabled the visualization of biomolecules in living systems. Such activatable probes with near-infrared (NIR) emission would be ideal for in vivo imaging but have proven difficult to engineer. We present the development of NIR fluorogenic azide probes based on the Si-rhodamine scaffold that undergo a fluorescence enhancement of up to 48-fold upon reaction with terminal or strained alkynes. We used the probes for mammalian cell surface imaging and, in conjunction with a new class of cyclooctyne D-amino acids, for visualization of bacterial peptidoglycan without the need to wash away unreacted probe.

    View details for DOI 10.1073/pnas.1322727111

    View details for Web of Science ID 000334288600021

    View details for PubMedID 24706769

  • Glycocalyx engineering reveals a Siglec-based mechanism for NK cell immunoevasion NATURE CHEMICAL BIOLOGY Hudak, J. E., Canham, S. M., Bertozzi, C. R. 2014; 10 (1): 69-U111

    Abstract

    The increase of cell surface sialic acid is a characteristic shared by many tumor types. A correlation between hypersialylation and immunoprotection has been observed, but few hypotheses have provided a mechanistic understanding of this immunosuppressive phenomenon. Here, we show that increasing sialylated glycans on cancer cells inhibits human natural killer (NK) cell activation through the recruitment of sialic acid-binding immunoglobulin-like lectin 7 (Siglec-7). Key to these findings was the use of glycopolymers end-functionalized with phospholipids, which enable the introduction of synthetically defined glycans onto cancer cell surfaces. Remodeling the sialylation status of cancer cells affected the susceptibility to NK cell cytotoxicity via Siglec-7 engagement in a variety of tumor types. These results support a model in which hypersialylation offers a selective advantage to tumor cells under pressure from NK immunosurveillance by increasing Siglec ligands. We also exploited this finding to protect allogeneic and xenogeneic primary cells from NK-mediated killing, suggesting the potential of Siglecs as therapeutic targets in cell transplant therapy.

    View details for DOI 10.1038/NCHEMBIO.1388

    View details for Web of Science ID 000328854900013

    View details for PubMedID 24292068

  • Osmosensory signaling in Mycobacterium tuberculosis mediated by a eukaryotic-like Ser/Thr protein kinase PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hatzios, S. K., Baer, C. E., Rustad, T. R., Siegrist, M. S., Pang, J. M., Ortega, C., Alber, T., Grundner, C., Sherman, D. R., Bertozzi, C. R. 2013; 110 (52): E5069-E5077

    Abstract

    Bacteria are able to adapt to dramatically different microenvironments, but in many organisms, the signaling pathways, transcriptional programs, and downstream physiological changes involved in adaptation are not well-understood. Here, we discovered that osmotic stress stimulates a signaling network in Mycobacterium tuberculosis regulated by the eukaryotic-like receptor Ser/Thr protein kinase PknD. Expression of the PknD substrate Rv0516c was highly induced by osmotic stress. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative ?-factor SigF. Phosphorylation of Rv0516c regulated the abundance of EspA, a virulence-associated substrate of the type VII ESX-1 secretion system. These findings identify an osmosensory pathway orchestrated by PknD, Rv0516c, and SigF that enables adaptation to osmotic stress through cell wall remodeling and virulence factor production. Given the widespread occurrence of eukaryotic-like Ser/Thr protein kinases in bacteria, these proteins may play a broad role in bacterial osmosensing.

    View details for DOI 10.1073/pnas.1321205110

    View details for Web of Science ID 000328858800008

    View details for PubMedID 24309377

  • Imaging the Glycosylation State of Cell Surface Glycoproteins by Two-Photon Fluorescence Lifetime Imaging Microscopy ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Belardi, B., de la Zerda, A., Spiciarich, D. R., Maund, S. L., Peehl, D. M., Bertozzi, C. R. 2013; 52 (52): 14045-14049

    View details for DOI 10.1002/anie.201307512

    View details for Web of Science ID 000328531100027

    View details for PubMedID 24259491

    View details for PubMedCentralID PMC3920747

  • A Chemical Glycoproteomics Platform Reveals O-GlcNAcylation of Mitochondrial Voltage-Dependent Anion Channel 2 CELL REPORTS Palaniappan, K. K., Hangauer, M. J., Smith, T. J., Smart, B. P., Pitcher, A. A., Cheng, E. H., Bertozzi, C. R., Boyce, M. 2013; 5 (2): 546-552

    Abstract

    Protein modification by O-linked ?-N-acetylglucosamine (O-GlcNAc) is a critical cell signaling modality, but identifying signal-specific O-GlcNAcylation events remains a significant experimental challenge. Here, we describe a method for visualizing and analyzing organelle- and stimulus-specific O-GlcNAcylated proteins and use it to identify the mitochondrial voltage-dependent anion channel 2 (VDAC2) as an O-GlcNAc substrate. VDAC2(-/-) cells resist the mitochondrial dysfunction and apoptosis caused by global O-GlcNAc perturbation, demonstrating a functional connection between O-GlcNAc signaling and mitochondrial physiology through VDAC2. More broadly, our method will enable the discovery of signal-specific O-GlcNAcylation events in a wide array of experimental contexts.

    View details for DOI 10.1016/j.celrep.2013.08.048

    View details for Web of Science ID 000328263000025

    View details for PubMedID 24120863

    View details for PubMedCentralID PMC3869705

  • Modulation of Ocular Surface Glycocalyx Barrier Function by a Galectin-3 N-terminal Deletion Mutant and Membrane-Anchored Synthetic Glycopolymers PLOS ONE Mauris, J., Mantelli, F., Woodward, A. M., Cao, Z., Bertozzi, C. R., Panjwani, N., Godula, K., Argueeso, P. 2013; 8 (8)
  • Sulfatase-activated fluorophores for rapid discrimination of mycobacterial species and strains PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Beatty, K. E., Williams, M., Carlson, B. L., Swarts, B. M., Warren, R. M., van Helden, P. D., Bertozzi, C. R. 2013; 110 (32): 12911-12916

    Abstract

    Most current diagnostic tests for tuberculosis do not reveal the species or strain of pathogen causing pulmonary infection, which can lead to inappropriate treatment regimens and the spread of disease. Here, we report an assay for mycobacterial strain assignment based on genetically conserved mycobacterial sulfatases. We developed a sulfatase-activated probe, 7-hydroxy-9H-(1,3-dichloro-9,9-dimethylacridin-2-one)-sulfate, that detects enzyme activity in native protein gels, allowing the rapid detection of sulfatases in mycobacterial lysates. This assay revealed that mycobacterial strains have distinct sulfatase fingerprints that can be used to judge both the species and lineage. Our results demonstrate the potential of enzyme-activated probes for rapid pathogen discrimination for infectious diseases.

    View details for DOI 10.1073/pnas.1222041110

    View details for Web of Science ID 000322771100029

    View details for PubMedID 23878250

    View details for PubMedCentralID PMC3740907

  • Synthesis and Reactivity of Dibenzoselenacycloheptynes ORGANIC LETTERS de Almeida, G., Townsend, L. C., Bertozzi, C. R. 2013; 15 (12): 3038-3041

    Abstract

    Dibenzoselenacycloheptynes were prepared in three steps from commercially available reagents and trapped in situ with benzyl azide to form the corresponding triazoles. Surprisingly, the dibenzoselenacycloheptynes also abstracted hydrogen atoms from solvents such as THF or toluene, forming dibenzoselenacycloheptene products. These alkenyl compounds arise from a hydrogen transfer reaction from solvent to the unisolable intermediate, and we postulate that the reaction proceeds via a radical mechanism originating from the strained alkynyl bond that has unusually high radical character.

    View details for DOI 10.1021/ol401225n

    View details for Web of Science ID 000320979000038

    View details for PubMedID 23734979

    View details for PubMedCentralID PMC3827634

  • Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging PLOS ONE Sogi, K. M., Gartner, Z. J., Breidenbach, M. A., Appel, M. J., Schelle, M. W., Bertozzi, C. R. 2013; 8 (6)

    Abstract

    The genome of Mycobacterium tuberculosis (Mtb) encodes nine putative sulfatases, none of which have a known function or substrate. Here, we characterize Mtb's single putative type II sulfatase, Rv3406, as a non-heme iron (II) and ?-ketoglutarate-dependent dioxygenase that catalyzes the oxidation and subsequent cleavage of alkyl sulfate esters. Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Using an in vitro biochemical assay, we confirmed that Rv3406 is a sulfatase with a preference for alkyl sulfate substrates similar to those processed by AtsK. We determined the crystal structure of the apo Rv3406 sulfatase at 2.5 Å. The active site residues of Rv3406 and AtsK are essentially superimposable, suggesting that the two sulfatases share the same catalytic mechanism. Finally, we generated an Rv3406 mutant (?rv3406) in Mtb to study the sulfatase's role in sulfate scavenging. The ?rv3406 strain did not replicate in minimal media with 2-ethyl hexyl sulfate as the sole sulfur source, in contrast to wild type Mtb or the complemented strain. We conclude that Rv3406 is an iron and ?-ketoglutarate-dependent sulfate ester dioxygenase that has unique substrate specificity that is likely distinct from other Mtb sulfatases.

    View details for DOI 10.1371/journal.pone.0065080

    View details for Web of Science ID 000321099000031

    View details for PubMedID 23762287

    View details for PubMedCentralID PMC3675115

  • Self-Assembly of "S-Bilayers", a Step Toward Expanding the Dimensionality of S-Layer Assemblies ACS NANO Shin, S., Comolli, L. R., Tscheliessnig, R., Wang, C., Ki Tae Nam, K. T., Hexemer, A., Siegerist, C. E., De Yoreo, J. J., Bertozzi, C. R. 2013; 7 (6): 4946-4953

    Abstract

    Protein-based assemblies with ordered nanometer-scale features in three dimensions are of interest as functional nanomaterials but are difficult to generate. Here we report that a truncated S-layer protein assembles into stable bilayers, which we characterized using cryogenic-electron microscopy, tomography, and X-ray spectroscopy. We find that emergence of this supermolecular architecture is the outcome of hierarchical processes; the proteins condense in solution to form 2-D crystals, which then stack parallel to one another to create isotropic bilayered assemblies. Within this bilayered structure, registry between lattices in two layers was disclosed, whereas the intrinsic symmetry in each layer was altered. Comparison of these data to images of wild-type SbpA layers on intact cells gave insight into the interactions responsible for bilayer formation. These results establish a platform for engineering S-layer assemblies with 3-D architecture.

    View details for DOI 10.1021/nn400263j

    View details for Web of Science ID 000321093800027

    View details for PubMedID 23705800

  • D-Amino Acid Chemical Reporters Reveal Peptidoglycan Dynamics of an Intracellular Pathogen ACS CHEMICAL BIOLOGY Siegrist, M. S., Whiteside, S., Jewett, J. C., Aditham, A., Cava, F., Bertozzi, C. R. 2013; 8 (3): 500-505

    Abstract

    Peptidoglycan (PG) is an essential component of the bacterial cell wall. Although experiments with organisms in vitro have yielded a wealth of information on PG synthesis and maturation, it is unclear how these studies translate to bacteria replicating within host cells. We report a chemical approach for probing PG in vivo via metabolic labeling and bioorthogonal chemistry. A wide variety of bacterial species incorporated azide and alkyne-functionalized d-alanine into their cell walls, which we visualized by covalent reaction with click chemistry probes. The d-alanine analogues were specifically incorporated into nascent PG of the intracellular pathogen Listeria monocytogenes both in vitro and during macrophage infection. Metabolic incorporation of d-alanine derivatives and click chemistry detection constitute a facile, modular platform that facilitates unprecedented spatial and temporal resolution of PG dynamics in vivo.

    View details for DOI 10.1021/cb3004995

    View details for Web of Science ID 000316375500003

    View details for PubMedID 23240806

    View details for PubMedCentralID PMC3601600

  • Strategy for Dual-Analyte Luciferin Imaging: In Vivo Bioluminescence Detection of Hydrogen Peroxide and Caspase Activity in a Murine Model of Acute Inflammation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Van de Bittner, G. C., Bertozzi, C. R., Chang, C. J. 2013; 135 (5): 1783-1795

    Abstract

    In vivo molecular imaging holds promise for understanding the underlying mechanisms of health, injury, aging, and disease, as it can detect distinct biochemical processes such as enzymatic activity, reactive small-molecule fluxes, or post-translational modifications. Current imaging techniques often detect only a single biochemical process, but, within whole organisms, multiple types of biochemical events contribute to physiological and pathological phenotypes. In this report, we present a general strategy for dual-analyte detection in living animals that employs in situ formation of firefly luciferin from two complementary caged precursors that can be unmasked by different biochemical processes. To establish this approach, we have developed Peroxy Caged Luciferin-2 (PCL-2), a H(2)O(2)-responsive boronic acid probe that releases 6-hydroxy-2-cyanobenzothiazole (HCBT) upon reacting with this reactive oxygen species, as well as a peptide-based probe, z-Ile-Glu-ThrAsp-D-Cys (IETDC), which releases D-cysteine in the presence of active caspase 8. Once released, HCBT and D-cysteine form firefly luciferin in situ, giving rise to a bioluminescent signal if and only if both chemical triggers proceed. This system thus constitutes an AND-type molecular logic gate that reports on the simultaneous presence of H(2)O(2) and caspase 8 activity. Using these probes, chemoselective imaging of either H(2)O(2) or caspase 8 activity was performed in vitro and in vivo. Moreover, concomitant use of PCL-2 and IETDC in vivo establishes a concurrent increase in both H(2)O(2) and caspase 8 activity during acute inflammation in living mice. Taken together, this method offers a potentially powerful new chemical tool for studying simultaneous oxidative stress and inflammation processes in living animals during injury, aging, and disease, as well as a versatile approach for concurrent monitoring of multiple analytes using luciferin-based bioluminescence imaging technologies.

    View details for DOI 10.1021/ja309078t

    View details for Web of Science ID 000314794400031

    View details for PubMedID 23347279

    View details for PubMedCentralID PMC3583381

  • A Pictet-Spengler ligation for protein chemical modification PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Agarwal, P., van der Weijden, J., Sletten, E. M., Rabuka, D., Bertozzi, C. R. 2013; 110 (1): 46-51

    Abstract

    Aldehyde- and ketone-functionalized proteins are appealing substrates for the development of chemically modified biotherapeutics and protein-based materials. Their reactive carbonyl groups are typically conjugated with ?-effect nucleophiles, such as substituted hydrazines and alkoxyamines, to generate hydrazones and oximes, respectively. However, the resulting C=N linkages are susceptible to hydrolysis under physiologically relevant conditions, which limits the utility of such conjugates in biological systems. Here we introduce a Pictet-Spengler ligation that is based on the classic Pictet-Spengler reaction of aldehydes and tryptamine nucleophiles. The ligation exploits the bioorthogonal reaction of aldehydes and alkoxyamines to form an intermediate oxyiminium ion; this intermediate undergoes intramolecular C-C bond formation with an indole nucleophile to form an oxacarboline product that is hydrolytically stable. We used the reaction for site-specific chemical modification of glyoxyl- and formylglycine-functionalized proteins, including an aldehyde-tagged variant of the therapeutic monoclonal antibody Herceptin. In conjunction with techniques for site-specific introduction of aldehydes into proteins, the Pictet-Spengler ligation offers a means to generate stable bioconjugates for medical and materials applications.

    View details for DOI 10.1073/pnas.1213186110

    View details for Web of Science ID 000313630300024

    View details for PubMedID 23237853

  • Real-Time Noninvasive Imaging of Fatty Acid Uptake in Vivo ACS CHEMICAL BIOLOGY Henkin, A. H., Cohen, A. S., Dubikovskaya, E. A., Park, H. M., Nikitin, G. F., Auzias, M. G., Kazantzis, M., Bertozzi, C. R., Stahl, A. 2012; 7 (11): 1884-1891

    Abstract

    Detection and quantification of fatty acid fluxes in animal model systems following physiological, pathological, or pharmacological challenges is key to our understanding of complex metabolic networks as these macronutrients also activate transcription factors and modulate signaling cascades including insulin sensitivity. To enable noninvasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. This imaging approach should further our understanding of basic metabolic processes and pathological alterations in multiple disease models.

    View details for DOI 10.1021/cb300194b

    View details for Web of Science ID 000311072900014

    View details for PubMedID 22928772

    View details for PubMedCentralID PMC3500440

  • Fluorogenic Azidofluoresceins for Biological Imaging JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shieh, P., Hangauer, M. J., Bertozzi, C. R. 2012; 134 (42): 17428-17431

    Abstract

    Fluorogenic probes activated by bioorthogonal chemical reactions can enable biomolecule imaging in situations where it is not possible to wash away unbound probe. One challenge for the development of such probes is the a priori identification of structures that will undergo a dramatic fluorescence enhancement by virtue of the chemical transformation. With the aid of density functional theory calculations reported previously by Nagano and co-workers, we identified azidofluorescein derivatives that were predicted to undergo an increase in fluorescence quantum yield upon Cu-catalyzed or Cu-free cycloaddition with linear or cyclic alkynes, respectively. Four derivatives were experimentally verified in model reactions, and one, a 4-azidonaphthylfluorescein analogue, was further shown to label alkyne-functionalized proteins in vitro and glycoproteins on cells with excellent selectivity. The azidofluorescein derivative also enabled cell imaging under no-wash conditions with good signal above background. This work establishes a platform for the rational design of fluorogenic azide probes with spectral properties tailored for biological imaging.

    View details for DOI 10.1021/ja308203h

    View details for Web of Science ID 000310103800025

    View details for PubMedID 23025473

    View details for PubMedCentralID PMC3596100

  • Probing the Mycobacterial Trehalome with Bioorthogonal Chemistry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Swarts, B. M., Holsclaw, C. M., Jewett, J. C., Alber, M., Fox, D. M., Siegrist, M. S., Leary, J. A., Kalscheuer, R., Bertozzi, C. R. 2012; 134 (39): 16123-16126

    Abstract

    Mycobacteria, including the pathogen Mycobacterium tuberculosis, use the non-mammalian disaccharide trehalose as a precursor for essential cell-wall glycolipids and other metabolites. Here we describe a strategy for exploiting trehalose metabolic pathways to label glycolipids in mycobacteria with azide-modified trehalose (TreAz) analogues. Subsequent bioorthogonal ligation with alkyne-functionalized probes enabled detection and visualization of cell-surface glycolipids. Characterization of the metabolic fates of four TreAz analogues revealed unique labeling routes that can be harnessed for pathway-targeted investigation of the mycobacterial trehalome.

    View details for DOI 10.1021/ja3062419

    View details for Web of Science ID 000309335000008

    View details for PubMedID 22978752

    View details for PubMedCentralID PMC3466019

  • Density Variant Glycan Microarray for Evaluating Cross-Linking of Mucin-like Glycoconjugates by Lectins JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Godula, K., Bertozzi, C. R. 2012; 134 (38): 15732-15742

    Abstract

    Interactions of mucin glycoproteins with cognate receptors are dictated by the structures and spatial organization of glycans that decorate the mucin polypeptide backbone. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. In this work, we employed a microarray platform comprising synthetic glycopolymers that emulate natural mucins arrayed at different surface densities to evaluate how glycan valency and spatial separation affect the preferential binding mode of a particular lectin. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for ?-N-acetylgalactosamine (?-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. While these lectins possess the ability to agglutinate A(1)-blood cells carrying the ?-GalNAc epitope and cross-link low valency glycoconjugates, only SBA showed a tendency to form intermolecular cross-links among the arrayed polyvalent mucin mimetics. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. Our findings indicate that glycan valency can set thresholds for cross-linking by lectins. More broadly, well-defined synthetic glycopolymers enable the integration of glycoconjugate structural and spatial diversity in a single microarray screening platform.

    View details for DOI 10.1021/ja302193u

    View details for Web of Science ID 000309099700030

    View details for PubMedID 22967056

    View details for PubMedCentralID PMC3458438

  • Direct observation of kinetic traps associated with structural transformations leading to multiple pathways of S-layer assembly PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shin, S., Chung, S., Sanii, B., Comolli, L. R., Bertozzi, C. R., De Yoreo, J. J. 2012; 109 (32): 12968-12973

    Abstract

    The concept of a folding funnel with kinetic traps describes folding of individual proteins. Using in situ Atomic Force Microscopy to investigate S-layer assembly on mica, we show this concept is equally valid during self-assembly of proteins into extended matrices. We find the S-layer-on-mica system possesses a kinetic trap associated with conformational differences between a long-lived transient state and the final stable state. Both ordered tetrameric states emerge from clusters of the monomer phase, however, they then track along two different pathways. One leads directly to the final low-energy state and the other to the kinetic trap. Over time, the trapped state transforms into the stable state. By analyzing the time and temperature dependencies of formation and transformation we find that the energy barriers to formation of the two states differ by only 0.7 kT, but once the high-energy state forms, the barrier to transformation to the low-energy state is 25 kT. Thus the transient state exhibits the characteristics of a kinetic trap in a folding funnel.

    View details for DOI 10.1073/pnas.1201504109

    View details for Web of Science ID 000307551700033

    View details for PubMedID 22822216

    View details for PubMedCentralID PMC3420203

  • Investigating Cell Surface Galectin-Mediated Cross-Linking on Glycoengineered Cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Belardi, B., O'Donoghue, G. P., Smith, A. W., Groves, J. T., Bertozzi, C. R. 2012; 134 (23): 9549-9552

    Abstract

    The galectin family of glycan-binding proteins is thought to mediate many cellular processes by oligomerizing cell surface glycoproteins and glycolipids into higher-order aggregates. This hypothesis reflects the known oligomeric states of the galectins themselves and their binding properties with multivalent ligands in vitro, but direct evidence of their ability to cross-link ligands on a cell surface is lacking. A major challenge in fundamental studies of galectin-ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. Consequently, there is no obvious means to selectively monitor the behaviors of natural galectin ligands on live cell surfaces. Here we describe an approach for probing the galectin-induced multimerization of glycoconjugates on cultured cells. Using RAFT polymerization, we synthesized well-defined glycopolymers (GPs) functionalized with galectin-binding glycans along the backbone, a lipid group on one end and a fluorophore on the other. After insertion into live cell membranes, the GPs' fluorescence lifetime and diffusion time were measured in the presence and absence of galectin-1. We observed direct evidence for galectin-1-mediated extended cross-linking on the engineered cells, a phenomenon that was dependent on glycan structure. This platform offers a new approach to exploring the "galectin lattice" hypothesis and to defining galectin ligand specificity in a physiologically relevant context.

    View details for DOI 10.1021/ja301694s

    View details for Web of Science ID 000305107800004

    View details for PubMedID 22540968

    View details for PubMedCentralID PMC3374418

  • Reactivity of Biarylazacyclooctynones in Copper-Free Click Chemistry JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Gordon, C. G., Mackey, J. L., Jewett, J. C., Sletten, E. M., Houk, K. N., Bertozzi, C. R. 2012; 134 (22): 9199-9208

    Abstract

    The 1,3-dipolar cycloaddition of cyclooctynes with azides, also called "copper-free click chemistry", is a bioorthogonal reaction with widespread applications in biological discovery. The kinetics of this reaction are of paramount importance for studies of dynamic processes, particularly in living subjects. Here we performed a systematic analysis of the effects of strain and electronics on the reactivity of cyclooctynes with azides through both experimental measurements and computational studies using a density functional theory (DFT) distortion/interaction transition state model. In particular, we focused on biarylazacyclooctynone (BARAC) because it reacts with azides faster than any other reported cyclooctyne and its modular synthesis facilitated rapid access to analogues. We found that substituents on BARAC's aryl rings can alter the calculated transition state interaction energy of the cycloaddition through electronic effects or the calculated distortion energy through steric effects. Experimental data confirmed that electronic perturbation of BARAC's aryl rings has a modest effect on reaction rate, whereas steric hindrance in the transition state can significantly retard the reaction. Drawing on these results, we analyzed the relationship between alkyne bond angles, which we determined using X-ray crystallography, and reactivity, quantified by experimental second-order rate constants, for a range of cyclooctynes. Our results suggest a correlation between decreased alkyne bond angle and increased cyclooctyne reactivity. Finally, we obtained structural and computational data that revealed the relationship between the conformation of BARAC's central lactam and compound reactivity. Collectively, these results indicate that the distortion/interaction model combined with bond angle analysis will enable predictions of cyclooctyne reactivity and the rational design of new reagents for copper-free click chemistry.

    View details for DOI 10.1021/ja3000936

    View details for Web of Science ID 000304837800041

    View details for PubMedID 22553995

  • Site-specific chemical protein conjugation using genetically encoded aldehyde tags NATURE PROTOCOLS Rabuka, D., Rush, J. S., dehart, G. W., Wu, P., Bertozzi, C. R. 2012; 7 (6): 1052-1067

    Abstract

    We describe a method for modifying proteins site-specifically using a chemoenzymatic bioconjugation approach. Formylglycine generating enzyme (FGE) recognizes a pentapeptide consensus sequence, CxPxR, and it specifically oxidizes the cysteine in this sequence to an unusual aldehyde-bearing formylglyine. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The conversion of cysteine to formylglycine is accomplished by co-overexpression of FGE, either transiently or as a stable cell line, and the resulting aldehyde can be selectively reacted with ?-nucleophiles to generate a site-selectively modified bioconjugate. This protocol outlines both the generation and the analysis of proteins aldehyde-tagged at their termini and the methods for chemical conjugation to the formylglycine. The process of generating aldehyde-tagged protein followed by chemical conjugation and purification takes 20 d.

    View details for DOI 10.1038/nprot.2012.045

    View details for Web of Science ID 000304720700005

    View details for PubMedID 22576105

    View details for PubMedCentralID PMC3498491

  • Mapping Yeast N-Glycosites with Isotopically Recoded Glycans MOLECULAR & CELLULAR PROTEOMICS Breidenbach, M. A., Palaniappan, K. K., Pitcher, A. A., Bertozzi, C. R. 2012; 11 (6)

    Abstract

    Asparagine-linked glycosylation is a common post-translational modification of proteins; in addition to participating in key macromolecular interactions, N-glycans contribute to protein folding, trafficking, and stability. Despite their importance, few N-glycosites have been experimentally mapped in the Saccharomyces cerevisiae proteome. Factors including glycan heterogeneity, low abundance, and low occupancy can complicate site mapping. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Isotopic recoding is achieved via metabolic incorporation of a defined mixture of N-acetylglucosamine isotopologs into N-glycans. Peptides bearing the recoded envelopes are specifically targeted for fragmentation, facilitating high confidence site mapping. This strategy requires no chemical modification of the N-glycans or stringent sample enrichment. Further, enzymatically simplified N-glycans are preserved on peptides. Using this approach, we identify 133 N-glycosites spanning 58 proteins, nearly doubling the number of experimentally observed N-glycosites in the yeast proteome.

    View details for DOI 10.1074/mcp.M111.015339

    View details for Web of Science ID 000306408500017

    View details for PubMedID 22261724

  • Cellular Microfabrication: Observing Intercellular Interactions Using Lithographically-Defined DNA Capture Sequences LANGMUIR Onoe, H., Hsiao, S. C., Douglas, E. S., Gartner, Z. J., Bertozzi, C. R., Francis, M. B., Mathies, R. A. 2012; 28 (21): 8120-8126

    Abstract

    Previous reports have shown that synthetic DNA strands can be attached to the plasma membrane of living cells to equip them with artificial adhesion "receptors" that bind to complementary strands extending from material surfaces. This approach is compatible with a wide range of cell types, offers excellent capture efficiency, and can potentially be used to create complex multicellular arrangements through the use of multiple capture sequences. In this work, we apply an aluminum "lift off" lithography method to allow the efficient generation of complex patterns comprising different DNA sequences. The resulting surfaces are then demonstrated to be able to capture up to three distinct types of living cells in specific locations. The utility of this approach is demonstrated through the observation of patterned cells as they communicate by diffusion-based paracrine signaling. It is anticipated that the ability of this technique to create virtually any type of 2D heterogeneous cell pattern should prove highly useful for the examination of key questions in cell signaling, including stem cell differentiation and cancer metastasis.

    View details for DOI 10.1021/la204863s

    View details for Web of Science ID 000304492700020

    View details for PubMedID 22512362

  • Sulfolipid-1 Biosynthesis Restricts Mycobacterium tuberculosis Growth in Human Macrophages ACS CHEMICAL BIOLOGY Gilmore, S. A., Schelle, M. W., Holsclaw, C. M., Leigh, C. D., Jain, M., Cox, J. S., Leary, J. A., Bertozzi, C. R. 2012; 7 (5): 863-870

    Abstract

    Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, is a highly evolved human pathogen characterized by its formidable cell wall. Many unique lipids and glycolipids from the Mtb cell wall are thought to be virulence factors that mediate host-pathogen interactions. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Previously, we described the biochemical activity of the sulfotransferase Stf0 that initiates SL-1 biosynthesis. Here we show that a stf0-deletion mutant exhibits augmented survival in human but not murine macrophages, suggesting that SL-1 negatively regulates the intracellular growth of Mtb in a species-specific manner. Furthermore, we demonstrate that SL-1 plays a role in mediating the susceptibility of Mtb to a human cationic antimicrobial peptide in vitro, despite being dispensable for maintaining overall cell envelope integrity. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages.

    View details for DOI 10.1021/cb200311s

    View details for Web of Science ID 000304129200013

    View details for PubMedID 22360425

    View details for PubMedCentralID PMC3355658

  • Extensive Determination of Glycan Heterogeneity Reveals an Unusual Abundance of High Mannose Glycans in Enriched Plasma Membranes of Human Embryonic Stem Cells MOLECULAR & CELLULAR PROTEOMICS An, H. J., Gip, P., Kim, J., Wu, S., Park, K. W., McVaugh, C. T., Schaffer, D. V., Bertozzi, C. R., Lebrilla, C. B. 2012; 11 (4)

    Abstract

    Most cell membrane proteins are known or predicted to be glycosylated in eukaryotic organisms, where surface glycans are essential in many biological processes including cell development and differentiation. Nonetheless, the glycosylation on cell membranes remains not well characterized because of the lack of sensitive analytical methods. This study introduces a technique for the rapid profiling and quantitation of N- and O-glycans on cell membranes using membrane enrichment and nanoflow liquid chromatography/mass spectrometry of native structures. Using this new method, the glycome analysis of cell membranes isolated from human embryonic stem cells and somatic cell lines was performed. Human embryonic stem cells were found to have high levels of high mannose glycans, which contrasts with IMR-90 fibroblasts and a human normal breast cell line, where complex glycans are by far the most abundant and high mannose glycans are minor components. O-Glycosylation affects relatively minor components of cell surfaces. To verify the quantitation and localization of glycans on the human embryonic stem cell membranes, flow cytometry and immunocytochemistry were performed. Proteomics analyses were also performed and confirmed enrichment of plasma membrane proteins with some contamination from endoplasmic reticulum and other membranes. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. High mannose glycans are not commonly presented on the surfaces of mammalian cells or in serum yet may play important roles in stem cell biology. The results also mean that distinguishing stem cells from other mammalian cells may be facilitated by the major difference in the glycosylation of the cell membrane. The deep structural analysis enabled by this new method will enable future mechanistic studies on the biological significance of high mannose glycans on stem cell membranes and provide a general tool to examine cell surface glycosylation.

    View details for DOI 10.1074/mcp.M111.010660

    View details for Web of Science ID 000302786500006

    View details for PubMedID 22147732

    View details for PubMedCentralID PMC3322563

  • Metabolic labeling enables selective photocrosslinking of O-GlcNAc-modified proteins to their binding partners PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Yu, S., Boyce, M., Wands, A. M., Bond, M. R., Bertozzi, C. R., Kohler, J. J. 2012; 109 (13): 4834-4839

    Abstract

    O-linked ?-N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification found on hundreds of nuclear and cytoplasmic proteins in higher eukaryotes. Despite its ubiquity and essentiality in mammals, functional roles for the O-GlcNAc modification remain poorly defined. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. Irradiation of cells with UV light activated the crosslinker, resulting in formation of covalent bonds between O-GlcNAc-modified proteins and neighboring molecules, which could be identified by mass spectrometry. We used this method to identify interaction partners for the O-GlcNAc-modified FG-repeat nucleoporins. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Further, we propose that the method reported here could find widespread use in investigating the functional consequences of O-GlcNAcylation.

    View details for DOI 10.1073/pnas.1114356109

    View details for Web of Science ID 000302164200031

    View details for PubMedID 22411826

    View details for PubMedCentralID PMC3323966

  • Elucidation and Chemical Modulation of Sulfolipid-1 Biosynthesis in Mycobacterium tuberculosis JOURNAL OF BIOLOGICAL CHEMISTRY Seeliger, J. C., Holsclaw, C. M., Schelle, M. W., Botyanszki, Z., Gilmore, S. A., Tully, S. E., Niederweis, M., Cravatt, B. F., Leary, J. A., Bertozzi, C. R. 2012; 287 (11): 7990-8000

    Abstract

    Mycobacterium tuberculosis possesses unique cell-surface lipids that have been implicated in virulence. One of the most abundant is sulfolipid-1 (SL-1), a tetraacyl-sulfotrehalose glycolipid. Although the early steps in SL-1 biosynthesis are known, the machinery underlying the final acylation reactions is not understood. We provide genetic and biochemical evidence for the activities of two proteins, Chp1 and Sap (corresponding to gene loci rv3822 and rv3821), that complete this pathway. The membrane-associated acyltransferase Chp1 accepts a synthetic diacyl sulfolipid and transfers an acyl group regioselectively from one donor substrate molecule to a second acceptor molecule in two successive reactions to yield a tetraacylated product. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. We also show that the integral membrane protein Sap and MmpL8 are both essential for sulfolipid transport. Finally, the lipase inhibitor tetrahydrolipstatin disrupts Chp1 activity in M. tuberculosis, suggesting an avenue for perturbing SL-1 biosynthesis in vivo. These data complete the SL-1 biosynthetic pathway and corroborate a model in which lipid biosynthesis and transmembrane transport are coupled at the membrane-cytosol interface through the activity of multiple proteins, possibly as a macromolecular complex.

    View details for DOI 10.1074/jbc.M111.315473

    View details for Web of Science ID 000301349400015

    View details for PubMedID 22194604

  • Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Yao, J. Z., Uttamapinant, C., Poloukhtine, A., Baskin, J. M., Codelli, J. A., Sletten, E. M., Bertozzi, C. R., Popik, V. V., Ting, A. Y. 2012; 134 (8): 3720-3728

    Abstract

    Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

    View details for DOI 10.1021/ja208090p

    View details for Web of Science ID 000301161600025

    View details for PubMedID 22239252

    View details for PubMedCentralID PMC3306817

  • Cholesterol Catabolism by Mycobacterium tuberculosis Requires Transcriptional and Metabolic Adaptations CHEMISTRY & BIOLOGY Griffin, J. E., Pandey, A. K., Gilmore, S. A., Mizrahi, V., McKinney, J. D., Bertozzi, C. R., Sassetti, C. M. 2012; 19 (2): 218-227

    Abstract

    To understand the adaptation of Mycobacterium tuberculosis to the intracellular environment, we used comprehensive metabolite profiling to identify the biochemical pathways utilized during growth on cholesterol, a critical carbon source during chronic infection. Metabolic alterations observed during cholesterol catabolism centered on propionyl-CoA and pyruvate pools. Consequently, growth on this substrate required the transcriptional induction of the propionyl-CoA-assimilating methylcitrate cycle (MCC) enzymes, via the Rv1129c regulatory protein. We show that both Rv1129c and the MCC enzymes are required for intracellular growth in macrophages and that the growth defect of MCC mutants is largely attributable to the degradation of host-derived cholesterol. Together, these observations define a coordinated transcriptional and metabolic adaptation that is required for scavenging carbon during intracellular growth.

    View details for DOI 10.1016/j.chembiol.2011.12.016

    View details for Web of Science ID 000300917300009

    View details for PubMedID 22365605

    View details for PubMedCentralID PMC3292763

  • Imaging the Sialome during Zebrafish Development with Copper-Free Click Chemistry CHEMBIOCHEM Dehnert, K. W., Baskin, J. M., Laughlin, S. T., Beahm, B. J., Naidu, N. N., Amacher, S. L., Bertozzi, C. R. 2012; 13 (3): 353-357

    Abstract

    The sialome comprises sialylated glycoproteins and glycolipids that play essential roles in cell-cell communication. Using azide-modified molecular precursors of sialic acids and copper-free click chemistry, we visualized the spatiotemporal dynamics of the sialome in live zebrafish embryos.

    View details for DOI 10.1002/cbic.201100649

    View details for Web of Science ID 000299834900004

    View details for PubMedID 22262667

    View details for PubMedCentralID PMC3385855

  • A Riboswitch-Based Inducible Gene Expression System for Mycobacteria PLOS ONE Seeliger, J. C., Topp, S., Sogi, K. M., Previti, M. L., Gallivan, J. P., Bertozzi, C. R. 2012; 7 (1)

    Abstract

    Research on the human pathogen Mycobacterium tuberculosis (Mtb) would benefit from novel tools for regulated gene expression. Here we describe the characterization and application of a synthetic riboswitch-based system, which comprises a mycobacterial promoter for transcriptional control and a riboswitch for translational control. The system was used to induce and repress heterologous protein overexpression reversibly, to create a conditional gene knockdown, and to control gene expression in a macrophage infection model. Unlike existing systems for controlling gene expression in Mtb, the riboswitch does not require the co-expression of any accessory proteins: all of the regulatory machinery is encoded by a short DNA segment directly upstream of the target gene. The inducible riboswitch platform has the potential to be a powerful general strategy for creating customized gene regulation systems in Mtb.

    View details for DOI 10.1371/journal.pone.0029266

    View details for Web of Science ID 000299771900011

    View details for PubMedID 22279533

    View details for PubMedCentralID PMC3261144

  • Thiacycloalkynes for Copper-Free Click Chemistry ANGEWANDTE CHEMIE-INTERNATIONAL EDITION de Almeida, G., Sletten, E. M., Nakamura, H., Palaniappan, K. K., Bertozzi, C. R. 2012; 51 (10): 2443-2447

    View details for DOI 10.1002/anie.201106325

    View details for Web of Science ID 000300934700029

    View details for PubMedID 22282228

    View details for PubMedCentralID PMC3384729

  • Synthesis of Heterobifunctional Protein Fusions Using Copper-Free Click Chemistry and the Aldehyde Tag ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Hudak, J. E., Barfield, R. M., de Hart, G. W., Grob, P., Nogales, E., Bertozzi, C. R., Rabuka, D. 2012; 51 (17): 4161-4165

    View details for DOI 10.1002/anie.201108130

    View details for Web of Science ID 000303001000032

    View details for PubMedID 22407566

  • Synthesis of a Fluorogenic Cyclooctyne Activated by Cu-Free Click Chemistry ORGANIC LETTERS Jewett, J. C., Bertozzi, C. R. 2011; 13 (22): 5937-5939

    Abstract

    Cyclooctyne-based probes that become fluorescent upon reaction with azides are important targets for real-time imaging of azide-labeled biomolecules. The concise synthesis of a coumarin-conjugated cyclooctyne, coumBARAC, that undergoes a 10-fold enhancement in fluorescence quantum yield upon triazole formation with organic azides is reported. The design principles embodied in coumBARAC establish a platform for generating fluorogenic cyclooctynes suited for biological imaging.

    View details for DOI 10.1021/ol2025026

    View details for Web of Science ID 000296756600001

    View details for PubMedID 22029411

    View details for PubMedCentralID PMC3219546

  • A Bioorthogonal Quadricyclane Ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sletten, E. M., Bertozzi, C. R. 2011; 133 (44): 17570-17573

    Abstract

    New additions to the bioorthogonal chemistry compendium can advance biological research by enabling multiplexed analysis of biomolecules in complex systems. Here we introduce the quadricyclane ligation, a new bioorthogonal reaction between the highly strained hydrocarbon quadricyclane and Ni bis(dithiolene) reagents. This reaction has a second-order rate constant of 0.25 M(-1) s(-1), on par with fast bioorthogonal reactions of azides, and proceeds readily in aqueous environments. Ni bis(dithiolene) probes selectively labeled quadricyclane-modified bovine serum albumin, even in the presence of cell lysate. We have demonstrated that the quadricyclane ligation is compatible with, and orthogonal to, strain-promoted azide-alkyne cycloaddition and oxime ligation chemistries by performing all three reactions in one pot on differentially functionalized protein substrates. The quadricyclane ligation joins a small but growing list of tools for the selective covalent modification of biomolecules.

    View details for DOI 10.1021/ja2072934

    View details for Web of Science ID 000296312200014

    View details for PubMedID 21962173

  • Protein Glycoengineering Enabled by the Versatile Synthesis of Aminooxy Glycans and the Genetically Encoded Aldehyde Tag JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hudak, J. E., Yu, H. H., Bertozzi, C. R. 2011; 133 (40): 16127-16135

    Abstract

    Homogeneously glycosylated proteins are important targets for fundamental research and for biopharmaceutical development. The use of unnatural protein-glycan linkages bearing structural similarity to their native counterparts can accelerate the synthesis of glycoengineered proteins. Here we report an approach toward generating homogeneously glycosylated proteins that involves chemical attachment of aminooxy glycans to recombinantly produced proteins via oxime linkages. We employed the recently introduced aldehyde tag method to obtain a recombinant protein with the aldehyde-bearing formylglycine residue at a specific site. Complex aminooxy glycans were synthesized using a new route that features N-pentenoyl hydroxamates as key intermediates that can be readily elaborated chemically and enzymatically. We demonstrated the method by constructing site-specifically glycosylated variants of the human growth hormone.

    View details for DOI 10.1021/ja206023e

    View details for Web of Science ID 000296036700058

    View details for PubMedID 21866965

    View details for PubMedCentralID PMC3187659

  • Cell surface glycoproteomic analysis of prostate cancer-derived PC-3 cells BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Hubbard, S. C., Boyce, M., McVaugh, C. T., Peehl, D. M., Bertozzi, C. R. 2011; 21 (17): 4945-4950

    Abstract

    Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. PC-3 cells and primary human prostate cancer tissue were treated with peracetylated N-azidoacetylgalactosamine, resulting in metabolic labeling of cell surface glycans with the azidosugar. We used mass spectrometry to identify over 70 cell surface glycoproteins and biochemically validated CD146 and integrin beta-4, both of which are known to promote metastatic behavior. These results establish cell-surface glycoproteomics as an effective technique for discovery of cancer biomarkers.

    View details for DOI 10.1016/j.bmcl.2011.05.045

    View details for Web of Science ID 000293884100002

    View details for PubMedID 21798741

    View details for PubMedCentralID PMC3341932

  • From Mechanism to Mouse: A Tale of Two Bioorthogonal Reactions ACCOUNTS OF CHEMICAL RESEARCH Sletten, E. M., Bertozzi, C. R. 2011; 44 (9): 666-676

    Abstract

    Bioorthogonal reactions are chemical reactions that neither interact with nor interfere with a biological system. The participating functional groups must be inert to biological moieties, must selectively reactive with each other under biocompatible conditions, and, for in vivo applications, must be nontoxic to cells and organisms. Additionally, it is helpful if one reactive group is small and therefore minimally perturbing of a biomolecule into which it has been introduced either chemically or biosynthetically. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an analysis of functional group and reactivity space outside those defined by Nature. Issues such as stability of reactants and products (particularly in water), kinetics, and unwanted side reactivity with biofunctionalities must be addressed, ideally guided by detailed mechanistic studies. Finally, the reaction must be tested in a variety of environments, escalating from aqueous media to biomolecule solutions to cultured cells and, for the most optimized transformations, to live organisms. Work in our laboratory led to the development of two bioorthogonal transformations that exploit the azide as a small, abiotic, and bioinert reaction partner: the Staudinger ligation and strain-promoted azide-alkyne cycloaddition. The Staudinger ligation is based on the classic Staudinger reduction of azides with triarylphosphines first reported in 1919. In the ligation reaction, the intermediate aza-ylide undergoes intramolecular reaction with an ester, forming an amide bond faster than aza-ylide hydrolysis would otherwise occur in water. The Staudinger ligation is highly selective and reliably forms its product in environs as demanding as live mice. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddition reactions. In 1961, Wittig and Krebs noted that the strained, cyclic alkyne cyclooctyne reacts violently when combined neat with phenyl azide, forming a triazole product by 1,3-dipolar cycloaddition. This observation stood in stark contrast to the slow kinetics associated with 1,3-dipolar cycloaddition of azides with unstrained, linear alkynes, the conventional Huisgen process. Notably, the reaction of azides with terminal alkynes can be accelerated dramatically by copper catalysis (this highly popular Cu-catalyzed azide-alkyne cycloaddition (CuAAC) is a quintessential "click" reaction). However, the copper catalysts are too cytotoxic for long-term exposure with live cells or organisms. Thus, for applications of bioorthogonal chemistry in living systems, we built upon Wittig and Krebs' observation with the design of cyclooctyne reagents that react rapidly and selectively with biomolecule-associated azides. This strain-promoted azide-alkyne cycloaddition is often referred to as "Cu-free click chemistry". Mechanistic and theoretical studies inspired the design of a series of cyclooctyne compounds bearing fluorine substituents, fused rings, and judiciously situated heteroatoms, with the goals of optimizing azide cycloaddition kinetics, stability, solubility, and pharmacokinetic properties. Cyclooctyne reagents have now been used for labeling azide-modified biomolecules on cultured cells and in live Caenorhabditis elegans, zebrafish, and mice. As this special issue testifies, the field of bioorthogonal chemistry is firmly established as a challenging frontier of reaction methodology and an important new instrument for biological discovery. The above reactions, as well as several newcomers with bioorthogonal attributes, have enabled the high-precision chemical modification of biomolecules in vitro, as well as real-time visualization of molecules and processes in cells and live organisms. The consequence is an impressive body of new knowledge and technology, amassed using a relatively small bioorthogonal reaction compendium. Expansion of this toolkit, an effort that is already well underway, is an important objective for chemists and biologists alike.

    View details for DOI 10.1021/ar200148z

    View details for Web of Science ID 000296075300003

    View details for PubMedID 21838330

  • The Mycobacterium tuberculosis CysQ phosphatase modulates the biosynthesis of sulfated glycolipids and bacterial growth BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Hatzios, S. K., Schelle, M. W., Newton, G. L., Sogi, K. M., Holsclaw, C. M., Fahey, R. C., Bertozzi, C. R. 2011; 21 (17): 4956-4959

    Abstract

    CysQ is a 3'-phosphoadenosine-5'-phosphatase that dephosphorylates intermediates from the sulfate assimilation pathway of Mycobacterium tuberculosis (Mtb). Here, we demonstrate that cysQ disruption attenuates Mtb growth in vitro and decreases the biosynthesis of sulfated glycolipids but not major thiols, suggesting that the encoded enzyme specifically regulates mycobacterial sulfation.

    View details for DOI 10.1016/j.bmcl.2011.06.057

    View details for Web of Science ID 000293884100004

    View details for PubMedID 21795043

    View details for PubMedCentralID PMC3184767

  • Bringing chemistry to life NATURE METHODS Boyce, M., Bertozzi, C. R. 2011; 8 (8): 638-642

    View details for DOI 10.1038/nmeth.1657

    View details for Web of Science ID 000293220600016

    View details for PubMedID 21799498

    View details for PubMedCentralID PMC3184769

  • Isotopic Signature Transfer and Mass Pattern Prediction (IsoStamp): An Enabling Technique for Chemically-Directed Proteomics ACS CHEMICAL BIOLOGY Palaniappan, K. K., Pitcher, A. A., Smart, B. P., Spiciarich, D. R., Iavarone, A. T., Bertozzi, C. R. 2011; 6 (8): 829-836

    Abstract

    Directed proteomics applies mass spectrometry analysis to a subset of information-rich proteins. Here we describe a method for targeting select proteins by chemical modification with a tag that imparts a distinct isotopic signature detectable in a full-scan mass spectrum. Termed isotopic signature transfer and mass pattern prediction (IsoStamp), the technique exploits the perturbing effects of a dibrominated chemical tag on a peptide's mass envelope, which can be detected with high sensitivity and fidelity using a computational method. Applying IsoStamp, we were able to detect femtomole quantities of a single tagged protein from total mammalian cell lysates at signal-to-noise ratios as low as 2.5:1. To identify a tagged-peptide's sequence, we performed an inclusion list-driven shotgun proteomics experiment where peptides bearing a recoded mass envelope were targeted for fragmentation, allowing for direct site mapping. Using this approach, femtomole quantities of several targeted peptides were identified in total mammalian cell lysate, while traditional data-dependent methods were unable to identify as many peptides. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). IsoStamp has the potential to enhance any proteomics platform that employs chemical labeling for targeted protein identification, including isotope coded affinity tagging, isobaric tagging for relative and absolute quantitation, and chemical tagging strategies for posttranslational modification.

    View details for DOI 10.1021/cb100338x

    View details for Web of Science ID 000294081900008

    View details for PubMedID 21604797

  • The Regulation of Sulfur Metabolism in Mycobacterium tuberculosis PLOS PATHOGENS Hatzios, S. K., Bertozzi, C. R. 2011; 7 (7)

    Abstract

    Mycobacterium tuberculosis (Mtb) has evolved into a highly successful human pathogen. It deftly subverts the bactericidal mechanisms of alveolar macrophages, ultimately inducing granuloma formation and establishing long-term residence in the host. These hallmarks of Mtb infection are facilitated by the metabolic adaptation of the pathogen to its surrounding environment and the biosynthesis of molecules that mediate its interactions with host immune cells. The sulfate assimilation pathway of Mtb produces a number of sulfur-containing metabolites with important contributions to pathogenesis and survival. This pathway is regulated by diverse environmental cues and regulatory proteins that mediate sulfur transactions in the cell. Here, we discuss the transcriptional and biochemical mechanisms of sulfur metabolism regulation in Mtb and potential small molecule regulators of the sulfate assimilation pathway that are collectively poised to aid this intracellular pathogen in its expert manipulation of the host. From this global analysis, we have identified a subset of sulfur-metabolizing enzymes that are sensitive to multiple regulatory cues and may be strong candidates for therapeutic intervention.

    View details for DOI 10.1371/journal.ppat.1002036

    View details for Web of Science ID 000293339300002

    View details for PubMedID 21811406

    View details for PubMedCentralID PMC3141025

  • Metabolic Labeling of Fucosylated Glycans in Developing Zebrafish ACS CHEMICAL BIOLOGY Dehnert, K. W., Beahm, B. J., Huynh, T. T., Baskin, J. M., Laughlin, S. T., Wang, W., Wu, P., Amacher, S. L., Bertozzi, C. R. 2011; 6 (6): 547-552

    Abstract

    Many developmental processes depend on proper fucosylation, but this post-translational modification is difficult to monitor in vivo. Here we applied a chemical reporter strategy to visualize fucosylated glycans in developing zebrafish. Using azide-derivatized analogues of fucose, we metabolically labeled cell-surface glycans and then detected the incorporated azides via copper-free click chemistry with a difluorinated cyclooctyne probe. We found that the fucose salvage pathway enzymes are expressed during zebrafish embryogenesis but that they process the azide-modified substrates inefficiently. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. This nucleotide sugar was readily accepted by fucosyltransferases and provided robust cell-surface labeling of fucosylated glycans, as determined by flow cytometry and confocal microscopy analysis. We used this technique to image fucosylated glycans in the enveloping layer of zebrafish embryos during the first 5 days of development. This work provides a method to study the biosynthesis of fucosylated glycans in vivo.

    View details for DOI 10.1021/cb100284d

    View details for Web of Science ID 000291896400004

    View details for PubMedID 21425872

    View details for PubMedCentralID PMC3117394

  • Metabolic cross-talk allows labeling of O-linked beta-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Boyce, M., Carrico, I. S., Ganguli, A. S., Yu, S., Hangauer, M. J., Hubbard, S. C., Kohler, J. J., Bertozzi, C. R. 2011; 108 (8): 3141-3146

    Abstract

    Hundreds of mammalian nuclear and cytoplasmic proteins are reversibly glycosylated by O-linked ?-N-acetylglucosamine (O-GlcNAc) to regulate their function, localization, and stability. Despite its broad functional significance, the dynamic and posttranslational nature of O-GlcNAc signaling makes it challenging to study using traditional molecular and cell biological techniques alone. Here, we report that metabolic cross-talk between the N-acetylgalactosamine salvage and O-GlcNAcylation pathways can be exploited for the tagging and identification of O-GlcNAcylated proteins. We found that N-azidoacetylgalactosamine (GalNAz) is converted by endogenous mammalian biosynthetic enzymes to UDP-GalNAz and then epimerized to UDP-N-azidoacetylglucosamine (GlcNAz). O-GlcNAc transferase accepts UDP-GlcNAz as a nucleotide-sugar donor, appending an azidosugar onto its native substrates, which can then be detected by covalent labeling using azide-reactive chemical probes. In a proof-of-principle proteomics experiment, we used metabolic GalNAz labeling of human cells and a bioorthogonal chemical probe to affinity-purify and identify numerous O-GlcNAcylated proteins. Our work provides a blueprint for a wide variety of future chemical approaches to identify, visualize, and characterize dynamic O-GlcNAc signaling.

    View details for DOI 10.1073/pnas.1010045108

    View details for Web of Science ID 000287580400017

    View details for PubMedID 21300897

  • A Chemical Method for Labeling Lysine Methyltransferase Substrates CHEMBIOCHEM Binda, O., Boyce, M., Rush, J. S., Palaniappan, K. K., Bertozzi, C. R., Gozani, O. 2011; 12 (2): 330-334

    Abstract

    Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. PKMTs are likely to have many additional substrates in addition to histones, but relatively few nonhistone substrates have been characterized, and the substrate specificity for many PKMTs has yet to be defined. Thus, new unbiased methods are needed to find PKMT substrates. Here, we describe a chemical biology approach for unbiased, proteome-wide identification of novel PKMT substrates. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Such labeled proteins can then be treated with azide-functionalized probes to ligate affinity handles or fluorophores to the PKMT substrates. As a proof-of-concept, we have used SETDB1 to transfer the alkyne moiety from the SAM analogue onto a recombinant histone H3 substrate. We anticipate that this chemical method will find broad use in epigenetics to enable unbiased searches for new PKMT substrates by using recombinant enzymes and unnatural SAM cofactors to label and purify many substrates simultaneously from complex organelle or cell extracts.

    View details for DOI 10.1002/cbic.201000433

    View details for Web of Science ID 000286433800014

    View details for PubMedID 21243721

    View details for PubMedCentralID PMC3056122

  • Organelle Membrane Proteomics Reveals Differential Influence of Mycobacterial Lipoglycans on Macrophage Phagosome Maturation and Autophagosome Accumulation JOURNAL OF PROTEOME RESEARCH Shui, W., Petzold, C. J., Redding, A., Liu, J., Pitcher, A., Sheu, L., Hsieh, T., Keasling, J. D., Bertozzi, C. R. 2011; 10 (1): 339-348

    Abstract

    The mycobacterial cell wall component lipoarabinomannan (LAM) has been described as one of the key virulence factors of Mycobacterium tuberculosis. Modification of the terminal arabinan residues of this lipoglycan with mannose caps in M. tuberculosis or with phosphoinositol caps in Mycobacterium smegmatis results in distinct host immune responses. Given that M. tuberculosis typically persists in the phagosomal vacuole after being phagocytosed by macrophages, we performed a proteomic analysis of that organelle after treatment of macrophages with LAMs purified from the two mycobacterial species. The quantitative changes in phagosomal proteins suggested a distinct role for mannose-capped LAM in modulating protein trafficking pathways that contribute to the arrest of phagosome maturation. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid.

    View details for DOI 10.1021/pr100688h

    View details for Web of Science ID 000285812000039

    View details for PubMedID 21105745

    View details for PubMedCentralID PMC3018347

  • In vivo imaging of hydrogen peroxide production in a murine tumor model with a chemoselective bioluminescent reporter PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Van de Bittner, G. C., Dubikovskaya, E. A., Bertozzi, C. R., Chang, C. J. 2010; 107 (50): 21316-21321

    Abstract

    Living organisms produce hydrogen peroxide (H(2)O(2)) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. The effects of H(2)O(2) on the overall health of living animals remain elusive, in part owing to a dearth of methods for studying this transient small molecule in vivo. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H(2)O(2) within living animals. PCL-1 is a boronic acid-caged firefly luciferin molecule that selectively reacts with H(2)O(2) to release firefly luciferin, which triggers a bioluminescent response in the presence of firefly luciferase. The high sensitivity and selectivity of PCL-1 for H(2)O(2), combined with the favorable properties of bioluminescence for in vivo imaging, afford a unique technology for real-time detection of basal levels of H(2)O(2) generated in healthy, living mice. Moreover, we demonstrate the efficacy of PCL-1 for monitoring physiological fluctuations in H(2)O(2) levels by directly imaging elevations in H(2)O(2) within testosterone-stimulated tumor xenografts in vivo. The ability to chemoselectively monitor H(2)O(2) fluxes in real time in living animals offers opportunities to dissect H(2)O(2)'s disparate contributions to health, aging, and disease.

    View details for DOI 10.1073/pnas.1012864107

    View details for Web of Science ID 000285521500020

    View details for PubMedID 21115844

    View details for PubMedCentralID PMC3003011

  • Synthetic Riboswitches That Induce Gene Expression in Diverse Bacterial Species APPLIED AND ENVIRONMENTAL MICROBIOLOGY Topp, S., Reynoso, C. M., Seeliger, J. C., Goldlust, I. S., Desai, S. K., Murat, D., Shen, A., Puri, A. W., Komeili, A., Bertozzi, C. R., Scott, J. R., Gallivan, J. P. 2010; 76 (23): 7881-7884

    Abstract

    We developed a series of ligand-inducible riboswitches that control gene expression in diverse species of Gram-negative and Gram-positive bacteria, including human pathogens that have few or no previously reported inducible expression systems. We anticipate that these riboswitches will be useful tools for genetic studies in a wide range of bacteria.

    View details for DOI 10.1128/AEM.01537-10

    View details for Web of Science ID 000284310500026

    View details for PubMedID 20935124

    View details for PubMedCentralID PMC2988590

  • Live-Cell Imaging of Cellular Proteins by a Strain-Promoted Azide-Alkyne Cycloaddition CHEMBIOCHEM Beatty, K. E., Fisk, J. D., Smart, B. P., Lu, Y. Y., Szychowski, J., Hangauer, M. J., Baskin, J. M., Bertozzi, C. R., Tirrell, D. A. 2010; 11 (15): 2092-2095

    View details for DOI 10.1002/cbic.201000419

    View details for Web of Science ID 000284050000005

    View details for PubMedID 20836119

    View details for PubMedCentralID PMC3069858

  • Bioluminescent Probes of Sulfatase Activity CHEMBIOCHEM Rush, J. S., Beatty, K. E., Bertozzi, C. R. 2010; 11 (15): 2096-2099

    View details for DOI 10.1002/cbic.201000115

    View details for Web of Science ID 000284050000006

    View details for PubMedID 20872389

    View details for PubMedCentralID PMC3069857

  • Self-catalyzed growth of S layers via an amorphous-to-crystalline transition limited by folding kinetics PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chung, S., Shin, S., Bertozzi, C. R., De Yoreo, J. J. 2010; 107 (38): 16536-16541

    Abstract

    The importance of nonclassical, multistage crystallization pathways is increasingly evident from theoretical studies on colloidal systems and experimental investigations of proteins and biomineral phases. Although theoretical predictions suggest that proteins follow these pathways as a result of fluctuations that create unstable dense-liquid states, microscopic studies indicate these states are long-lived. Using in situ atomic force microscopy to follow 2D assembly of S-layer proteins on supported lipid bilayers, we have obtained a molecular-scale picture of multistage protein crystallization that reveals the importance of conformational transformations in directing the pathway of assembly. We find that monomers with an extended conformation first form a mobile adsorbed phase, from which they condense into amorphous clusters. These clusters undergo a phase transition through S-layer folding into crystalline clusters composed of compact tetramers. Growth then proceeds by formation of new tetramers exclusively at cluster edges, implying tetramer formation is autocatalytic. Analysis of the growth kinetics leads to a quantitative model in which tetramer creation is rate limiting. However, the estimated barrier is much smaller than expected for folding of isolated S-layer proteins, suggesting an energetic rationale for this multistage pathway.

    View details for DOI 10.1073/pnas.1008280107

    View details for Web of Science ID 000282003700030

    View details for PubMedID 20823255

    View details for PubMedCentralID PMC2944705

  • Difluorobenzocyclooctyne: Synthesis, Reactivity, and Stabilization by beta-Cyclodextrin JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Sletten, E. M., Nakamura, H., Jewett, J. C., Bertozzi, C. R. 2010; 132 (33): 11799-11805

    Abstract

    Highly reactive cyclooctynes have been sought as substrates for Cu-free cycloaddition reactions with azides in biological systems. To elevate the reactivities of cyclooctynes, two strategies, LUMO lowering through propargylic fluorination and strain enhancement through fused aryl rings, have been explored. Here we report the facile synthesis of a difluorobenzocyclooctyne (DIFBO) that combines these modifications. DIFBO was so reactive that it spontaneously trimerized to form two asymmetric products that we characterized by X-ray crystallography. However, we were able to trap DIFBO by forming a stable inclusion complex with beta-cyclodextrin in aqueous media. This complex could be stored as a lyophilized powder and then dissociated in organic solvents to produce free DIFBO for in situ kinetic and spectroscopic analysis. Using this procedure, we found that the rate constant for the cycloaddition reaction of DIFBO with an azide exceeds those for difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne (DIBO). Cyclodextrin complexation is therefore a promising approach for stabilizing compounds that possess the high intrinsic reactivities desired for Cu-free click chemistry.

    View details for DOI 10.1021/ja105005t

    View details for Web of Science ID 000281066400069

    View details for PubMedID 20666466

    View details for PubMedCentralID PMC2923465

  • Synthesis of Glycopolymers for Microarray Applications via Ligation of Reducing Sugars to a Poly(acryloyl hydrazide) Scaffold JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Godula, K., Bertozzi, C. R. 2010; 132 (29): 9963-9965

    Abstract

    In this paper, we report on a general synthetic strategy for the assembly of glycopolymers that capitalizes on the intrinsic reactivity of reducing glycans toward hydrazides to form stable cyclic N-glycosides. We developed a poly(acryloyl hydrazide) (PAH) scaffold to which we conjugated a variety of reducing glycans ranging in structure from simple mono- and disaccharides to considerably more complex human milk and blood oligosaccharides. The conjugation proceeds under mild conditions with excellent ligation efficiencies and in a stereoselective manner, providing glycopolymers with pendant glycans accommodated mostly in their cyclic beta-glycosidic form. Utilizing a biotin-terminated PAH scaffold prepared via RAFT polymerization, we quickly assembled a panel of glycopolymers that we microarrayed on streptavidin-coated glass. We then demonstrated that in these microarrays, the glycopolymer ligands bind lectins according to the structures of their pendant glycans. Importantly, glycopolymers containing biologically relevant branched oligosaccharides, such as sialyl Lewis(x), as well as sulfated glycosaminoglycan-like epitopes can be readily prepared using our methodology.

    View details for DOI 10.1021/ja103009d

    View details for Web of Science ID 000280227700012

    View details for PubMedID 20608651

    View details for PubMedCentralID PMC2907714

  • A Strategy for the Selective Imaging of Glycans Using Caged Metabolic Precursors JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chang, P. V., Dube, D. H., Sletten, E. M., Bertozzi, C. R. 2010; 132 (28): 9516-9518

    Abstract

    Glycans can be imaged by metabolic labeling with azidosugars followed by chemical reaction with imaging probes; however, tissue-specific labeling is difficult to achieve. Here we describe a strategy for the use of a caged metabolic precursor that is activated for cellular metabolism by enzymatic cleavage. An N-azidoacetylmannosamine derivative caged with a peptide substrate for the prostate-specific antigen (PSA) protease was converted to cell-surface azido sialic acids in a PSA-dependent manner. The approach has applications in tissue-selective imaging of glycans for clinical and basic research purposes.

    View details for DOI 10.1021/ja101080y

    View details for Web of Science ID 000280086800002

    View details for PubMedID 20568764

    View details for PubMedCentralID PMC2907715

  • Progress and challenges for the bottom-up synthesis of carbon nanotubes with discrete chirality CHEMICAL PHYSICS LETTERS Jasti, R., Bertozzi, C. R. 2010; 494 (1-3): 1-7
  • Identification of glycoproteins targeted by Trypanosoma cruzi trans-sialidase, a virulence factor that disturbs lymphocyte glycosylation GLYCOBIOLOGY Muia, R. P., Yu, H., Prescher, J. A., Hellman, U., Chen, X., Bertozzi, C. R., Campetella, O. 2010; 20 (7): 833-842

    Abstract

    Trypanosoma cruzi, the agent of the American trypanosomiasis or Chagas disease, bypasses its lack of de novo synthesis of sialic acids by expressing a surface-anchored trans-sialidase. This enzyme transfers sialic acid residues from the host's sialylglycoconjugates to the parasite's galactosylglycoconjugates. In addition to carrying out a pivotal role in parasite persistence/replication within the infected mammal, the trans-sialidase is shed into the bloodstream and induces alterations in the host immune system by modifying the sialylation of the immune cells. A major obstacle to understand these events is the difficulty to identify the transferred sialic acid among all those naturally occurring on the cell surface. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. In living parasites, we readily observed the transfer of azido-sialic acid to surface mucins. When evaluating mouse thymocytes and splenocytes as acceptors of the azido-sugar, a complex pattern of efficiently tagged glycoproteins was revealed. In both leukocyte populations, the main proteins labeled were identified as different CD45 isoforms. Disruption of the cell architecture increased the number and the molecular weight distribution of azido-sialic acid tagged proteins. Nevertheless, CD45 remained to be the main acceptor. Mass spectrometry assays allowed us to identify other acceptors, mainly integrins. The findings reported here provide a molecular basis to understand the abnormalities induced in the immune system by the trans-sialidase during T. cruzi infection.

    View details for DOI 10.1093/glycob/cwq037

    View details for Web of Science ID 000278437800004

    View details for PubMedID 20354005

    View details for PubMedCentralID PMC2900898

  • Real-Time Bioluminescence Imaging of Glycans on Live Cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cohen, A. S., Dubikovskaya, E. A., Rush, J. S., Bertozzi, C. R. 2010; 132 (25): 8563-?

    Abstract

    Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In this paper, we describe the design, synthesis, and biological application of a new phosphine probe for real-time imaging of cell-surface glycans using bioluminescence. To accomplish this goal, we took advantage of the bioorthogonal chemical reporter technique. This strategy uses a two-step labeling procedure in which an unnatural sugar analogue containing a functional handle is (1) incorporated into sugar-bearing proteins via the cell's own biosynthetic machinery and then (2) detected with an exogenously added probe. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. We chose to use a phosphine probe because, despite their slow reaction kinetics, they remain the best-performing reagents for tagging azidosugars in mice. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. In this work, we synthesized the first phosphine-luciferin probe for use in real-time BLI and demonstrated that azide-labeled cell-surface glycans can be imaged with 1 using concentrations as low as single digit nanomolar and times as little as 5 min, a feat that cannot be matched by any previous fluorescent phosphine probes. Even though we have only demonstrated its use in visualizing glycans, it can be envisioned that this probe could also be used for bioluminescence imaging of any azide-containing biomolecule, such as proteins and lipids, since azides have been previously incorporated into these molecules. The phosphine-luciferin probe is therefore poised for many applications in real-time imaging in cells and whole animals. These studies are currently in progress in our laboratory.

    View details for DOI 10.1021/ja101766r

    View details for Web of Science ID 000279196500017

    View details for PubMedID 20527879

    View details for PubMedCentralID PMC2890245

  • Visualizing enveloping layer glycans during zebrafish early embryogenesis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baskin, J. M., Dehnert, K. W., Laughlin, S. T., Amacher, S. L., Bertozzi, C. R. 2010; 107 (23): 10360-10365

    Abstract

    Developmental events can be monitored at the cellular and molecular levels by using noninvasive imaging techniques. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. We previously described a chemical method to image glycans during zebrafish larval development; however, we were unable to detect glycans during the first 24 hours of embryogenesis, a very dynamic period in development. Here we report an approach to the imaging of glycans that enables their visualization in the enveloping layer during the early stages of zebrafish embryogenesis. We microinjected embryos with azidosugars at the one-cell stage, allowed the zebrafish to develop, and detected the metabolically labeled glycans with copper-free click chemistry. Mucin-type O-glycans could be imaged as early as 7 hours postfertilization, during the gastrula stage of development. Additionally, we used a nonmetabolic approach to label sialylated glycans with an independent chemistry, enabling the simultaneous imaging of these two distinct classes of glycans. Imaging analysis of glycan trafficking revealed dramatic reorganization of glycans on the second time scale, including rapid migration to the cleavage furrow of mitotic cells. These studies yield insight into the biosynthesis and dynamics of glycans in the enveloping layer during embryogenesis and provide a platform for imaging other biomolecular targets by microinjection of appropriately functionalized biosynthetic precursors.

    View details for DOI 10.1073/pnas.0912081107

    View details for Web of Science ID 000278549300009

    View details for PubMedID 20489181

    View details for PubMedCentralID PMC2890823

  • Rapid and selective detection of fatty acylated proteins using omega-alkynyl-fatty acids and click chemistry JOURNAL OF LIPID RESEARCH Yap, M. C., Kostiuk, M. A., Martin, D. D., Perinpanayagam, M. A., Hak, P. G., Siddam, A., Majjigapu, J. R., Rajaiah, G., Keller, B. O., Prescher, J. A., Wu, P., Bertozzi, C. R., Falck, J. R., Berthiaume, L. G. 2010; 51 (6): 1566-1580

    Abstract

    Progress in understanding the biology of protein fatty acylation has been impeded by the lack of rapid direct detection and identification methods. We first report that a synthetic omega-alkynyl-palmitate analog can be readily and specifically incorporated into GAPDH or mitochondrial 3-hydroxyl-3-methylglutaryl-CoA synthase in vitro and reacted with an azido-biotin probe or the fluorogenic probe 3-azido-7-hydroxycoumarin using click chemistry for rapid detection by Western blotting or flat bed fluorescence scanning. The acylated cysteine residues were confirmed by MS. Second, omega-alkynyl-palmitate is preferentially incorporated into transiently expressed H- or N-Ras proteins (but not nonpalmitoylated K-Ras), compared with omega-alkynyl-myristate or omega-alkynyl-stearate, via an alkali sensitive thioester bond. Third, omega-alkynyl-myristate is specifically incorporated into endogenous co- and posttranslationally myristoylated proteins. The competitive inhibitors 2-bromopalmitate and 2-hydroxymyristate prevented incorporation of omega-alkynyl-palmitate and omega-alkynyl-myristate into palmitoylated and myristoylated proteins, respectively. Labeling cells with omega-alkynyl-palmitate does not affect membrane association of N-Ras. Furthermore, the palmitoylation of endogenous proteins including H- and N-Ras could be easily detected using omega-alkynyl-palmitate as label in cultured HeLa, Jurkat, and COS-7 cells, and, promisingly, in mice. The omega-alkynyl-myristate and -palmitate analogs used with click chemistry and azido-probes will be invaluable to study protein acylation in vitro, in cells, and in vivo.

    View details for DOI 10.1194/jlr.D002790

    View details for Web of Science ID 000277564700033

    View details for PubMedID 20028662

    View details for PubMedCentralID PMC3035521

  • Rapid Cu-Free Click Chemistry with Readily Synthesized Biarylazacyclooctynones JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Jewett, J. C., Sletten, E. M., Bertozzi, C. R. 2010; 132 (11): 3688-?

    Abstract

    Bioorthogonal chemical reactions, those that do not interact or interfere with biology, have allowed for exploration of numerous biological processes that were previously difficult to study. The reaction of azides with strained alkynes, such as cyclooctynes, readily forms a triazole product without the need for a toxic catalyst. Here we describe a biarylazacyclooctynone (BARAC) that has exceptional reaction kinetics and whose synthesis is designed to be both modular and scalable. We employed BARAC for live cell fluorescence imaging of azide-labeled glycans. The high signal-to-background ratio obtained using nanomolar concentrations of BARAC obviated the need for washing steps. Thus, BARAC is a promising reagent for in vivo imaging.

    View details for DOI 10.1021/ja100014q

    View details for Web of Science ID 000275868700024

    View details for PubMedID 20187640

    View details for PubMedCentralID PMC2840677

  • Targeted metabolic labeling of yeast N-glycans with unnatural sugars PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Breidenbach, M. A., Gallagher, J. E., King, D. S., Smart, B. P., Wu, P., Bertozzi, C. R. 2010; 107 (9): 3988-3993

    Abstract

    Metabolic labeling of glycans with synthetic sugar analogs has emerged as an attractive means for introducing nonnatural chemical functionality into glycoproteins. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. Here, we demonstrate that the conserved N-acetyglucosamine (GlcNAc) residues within chitobiose cores of N-glycans in the model organism Saccharomyces cerevisiae can be specifically targeted for metabolic replacement by unnatural sugars. We introduced an exogenous GlcNAc salvage pathway into yeast, allowing cells to metabolize GlcNAc provided as a supplement to the culture medium. We then rendered the yeast auxotrophic for production of the donor nucleotide-sugar uridine-diphosphate-GlcNAc (UDP-GlcNAc) by deletion of the essential gene GNA1. We demonstrate that gna1Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. To verify incorporation of the nonnatural sugars at N-glycan core positions, endoglycosidase H (endoH)-digested peptides from a purified secretory glycoprotein, Ygp1, were analyzed by mass spectrometry. Multiple Ygp1 N-glycosylation sites bearing GlcNAc, isotopically labeled GlcNAc, or GlcNAz were identified; these modifications were dependent on the supplement added to the culture medium. This system enables the production of glycoproteins that are functionalized for specific chemical modifications at their glycosylation sites.

    View details for DOI 10.1073/pnas.0911247107

    View details for Web of Science ID 000275131100011

    View details for PubMedID 20142501

    View details for PubMedCentralID PMC2840165

  • Copper-free click chemistry in living animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chang, P. V., Prescher, J. A., Sletten, E. M., Baskin, J. M., Miller, I. A., Agard, N. J., Lo, A., Bertozzi, C. R. 2010; 107 (5): 1821-1826

    Abstract

    Chemical reactions that enable selective biomolecule labeling in living organisms offer a means to probe biological processes in vivo. Very few reactions possess the requisite bioorthogonality, and, among these, only the Staudinger ligation between azides and triarylphosphines has been employed for direct covalent modification of biomolecules with probes in the mouse, an important model organism for studies of human disease. Here we explore an alternative bioorthogonal reaction, the 1,3-dipolar cycloaddition of azides and cyclooctynes, also known as "Cu-free click chemistry," for labeling biomolecules in live mice. Mice were administered peracetylated N-azidoacetylmannosamine (Ac(4)ManNAz) to metabolically label cell-surface sialic acids with azides. After subsequent injection with cyclooctyne reagents, glycoconjugate labeling was observed on isolated splenocytes and in a variety of tissues including the intestines, heart, and liver, with no apparent toxicity. The cyclooctynes tested displayed various labeling efficiencies that likely reflect the combined influence of intrinsic reactivity and bioavailability. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse.

    View details for DOI 10.1073/pnas.0911116107

    View details for Web of Science ID 000274296300006

    View details for PubMedID 20080615

    View details for PubMedCentralID PMC2836626

  • Cu-free click cycloaddition reactions in chemical biology CHEMICAL SOCIETY REVIEWS Jewett, J. C., Bertozzi, C. R. 2010; 39 (4): 1272-1279

    Abstract

    Bioorthogonal chemical reactions are paving the way for new innovations in biology. These reactions possess extreme selectivity and biocompatibility, such that their participating reagents can form covalent bonds within richly functionalized biological systems--in some cases, living organisms. This tutorial review will summarize the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddition reactions.

    View details for DOI 10.1039/b901970g

    View details for Web of Science ID 000275864500006

    View details for PubMedID 20349533

    View details for PubMedCentralID PMC2865253

  • Copper-Free Click Chemistry: Bioorthogonal Reagents for Tagging Azides ALDRICHIMICA ACTA Baskin, J. M., Bertozzi, C. R. 2010; 43 (1): 15-23
  • Symbol nomenclature for glycan representation PROTEOMICS Varki, A., Cummings, R. D., Esko, J. D., Freeze, H. H., Stanley, P., Marth, J. D., Bertozzi, C. R., Hart, G. W., Etzler, M. E. 2009; 9 (24): 5398-5399

    Abstract

    The glycan symbol nomenclature proposed by Harvey et al. in these pages has relative advantages and disadvantages. The use of symbols to depict glycans originated from Kornfeld in 1978, was systematized in the First Edition of "Essentials of Glycobiology" and updated for the second edition, with input from relevant organizations such as the Consortium for Functional Glycomics. We also note that >200 illustrations in the second edition have already been published using our nomenclature and are available for download at PubMed.

    View details for DOI 10.1002/pmic.200900708

    View details for Web of Science ID 000273515000001

    View details for PubMedID 19902428

    View details for PubMedCentralID PMC2882983

  • In Vivo Imaging of Caenorhabditis elegans Glycans ACS CHEMICAL BIOLOGY Laughlin, S. T., Bertozzi, C. R. 2009; 4 (12): 1068-1072

    Abstract

    The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. We treated worms with azidosugar variants of N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GalNAc), and N-acetylmannosamine (ManNAc), resulting in the metabolic labeling of their cell-surface glycans with azides. Subsequently, the worms were reacted via copper-free click reaction with fluorophore-conjugated difluorinated cyclooctyne (DIFO) reagents. We identified prominent localization of mucins in the pharynx of all four larval stages, in the adult hermaphrodite pharynx, vulva and anus, and in the tail of the adult male. Using a multicolor, time-resolved imaging strategy, we found that the distribution and dynamics of the glycans varied anatomically and with respect to developmental stage.

    View details for DOI 10.1021/cb900254y

    View details for Web of Science ID 000272845900011

    View details for PubMedID 19954190

    View details for PubMedCentralID PMC2807738

  • Control of the Molecular Orientation of Membrane-Anchored Biomimetic Glycopolymers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Godula, K., Umbel, M. L., Rabuka, D., Botyanszki, Z., Bertozzi, C. R., Parthasarathy, R. 2009; 131 (29): 10263-10268

    Abstract

    Quantifying and controlling the orientation of surface-bound macromolecules is crucial to a wide range of processes in areas as diverse as biology, materials science, and nanotechnology. Methods capable of directing orientation, as well as an understanding of the underlying physical mechanisms are, however, lacking. In this paper, we describe experiments in which the conformations of structurally well-defined polymers anchored to fluid lipid membranes were probed using Fluorescence Interference Contrast Microscopy (FLIC), an optical technique that provides topographic information with few-nanometer precision. The novel rodlike polymers mimic the architecture of mucin glycoproteins and feature a phospholipid tail for membrane incorporation and a fluorescent optical probe for FLIC imaging situated at the opposite termini of the densely glycosylated polymeric backbones. We find that the orientation of the rigid, approximately 30 nm long glycopolymers depends profoundly on the properties of the optical reporter. Molecules terminated with Alexa Fluor 488 projected away from the lipid bilayer by 11 +/- 1 nm, consistent with entropy-dominated sampling of the membrane-proximal space. Molecules terminated with Texas Red lie flat at the membrane (height, 0 +/- 2 nm), implying that interactions between Texas Red and the bilayer dominate the polymers' free energy. These results demonstrate the design of macromolecules with specific orientational preferences, as well as nanometer-scale measurement of their orientation. Importantly, they reveal that seemingly minute changes in molecular structure, in this case fluorophores that comprise only 2% of the total molecular weight, can significantly alter the molecule's presentation to the surrounding environment.

    View details for DOI 10.1021/ja903114g

    View details for Web of Science ID 000268395000075

    View details for PubMedID 19580278

    View details for PubMedCentralID PMC2716393

  • Glycopeptide-preferring Polypeptide GalNAc Transferase 10 (ppGalNAc T10), Involved in Mucin-type O-Glycosylation, Has a Unique GalNAc-O-Ser/Thr-binding Site in Its Catalytic Domain Not Found in ppGalNAc T1 or T2 JOURNAL OF BIOLOGICAL CHEMISTRY Perrine, C. L., Ganguli, A., Wu, P., Bertozzi, C. R., Fritz, T. A., Raman, J., Tabak, L. A., Gerken, T. A. 2009; 284 (30): 20387-20397

    Abstract

    Mucin-type O-gly co sy la tion is initiated by a large family of UDP-GalNAc:polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAc Ts) that transfer GalNAc from UDP-GalNAc to the Ser and Thr residues of polypeptide acceptors. Some members of the family prefer previously gly co sylated peptides (ppGalNAc T7 and T10), whereas others are inhibited by neighboring gly co sy la tion (ppGalNAc T1 and T2). Characterizing their peptide and glycopeptide substrate specificity is critical for understanding the biological role and significance of each isoform. Utilizing a series of random peptide and glycopeptide substrates, we have obtained the peptide and glycopeptide specificities of ppGalNAc T10 for comparison with ppGalNAc T1 and T2. For the glycopeptide substrates, ppGalNAc T10 exhibited a single large preference for Ser/Thr-O-GalNAc at the +1 (C-terminal) position relative to the Ser or Thr acceptor site. ppGalNAc T1 and T2 revealed no significant enhancements suggesting Ser/Thr-O-GalNAc was inhibitory at most positions for these isoforms. Against random peptide substrates, ppGalNAc T10 revealed no significant hydrophobic or hydrophilic residue enhancements, in contrast to what has been reported previously for ppGalNAc T1 and T2. Our results reveal that these transferases have unique peptide and glycopeptide preferences demonstrating their substrate diversity and their likely roles ranging from initiating transferases to filling-in transferases.

    View details for DOI 10.1074/jbc.M109.017236

    View details for Web of Science ID 000268097400064

    View details for PubMedID 19460755

    View details for PubMedCentralID PMC2740463

  • Polysialic acid governs T-cell development by regulating progenitor access to the thymus PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Drake, P. M., Stock, C. M., Nathan, J. K., Gip, P., Golden, K. P., Weinhold, B., Gerardy-Schahn, R., Bertozzi, C. R. 2009; 106 (29): 11995-12000

    Abstract

    Although the polysialyltransferase ST8Sia IV is expressed in both primary and secondary human lymphoid organs, its product, polysialic acid (polySia), has been largely overlooked by immunologists. In contrast, polySia expression and function in the nervous system has been well characterized. In this context, polySia modulates cellular adhesion, migration, cytokine response, and contact-dependent differentiation. Provocatively, these same processes are vital components of immune development and function. We previously established that mouse multipotent hematopoietic progenitors use ST8Sia IV to express polySia on their cell surfaces. Here, we demonstrate that, relative to wild-type controls, ST8Sia IV(-/-) mice have a 30% reduction in total thymocytes and a concomitant deficiency in the earliest thymocyte precursors. T-cell progenitors originate in the bone marrow and are mobilized to the blood at regular intervals by unknown signals. We performed in vivo reconstitution experiments in which ST8Sia IV(-/-) progenitors competed with wild-type cells to repopulate depleted or deficient immune subsets. Progenitors lacking polySi exhibited a specific defect in T-cell development because of an inability to access the thymus. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. Collectively, these results provide evidence that polySia is involved in hematopoietic development.

    View details for DOI 10.1073/pnas.0905188106

    View details for Web of Science ID 000268178400034

    View details for PubMedID 19587240

    View details for PubMedCentralID PMC2715481

  • Selective Enrichment of Azide-Containing Peptides from Complex Mixtures JOURNAL OF PROTEOME RESEARCH Nessen, M. A., Kramer, G., Back, J., Baskin, J. M., Smeenk, L. E., de Koning, L. J., van Maarseveen, J. H., de Jong, L., Bertozzi, C. R., Hiemstra, H., de Koster, C. G. 2009; 8 (7): 3702-3711

    Abstract

    A general method is described to sequester peptides containing azides from complex peptide mixtures, aimed at facilitating mass spectrometric analysis to study different aspects of proteome dynamics. The enrichment method is based on covalent capture of azide-containing peptides by the azide-reactive cyclooctyne (ARCO) resin and is demonstrated for two different applications. Enrichment of peptides derived from cytochrome c treated with the azide-containing cross-linker bis(succinimidyl)-3-azidomethyl glutarate (BAMG) shows several cross-link containing peptides. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Furthermore, the method is found to be very specific, as after enrichment over 87% of all peptides contain (modified) azidohomoalanine.

    View details for DOI 10.1021/pr900257z

    View details for Web of Science ID 000267694600045

    View details for PubMedID 19402736

    View details for PubMedCentralID PMC2761887

  • Direct Cell Surface Modification with DNA for the Capture of Primary Cells and the Investigation of Myotube Formation on Defined Patterns LANGMUIR Hsiao, S. C., Shum, B. J., Onoe, H., Douglas, E. S., Gartner, Z. J., Mathies, R. A., Bertozzi, C. R., Francis, M. B. 2009; 25 (12): 6985-6991

    Abstract

    Previously, we reported a method for the attachment of living cells to surfaces through the hybridization of synthetic DNA strands attached to their plasma membrane. The oligonucleotides were introduced using metabolic carbohydrate engineering, which allowed reactive tailoring of the cell surface glycans for chemoselective bioconjugation. While this method is highly effective for cultured mammalian cells, we report here a significant improvement of this technique that allows the direct modification of cell surfaces with NHS-DNA conjugates. This method is rapid and efficient, allowing virtually any mammalian cell to be patterned on surfaces bearing complementary DNA in under 1 h. We demonstrate this technique using several types of cells that are generally incompatible with integrin-targeting approaches, including red blood cells and primary T-cells. Cardiac myoblasts were also captured. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. The availability of this new protocol greatly expands the applicability of the DNA-based attachment strategy for the generation of artificial tissues and the incorporation of living cells into device settings.

    View details for DOI 10.1021/la900150n

    View details for Web of Science ID 000266929900058

    View details for PubMedID 19505164

    View details for PubMedCentralID PMC2812030

  • Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications JOURNAL OF BIOMEDICAL MATERIALS RESEARCH PART A Song, J., Xu, J., Filion, T., Saiz, E., Tomsia, A. P., Lian, J. B., Stein, G. S., Ayers, D. C., Bertozzi, C. R. 2009; 89A (4): 1098-1107
  • Chemical Approaches To Perturb, Profile, and Perceive Glycans ACCOUNTS OF CHEMICAL RESEARCH Agard, N. J., Bertozzi, C. R. 2009; 42 (6): 788-797

    Abstract

    Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice. In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Though not directly subject to mutation, we can determine glycan structure-function relationships by synthesizing defined glycoconjugates or by altering natural glycosylation pathways. Chemical syntheses of uniform glycoproteins and polymeric glycoprotein mimics have facilitated the study of individual glycoconjugates in the absence of glycan microheterogeneity. Alternatively, selective inhibition or activation of glycosyltransferases or glycosidases can define the biological roles of the corresponding glycans. Investigators have developed tools including small molecule inhibitors, decoy substrates, and engineered proteins to modify cellular glycans. Current approaches offer a precision approaching that of genetic control. Genomic and proteomic profiling form a basis for biological discovery. Glycans also present a rich matrix of information that adapts rapidly to changing environs. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. These approaches have already identified several cancer biomarkers. Metabolic labeling can identify recently synthesized glycans and thus directly track glycan dynamics. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. Together, glycomic and metabolic labeling techniques provide a comprehensive description of glycosylation as a foundation for hypothesis generation. Direct visualization of proteins via the green fluorescent protein (GFP) and its congeners has revolutionized the field of protein dynamics. Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. These results have highlighted the need for additional imaging probes.

    View details for DOI 10.1021/ar800267j

    View details for Web of Science ID 000267049000011

    View details for PubMedID 19361192

    View details for PubMedCentralID PMC2697281

  • PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis JOURNAL OF BIOLOGICAL CHEMISTRY Hatzios, S. K., Schelle, M. W., Holsclaw, C. M., Behrens, C. R., Botyanszki, Z., Lin, F. L., Carlson, B. L., Kumar, P., Leary, J. A., Bertozzi, C. R. 2009; 284 (19): 12745-12751

    Abstract

    Mycobacterium tuberculosis possesses an unusual cell wall that is replete with virulence-enhancing lipids. One cell wall molecule unique to pathogenic M. tuberculosis is polyacyltrehalose (PAT), a pentaacylated, trehalose-based glycolipid. Little is known about the biosynthesis of PAT, although its biosynthetic gene cluster has been identified and found to resemble that of the better studied M. tuberculosis cell wall component sulfolipid-1. In this study, we sought to elucidate the function of papA3, a gene from the PAT locus encoding a putative acyltransferase. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. The purified enzyme catalyzed the sequential esterification of trehalose with two palmitoyl groups, generating a diacylated product similar to the 2,3-diacyltrehalose glycolipids of M. tuberculosis. Notably, PapA3 was selective for trehalose; no activity was observed with other structurally related disaccharides. Disruption of the papA3 gene from M. tuberculosis resulted in the loss of PAT from bacterial lipid extracts. Complementation of the mutant strain restored PAT production, demonstrating that PapA3 is essential for the biosynthesis of this glycolipid in vivo. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures.

    View details for DOI 10.1074/jbc.M809088200

    View details for Web of Science ID 000265688300019

    View details for PubMedID 19276083

    View details for PubMedCentralID PMC2676004

  • Synthetic Trehalose Glycolipids Confer Desiccation Resistance to Supported Lipid Monolayers LANGMUIR Harland, C. W., Botyanszki, Z., Rabuka, D., Bertozzi, C. R., Parthasarathy, R. 2009; 25 (9): 5193-5198

    Abstract

    Lipid-derived desiccation resistance in membranes is a rare, unique ability previously observed only with trehalose dimycolate (TDM), an abundant mycobacterial glycolipid. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. The synthetic glycolipids consist of a simple trehalose disaccharide headgroup, similar to TDM, with hydrophobic tail groups of two 15- or 18-carbon chains. The synthetic trehalose glycolipids protected supported monolayers of phospholipids against dehydration even as minority components of the overall membrane, down to as little as 20 mol % trehalose glycolipid as assessed by assays of membrane fluidity. The dependence of the desiccation protection on the synthetic trehalose glycolipid fraction is nearly identical to that of TDM. The striking similarity of the desiccation resistance observed with TDM and the synthetic trehalose glycolipids, despite the variety of hydrophobic tail structures employed, suggests that interactions between the trehalose headgroup and surrounding molecules are the determining factor in dehydration protection.

    View details for DOI 10.1021/la804007a

    View details for Web of Science ID 000265528600051

    View details for PubMedID 19323499

    View details for PubMedCentralID PMC2699213

  • Crosslinking Studies of Protein-Protein Interactions in Nonribosomal Peptide Biosynthesis CHEMISTRY & BIOLOGY Hur, G. H., Meier, J. L., Baskin, J., Codelli, J. A., Bertozzi, C. R., Marahiel, M. A., Burkart, M. D. 2009; 16 (4): 372-381

    Abstract

    Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. In this study, we developed a crosslinking assay, utilizing bioorthogonal probes compatible with carrier protein modification, for probing the protein interactions between COM domains of NRPS enzymes. Employing the Huisgen 1,3-dipolar cycloaddition of azides and alkynes, we examined crosslinking of cognate NRPS modules within the tyrocidine pathway and demonstrated the sensitivity of our panel of crosslinking probes toward the selective protein interactions of compatible COM domains. These studies indicate that copper-free crosslinking substrates uniquely offer a diagnostic probe for protein-protein interactions. Likewise, these crosslinking probes serve as ideal chemical tools for structural studies between NRPS modules where functional assays are lacking.

    View details for DOI 10.1016/j.chembiol.2009.02.009

    View details for Web of Science ID 000265816900004

    View details for PubMedID 19345117

    View details for PubMedCentralID PMC2743379

  • Uridine-Based Inhibitors as New Leads for Antibiotics Targeting Escherichia coli LpxC BIOCHEMISTRY Barb, A. W., Leavy, T. M., Robins, L. I., Guan, Z., Six, D. A., Zhou, P., Bertozzi, C. R., Raetz, C. R. 2009; 48 (14): 3068-3077

    Abstract

    The UDP-3-O-(R-3-hydroxyacyl)-N-acetylglucosamine deacetylase LpxC catalyzes the committed reaction of lipid A (endotoxin) biosynthesis in Gram-negative bacteria and is a validated antibiotic target. Although several previously described compounds bind to the unique acyl chain binding passage of LpxC with high affinity, strategies to target the enzyme's UDP-binding site have not been reported. Here the identification of a series of uridine-based LpxC inhibitors is presented. The most potent examined, 1-68A, is a pH-dependent, two-step, covalent inhibitor of Escherichia coli LpxC that competes with UDP to bind the enzyme in the first step of inhibition. Compound 1-68A exhibits a K(I) of 54 muM and a maximal rate of inactivation (k(inact)) of 1.7 min(-1) at pH 7.4. Dithiothreitol, glutathione and the C207A mutant of E. coli LpxC prevent the formation of a covalent complex by 1-68A, suggesting a role for Cys-207 in inhibition. The inhibitory activity of 1-68A and a panel of synthetic analogues identified moieties necessary for inhibition. 1-68A and a 2-dehydroxy analogue, 1-68Aa, inhibit several purified LpxC orthologues. These compounds may provide new scaffolds for extension of existing LpxC-inhibiting antibiotics to target the UDP binding pocket.

    View details for DOI 10.1021/bi900167q

    View details for Web of Science ID 000264983800005

    View details for PubMedID 19256534

    View details for PubMedCentralID PMC2709817

  • Programmed assembly of 3-dimensional microtissues with defined cellular connectivity PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Gartner, Z. J., Bertozzi, C. R. 2009; 106 (12): 4606-4610

    Abstract

    Multicellular organs comprise differentiated cell types with discrete yet interdependent functions. The cells' spatial arrangements and interconnectivities, both critical elements of higher-order function, derive from complex developmental programs in vivo and are often difficult or impossible to emulate in vitro. Here, we report the bottom-up synthesis of microtissues composed of multiple cell types with programmed connectivity. We functionalized cells with short oligonucleotides to impart specific adhesive properties. Hybridization of complementary DNA sequences enabled the assembly of multicellular structures with defined cell-cell contacts. We demonstrated that the kinetic parameters of the assembly process depend on DNA sequence complexity, density, and total cell concentration. Thus, cell assembly can be highly controlled, enabling the design of microtissues with defined cell composition and stoichiometry. We used this strategy to construct a paracrine signaling network in isolated 3-dimensional microtissues.

    View details for DOI 10.1073/pnas.0900717106

    View details for Web of Science ID 000264522600009

    View details for PubMedID 19273855

    View details for PubMedCentralID PMC2660766

  • Site-specific chemical modification of recombinant proteins produced in mammalian cells by using the genetically encoded aldehyde tag PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Wu, P., Shui, W., Carlson, B. L., Hu, N., Rabuka, D., Lee, J., Bertozzi, C. R. 2009; 106 (9): 3000-3005

    Abstract

    The properties of therapeutic proteins can be enhanced by chemical modification. Methods for site-specific protein conjugation are critical to such efforts. Here, we demonstrate that recombinant proteins expressed in mammalian cells can be site-specifically modified by using a genetically encoded aldehyde tag. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Cotranslational modification of the proteins by FGE produced products bearing a unique aldehyde group. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. We applied the technique to site-specific modification of monoclonal antibodies, the fastest growing class of biopharmaceuticals, as well as membrane-associated and cytosolic proteins expressed in mammalian cells.

    View details for DOI 10.1073/pnas.0807820106

    View details for Web of Science ID 000263844100008

    View details for PubMedID 19202059

    View details for PubMedCentralID PMC2651276

  • Boron Nitride Nanotubes Are Noncytotoxic and Can Be Functionalized for Interaction with Proteins and Cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, X., Wu, P., Rousseas, M., Okawa, D., Gartner, Z., Zettl, A., Bertozzi, C. R. 2009; 131 (3): 890-?

    Abstract

    We report the discovery that boron nitride nanotubes (BNNTs), isosteres of CNTs with unique physical properties, are inherently noncytotoxic. Furthermore, we developed a biomemetic coating strategy to interface BNNTs with proteins and cells. Finally, we showed that BNNTs can deliver DNA oligomers to the interior of cells with no apparent toxicity. This work suggests that BNNTs may be superior to CNTs for use as biological probes and in biomaterials.

    View details for DOI 10.1021/ja807334b

    View details for Web of Science ID 000264791600009

    View details for PubMedID 19119844

    View details for PubMedCentralID PMC2657038

  • Imaging the glycome PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Laughlin, S. T., Bertozzi, C. R. 2009; 106 (1): 12-17

    Abstract

    Molecular imaging enables visualization of specific molecules in vivo and without substantial perturbation to the target molecule's environment. Glycans are appealing targets for molecular imaging but are inaccessible with conventional approaches. Classic methods for monitoring glycans rely on molecular recognition with probe-bearing lectins or antibodies, but these techniques are not well suited to in vivo imaging. In an emerging strategy, glycans are imaged by metabolic labeling with chemical reporters and subsequent ligation to fluorescent probes. This technique has enabled visualization of glycans in living cells and in live organisms such as zebrafish. Molecular imaging with chemical reporters offers a new avenue for probing changes in the glycome that accompany development and disease.

    View details for DOI 10.1073/pnas.0811481106

    View details for Web of Science ID 000262263900006

    View details for PubMedID 19104067

    View details for PubMedCentralID PMC2629201

  • Metabolic Labeling of Sialic Acids in Living Animals with Alkynyl Sugars ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Chang, P. V., Chen, X., Smyrniotis, C., Xenakis, A., Hu, T., Bertozzi, C. R., Wu, P. 2009; 48 (22): 4030-4033

    Abstract

    Sialome sweet sialome: As sialic acids are involved in many host-pathogen recognition events and are markers of embryonic and malignant tissues, there is great interest in methods for the enrichment and identification of sialylated glycoproteins from complex tissues. Now N-(4-pentynoyl)mannosamine can be used to metabolically label sialylated glycoproteins in living animals, enabling future identification of new biomarkers.

    View details for DOI 10.1002/anie.200806319

    View details for Web of Science ID 000266415400022

    View details for PubMedID 19388017

    View details for PubMedCentralID PMC2868584

  • DNA-barcode directed capture and electrochemical metabolic analysis of single mammalian cells on a microelectrode array LAB ON A CHIP Douglas, E. S., Hsiao, S. C., Onoe, H., Bertozzi, C. R., Francis, M. B., Mathies, R. A. 2009; 9 (14): 2010-2015

    Abstract

    A microdevice is developed for DNA-barcode directed capture of single cells on an array of pH-sensitive microelectrodes for metabolic analysis. Cells are modified with membrane-bound single-stranded DNA, and specific single-cell capture is directed by the complementary strand bound in the sensor area of the iridium oxide pH microelectrodes within a microfluidic channel. This bifunctional microelectrode array is demonstrated for the pH monitoring and differentiation of primary T cells and Jurkat T lymphoma cells. Single Jurkat cells exhibited an extracellular acidification rate of 11 milli-pH min(-1), while primary T cells exhibited only 2 milli-pH min(-1). This system can be used to capture non-adherent cells specifically and to discriminate between visually similar healthy and cancerous cells in a heterogeneous ensemble based on their altered metabolic properties.

    View details for DOI 10.1039/b821690h

    View details for Web of Science ID 000267572000007

    View details for PubMedID 19568668

    View details for PubMedCentralID PMC2892333

  • Synthesis and Microcontact Printing of Dual End-Functionalized Mucin-like Glycopolymers for Microarray Applications ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Godula, K., Rabuka, D., Nam, K. T., Bertozzi, C. R. 2009; 48 (27): 4973-4976

    Abstract

    Click to view: Glycopolymers can be used to display glycans on microarrays in native-like architectures. The structurally uniform alkyne-terminated mucin mimetic glycopolymers (see picture; TR = fluorophore) were printed on azide-functionalized chips by microcontact printing in the presence of a copper catalyst. The surface-bound glycopolymers bind lectins in a ligand-specific manner.

    View details for DOI 10.1002/anie.200805756

    View details for Web of Science ID 000267713800017

    View details for PubMedID 19479916

    View details for PubMedCentralID PMC2735190

  • Quantitative Proteomic Profiling of Host-Pathogen Interactions: The Macrophage Response to Mycobacterium tuberculosis Lipids JOURNAL OF PROTEOME RESEARCH Shui, W., Gilmore, S. A., Sheu, L., Liu, J., Keasling, J. D., Bertozzi, C. R. 2009; 8 (1): 282-289

    Abstract

    Mycobacterium tuberculosis (M. tuberculosis) is an intracellular pathogen possessing a complex mixture of cell wall lipids that are thought to modulate the activities of host macrophages. In this study, we employed two state-of-the-art quantitative proteomic approaches, metabolic labeling SILAC and chemical isobaric tagging iTRAQ, to study changes in macrophage protein expression in response to exposure to M. tuberculosis lipids. From a total of 1286 proteins identified, 463 were discovered by both isotope-labeling strategies at a high consistency, and the rest of proteins were detected by only one of the two approaches. Upon exposure to mycobacterial cell wall lipids, 166 macrophage proteins showed differential expression. These included proteins involved in the immune response, oxidation and reduction, and vesicle transport, as well as other cellular processes. The response of the macrophage proteome to M. tuberculosis lipids reflects the cell's innate defense mechanisms as well as lipid-induced processes that may benefit the pathogen.

    View details for DOI 10.1021/pr800422e

    View details for Web of Science ID 000262171100029

    View details for PubMedID 19053526

    View details for PubMedCentralID PMC2655317

  • Synthesis, Characterization, and Theory of [9]-, [12]-, and [18]Cycloparaphenylene: Carbon Nanohoop Structures JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Jasti, R., Bhattacharjee, J., Neaton, J. B., Bertozzi, C. R. 2008; 130 (52): 17646-?

    Abstract

    The first synthesis and characterization of [9]-, [12]-, and [18]cycloparaphenylene was demonstrated utilizing a novel aromatization reaction. We refer to these fascinating structures as "carbon nanohoops" due to their structural similarity to carbon nanotubes. Additionally, we have utilized computational methods to understand the unique properties of these fully conjugated macrocycles.

    View details for DOI 10.1021/ja807126u

    View details for Web of Science ID 000263320900008

    View details for PubMedID 19055403

    View details for PubMedCentralID PMC2709987

  • Membrane proteomics of phagosomes suggests a connection to autophagy PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Shui, W., Sheu, L., Liu, J., Smart, B., Petzold, C. J., Hsieh, T., Pitcher, A., Keasling, J. D., Bertozzi, C. R. 2008; 105 (44): 16952-16957

    Abstract

    Phagocytosis is the central process by which macrophage cells internalize and eliminate infectious microbes as well as apoptotic cells. During maturation, phagosomes containing engulfed particles fuse with various endosomal compartments through the action of regulatory molecules on the phagosomal membrane. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. Many identified proteins were not previously known to reside in the phagosome. We characterized several proteins in LBC phagosomes that change in abundance on induction of autophagy, a process that has been previously implicated in the host defense against microbial pathogens. These observations suggest crosstalk between autophagy and phagocytosis that may be relevant to the innate immune response of macrophages.

    View details for DOI 10.1073/pnas.0809218105

    View details for Web of Science ID 000260913800030

    View details for PubMedID 18971338

    View details for PubMedCentralID PMC2579359

  • Conditional glycosylation in eukaryotic cells using a biocompatible chemical inducer of dimerization JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Czlapinski, J. L., Schelle, M. W., Miller, L. W., Laughlin, S. T., Kohler, J. J., Cornish, V. W., Bertozzi, C. R. 2008; 130 (40): 13186-13187

    Abstract

    Chemical inducers of dimerization (CIDs) are cell-permeable small molecules capable of dimerizing two protein targets. The most widely used CID, the natural product rapamycin and its relatives, is immunosuppressive due to interactions with endogenous targets and thus has limited utility in vivo. Here we report a new biocompatible CID, Tmp-SLF, which dimerizes E. coli DHFR and FKBP and has no endogenous mammalian targets that would lead to unwanted in vivo side effects. We employed Tmp-SLF to modulate gene expression in a yeast three-hybrid assay. Finally, we engineered the Golgi-resident glycosyltransferase FucT7 for tunable control by Tmp-SLF in mammalian cells.

    View details for DOI 10.1021/ja8037728

    View details for Web of Science ID 000259675500001

    View details for PubMedID 18788807

    View details for PubMedCentralID PMC2709988

  • Structural Characterization of a Novel Sulfated Menaquinone produced by stf3 from Mycobacterium tuberculosis ACS CHEMICAL BIOLOGY Hotsclaw, C. M., Sogi, K. M., Gilmore, S. A., Scheller, M. W., Leavell, M. D., Bertozzi, C. R., Leary, J. A. 2008; 3 (10): 619-624

    Abstract

    Mycobacterium tuberculosis, the causative agent of tuberculosis, produces unique sulfated metabolites associated with virulence. One such metabolite from M. tuberculosis lipid extracts, S881, has been shown to negatively regulate the virulence of M. tuberculosis in mouse infection studies, and its cell-surface localization suggests a role in modulating host-pathogen interactions. However, a detailed structural analysis of S881 has remained elusive. Here we use high-resolution, high-mass-accuracy, and tandem mass spectrometry to characterize the structure of S881. Exact mass measurements showed that S881 is highly unsaturated, tandem mass spectrometry indicated a polyisoprene-derived structure, and characterization of synthetic structural analogs confirmed that S881 is a previously undescribed sulfated derivative of dihydromenaquinone-9, the primary quinol electron carrier in M. tuberculosis. To our knowledge, this is the first example of a sulfated menaquinone produced in any prokaryote. Together with previous studies, these findings suggest that this redox cofactor may play a role in mycobacterial pathogenesis.

    View details for DOI 10.1021/cb800145r

    View details for Web of Science ID 000260193100005

    View details for PubMedID 18928249

    View details for PubMedCentralID PMC2680727

  • New aldehyde tag sequences identified by screening formylglycine generating enzymes in vitro and in vivo JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rush, J. S., Bertozzi, C. R. 2008; 130 (37): 12240-?

    Abstract

    Formylglycine generating enzyme (FGE) performs a critical posttranslational modification of type I sulfatases, converting cysteine within the motif CxPxR to the aldehyde-bearing residue formylglycine (FGly). This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. In this report, we screened FGEs from M. tuberculosis and S. coelicolor against synthetic peptide libraries and identified new substrate sequences that diverge from the canonical motif. We found that E. coli's FGE-like activity is similarly promiscuous, enabling the use of novel aldehyde tag sequences for in vivo modification of recombinant proteins.

    View details for DOI 10.1021/ja804530w

    View details for Web of Science ID 000259139900020

    View details for PubMedID 18722427

    View details for PubMedCentralID PMC2721638

  • Function and structure of a prokaryotic formylglycine-generating enzyme JOURNAL OF BIOLOGICAL CHEMISTRY Carlson, B. L., Ballister, E. R., Skordalakes, E., King, D. S., Breidenbach, M. A., Gilmore, S. A., Berger, J. M., Bertozzi, C. R. 2008; 283 (29): 20117-20125

    Abstract

    Type I sulfatases require an unusual co- or post-translational modification for their activity in hydrolyzing sulfate esters. In eukaryotic sulfatases, an active site cysteine residue is oxidized to the aldehyde-containing C(alpha)-formylglycine residue by the formylglycine-generating enzyme (FGE). The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Here we describe the identification of a prokaryotic FGE from Mycobacterium tuberculosis. In addition, we solved the crystal structure of the Streptomyces coelicolor FGE homolog to 2.1 A resolution. The prokaryotic homolog exhibits remarkable structural similarity to human FGE, including the position of catalytic cysteine residues. Both biochemical and structural data indicate the presence of an oxidized cysteine modification in the active site that may be relevant to catalysis. In addition, we generated a mutant M. tuberculosis strain lacking FGE. Although global sulfatase activity was reduced in the mutant, a significant amount of residual sulfatase activity suggests the presence of FGE-independent sulfatases in this organism.

    View details for DOI 10.1074/jbc.M800217200

    View details for Web of Science ID 000257565300028

    View details for PubMedID 18390551

    View details for PubMedCentralID PMC2459300

  • A hydrophilic azacyclooctyne for Cu-free click chemistry ORGANIC LETTERS Sletten, E. M., Bertozzi, C. R. 2008; 10 (14): 3097-3099

    Abstract

    Biomolecules labeled with azides can be detected through Cu-free click chemistry with cyclooctyne probes, but their intrinsic hydrophobicity can compromise bioavailability. Here, we report the synthesis and evaluation of a novel azacyclooctyne, 6,7-dimethoxyazacyclooct-4-yne (DIMAC). Generated in nine steps from a glucose analogue, DIMAC reacted with azide-labeled proteins and cells similarly to cyclooctynes. However, its superior polarity and water solubility reduced nonspecific binding, thereby improving the sensitivity of azide detection.

    View details for DOI 10.1021/ol801141k

    View details for Web of Science ID 000257629200046

    View details for PubMedID 18549231

    View details for PubMedCentralID PMC2664610

  • The glycosylphosphatidylinositol anchor: A complex membrane-anchoring structure for proteins BIOCHEMISTRY Paulick, M. G., Bertozzi, C. R. 2008; 47 (27): 6991-7000

    Abstract

    Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified protein in the outer leaflet of the cell membrane. The GPI anchor is a complex structure comprising a phosphoethanolamine linker, glycan core, and phospholipid tail. GPI-anchored proteins are structurally and functionally diverse and play vital roles in numerous biological processes. While several GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. This review discusses the structural diversity of the GPI anchor and its putative cellular functions, including involvement in lipid raft partitioning, signal transduction, targeting to the apical membrane, and prion disease pathogenesis. We specifically highlight studies in which chemically synthesized GPI anchors and analogues have been employed to study the roles of this unique posttranslational modification.

    View details for DOI 10.1021/bi8006324

    View details for Web of Science ID 000257257100003

    View details for PubMedID 18557633

    View details for PubMedCentralID PMC2663890

  • The Mycobacterium tuberculosis virulence factor trehalose dimycolate imparts desiccation resistance to model mycobacterial membranes BIOPHYSICAL JOURNAL Harland, C. W., Rabuka, D., Bertozzi, C. R., Parthasarathy, R. 2008; 94 (12): 4718-4724

    Abstract

    Mycobacteria, including persistent pathogens like Mycobacterium tuberculosis, have an unusual membrane structure in which, outside the plasma membrane, a nonfluid hydrophobic fatty acid layer supports a fluid monolayer rich in glycolipids such as trehalose 6,6'-dimycolate (TDM; cord factor). Given the abilities of mycobacteria to survive desiccation and trehalose in solution to protect biomolecules and whole organisms during freezing, drying, and other stresses, we hypothesized that TDM alone may suffice to confer dehydration resistance to the membranes of which it is a constituent. We devised an experimental model that mimics the structure of mycobacterial envelopes in which an immobile hydrophobic layer supports a TDM-rich, two-dimensionally fluid leaflet. We have found that TDM monolayers, in stark contrast to phospholipid membranes, can be dehydrated and rehydrated without loss of integrity, as assessed by fluidity and protein binding. Strikingly, this protection from dehydration extends to TDM-phospholipid mixtures with as little as 25 mol % TDM. The dependence of the recovery of membrane mobility upon rehydration on TDM fraction shows a functional form indicative of spatial percolation, implying that the connectivity of TDM plays a crucial role in membrane preservation. Our observations are the first reported instance of dehydration resistance provided by a membrane glycolipid.

    View details for DOI 10.1529/biophysj.107.125542

    View details for Web of Science ID 000256231700017

    View details for PubMedID 18326657

    View details for PubMedCentralID PMC2397374

  • Rv2131c from Mycobacterium tuberculosis is a CysQ 3 '-phosphoadenosine-5 '-phosphatase BIOCHEMISTRY Hatzios, S. K., Iavarone, A. T., Bertozzi, C. R. 2008; 47 (21): 5823-5831

    Abstract

    Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. These metabolites are products of the sulfate assimilation pathway. CysQ, a 3'-phosphoadenosine-5'-phosphatase, is considered an important regulator of this pathway in plants, yeast, and other bacteria. By controlling the pools of 3'-phosphoadenosine 5'-phosphate (PAP) and 3'-phosphoadenosine 5'-phosphosulfate (PAPS), CysQ has the potential to modulate flux in the biosynthesis of essential sulfur-containing metabolites. Bioinformatic analysis of the Mtb genome suggests the presence of a CysQ homologue encoded by the gene Rv2131c. However, a recent biochemical study assigned the protein's function as a class IV fructose-1,6-bisphosphatase. In the present study, we expressed Rv2131c heterologously and found that the protein dephosphorylates PAP in a magnesium-dependent manner, with optimal activity observed between pH 8.5 and pH 9.5 using 0.5 mM MgCl 2. A sensitive electrospray ionization mass spectrometry-based assay was used to extract the kinetic parameters for PAP, revealing a K m (8.1 +/- 3.1 microM) and k cat (5.4 +/- 1.1 s (-1)) comparable to those reported for other CysQ enzymes. The second-order rate constant for PAP was determined to be over 3 orders of magnitude greater than those determined for myo-inositol 1-phosphate (IMP) and fructose 1,6-bisphosphate (FBP), previously considered to be the primary substrates of this enzyme. Moreover, the ability of the Rv2131c-encoded enzyme to dephosphorylate PAP and PAPS in vivo was confirmed by functional complementation of an Escherichia coli Delta cysQ mutant. Taken together, these studies indicate that Rv2131c encodes a CysQ enzyme that may play a role in mycobacterial sulfur metabolism.

    View details for DOI 10.1021/bi702453s

    View details for Web of Science ID 000256043200016

    View details for PubMedID 18454554

    View details for PubMedCentralID PMC2711008

  • Noncovalent cell surface engineering: Incorporation of bioactive synthetic glycopolymers into cellular membranes JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rabuka, D., Forstner, M. B., Groves, J. T., Bertozzi, C. R. 2008; 130 (18): 5947-5953

    Abstract

    The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. Here we demonstrate that cell surfaces can be engineered to display synthetic bioactive polymers at defined densities by exogenous membrane insertion. The polymers were designed to mimic native cell-surface mucin glycoproteins, which are defined by their dense glycosylation patterns and rod-like structures. End-functionalization with a hydrophobic anchor permitted incorporation into the membranes of live cultured cells. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Their diffusion properties mirrored those of many natural membrane-associated biomolecules. Furthermore, the membrane-bound glycopolymers were internalized into early endosomes similarly to endogenous membrane components and were capable of specific interactions with protein receptors. This system provides a platform to study cell-surface phenomena with a degree of chemical control that cannot be achieved using conventional biological tools.

    View details for DOI 10.1021/ja710644g

    View details for Web of Science ID 000255629400034

    View details for PubMedID 18402449

    View details for PubMedCentralID PMC2724873

  • In vivo imaging of membrane-associated glycans in developing zebrafish SCIENCE Laughlin, S. T., Baskin, J. M., Amacher, S. L., Bertozzi, C. R. 2008; 320 (5876): 664-667

    Abstract

    Glycans are attractive targets for molecular imaging but have been inaccessible because of their incompatibility with genetically encoded reporters. We demonstrated the noninvasive imaging of glycans in live developing zebrafish, using a chemical reporter strategy. Zebrafish embryos were treated with an unnatural sugar to metabolically label their cell-surface glycans with azides. Subsequently, the embryos were reacted with fluorophore conjugates by means of copper-free click chemistry, enabling the visualization of glycans in vivo at subcellular resolution during development. At 60 hours after fertilization, we observed an increase in de novo glycan biosynthesis in the jaw region, pectoral fins, and olfactory organs. Using a multicolor detection strategy, we performed a spatiotemporal analysis of glycan expression and trafficking and identified patterns that would be undetectable with conventional molecular imaging approaches.

    View details for DOI 10.1126/science.1155106

    View details for Web of Science ID 000255454300046

    View details for PubMedID 18451302

  • Rapid detection, discovery, and identification of post-translationally myristoylated proteins during apoptosis using a bio-orthogonal azidomyristate analog FASEB JOURNAL Martin, D. D., Vilas, G. L., Prescher, J. A., Rajaiah, G., Falck, J. R., Bertozzi, C. R., Berthiaume, L. G. 2008; 22 (3): 797-806

    Abstract

    Myristoylation is the attachment of the 14-carbon fatty acid myristate to the N-terminal glycine residue of proteins. Typically a co-translational modification, myristoylation of proapoptotic cysteinyl-aspartyl proteases (caspase)-cleaved Bid and PAK2 was also shown to occur post-translationally and is essential for their proper localization and proapoptotic function. Progress in the identification and characterization of myristoylated proteins has been impeded by the long exposure times required to monitor incorporation of radioactive myristate into proteins (typically 1-3 months). Consequently, we developed a nonradioactive detection methodology in which a bio-orthogonal azidomyristate analog is specifically incorporated co- or post-translationally into proteins at N-terminal glycines, chemoselectively ligated to tagged triarylphosphines and detected by Western blotting with short exposure times (seconds to minutes). This represents over a million-fold signal amplification in comparison to using radioactive labeling methods. Using rational prediction analysis to recognize putative internal myristoylation sites in caspase-cleaved proteins combined with our nonradioactive chemical detection method, we identify 5 new post-translationally myristoylatable proteins (PKC epsilon, CD-IC2, Bap31, MST3, and the catalytic subunit of glutamate cysteine ligase). We also demonstrate that 15 proteins undergo post-translational myristoylation in apoptotic Jurkat T cells. This suggests that post-translational myristoylation of caspase-cleaved proteins represents a novel mechanism widely used to regulate cell death.

    View details for DOI 10.1096/fj.07-9198com

    View details for Web of Science ID 000254143700018

    View details for PubMedID 17932026

    View details for PubMedCentralID PMC2865240

  • Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue FASEB JOURNAL Kostiuk, M. A., Corvi, M. M., Keller, B. O., Plummer, G., Prescher, J. A., Hangauer, M. J., Bertozzi, C. R., Rajaiah, G., Falck, J. R., Berthiaume, L. G. 2008; 22 (3): 721-732

    Abstract

    Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. In addition to being a constituent of glycerolipids and a source of energy, palmitate also covalently attaches to numerous cellular proteins via a process named palmitoylation. Recognized for its roles in membrane tethering, cellular signaling, and protein trafficking, palmitoylation is also emerging as a potential regulator of metabolism. Indeed, we showed previously that the acylation of two mitochondrial proteins at their active site cysteine residues result in their inhibition. Herein, we sought to identify other palmitoylated proteins in mitochondria using a nonradioactive bio-orthogonal azido-palmitate analog that can be selectively derivatized with various tagged triarylphosphines. Our results show that, like palmitate, incorporation of azido-palmitate occurred on mitochondrial proteins via thioester bonds at sites that could be competed out by palmitoyl-CoA. Using this method, we identified 21 putative palmitoylated proteins in the rat liver mitochondrial matrix, a compartment not recognized for its content in palmitoylated proteins, and confirmed the palmitoylation of newly identified mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases.

    View details for DOI 10.1096/fj.07-9199com

    View details for Web of Science ID 000254143700011

    View details for PubMedID 17971398

    View details for PubMedCentralID PMC2860959

  • Synthetic studies toward Mycobacterium tuberculosis sulfolipid-I JOURNAL OF ORGANIC CHEMISTRY Leigh, C. D., Bertozzi, C. R. 2008; 73 (3): 1008-1017

    Abstract

    Sulfolipid-I (SL-I) is an abundant metabolite found in the cell wall of Mycobacterium tuberculosis that is comprised of a trehalose 2-sulfate core modified with four fatty acyl substituents. The correlation of its abundance with the virulence of clinical isolates suggests a role for SL-I in pathogenesis, although its biological functions remain unknown. Here we describe the synthesis of a SL-I analogue bearing unnatural lipid substituents. A key feature of the synthesis was application of an intramolecular aglycon delivery reaction to join two differentially protected glucose monomers, one prepared with a novel alpha-selective glycosylation. The route developed for the model compound can be readily extended to the synthesis of native SL-I as well as additional analogues for use in the investigation of SL-I's functions.

    View details for DOI 10.1021/jo702032c

    View details for Web of Science ID 000252686400026

    View details for PubMedID 18173284

    View details for PubMedCentralID PMC2735189

  • DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Hsiao, S. C., Crow, A. K., Lam, W. A., Bertozzi, C. R., Fletcher, D. A., Francis, M. B. 2008; 47 (44): 8473-8477

    View details for DOI 10.1002/anie.200802525

    View details for Web of Science ID 000260622700027

    View details for PubMedID 18798192

    View details for PubMedCentralID PMC2748828

  • Biocompatible carbon nanotubes generated by functionalization with glycodendrimers ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Wu, P., Chen, X., Hu, N., Tam, U. C., Blixt, O., Zettl, A., Bertozzi, C. R. 2008; 47 (27): 5022-5025

    View details for DOI 10.1002/anie.200705363

    View details for Web of Science ID 000257427000014

    View details for PubMedID 18509843

    View details for PubMedCentralID PMC2847391

  • Copper-free click chemistry for the in situ crosslinking of photodegradable star polymers CHEMICAL COMMUNICATIONS Johnson, J. A., Baskin, J. M., Bertozzi, C. R., Koberstein, J. T., Turro, N. J. 2008: 3064-3066

    Abstract

    Bifunctional, fluorinated cyclooctynes were used for the in situ "click" crosslinking of azide-terminated photodegradable star polymers, yielding photodegradable polymeric model networks with well-defined structures and tunable gelation times.

    View details for DOI 10.1039/b803043j

    View details for Web of Science ID 000257236600029

    View details for PubMedID 18688349

    View details for PubMedCentralID PMC2667816

  • A FRET-based fluorogenic phosphine for live-cell Imaging with the Staudinger ligation ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Hangauer, M. J., Bertozzi, C. R. 2008; 47 (13): 2394-2397

    View details for DOI 10.1002/anie.200704847

    View details for Web of Science ID 000254379500008

    View details for PubMedID 18306205

    View details for PubMedCentralID PMC2446402

  • A chemical approach to unraveling the biological function of the glycosylphosphatidylinositol anchor PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Paulick, M. G., Forstner, M. B., Groves, J. T., Bertozzi, C. R. 2007; 104 (51): 20332-20337

    Abstract

    The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. We previously synthesized a series of GPI anchor analogs with systematic deletions within the glycan core and coupled them to the GFP by a combination of expressed protein ligation and native chemical ligation [Paulick MG, Wise AR, Forstner MB, Groves JT, Bertozzi CR (2007) J Am Chem Soc 129:11543-11550]. Here we investigate the behavior of these GPI-protein analogs in living cells. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. The GPI-protein analogs also diffused freely in cellular membranes. However, changes in the glycan structure significantly affected membrane mobility, with the loss of monosaccharide units correlating to decreased diffusion. Thus, this cellular system provides a platform for dissecting the contributions of various GPI anchor components to their biological function.

    View details for DOI 10.1073/pnas.0710139104

    View details for Web of Science ID 000251885000034

    View details for PubMedID 18077333

    View details for PubMedCentralID PMC2154431

  • Bioorthogonal click chemistry: Covalent labeling in living systems QSAR & COMBINATORIAL SCIENCE Baskin, J. M., Bertozzi, C. R. 2007; 26 (11-12): 1211-1219
  • Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes NATURE BIOTECHNOLOGY Fernandez-Suarez, M., Baruah, H., Martinez-Hernandez, L., Xie, K. T., Baskin, J. M., Bertozzi, C. R., Ting, A. Y. 2007; 25 (12): 1483-1487

    Abstract

    Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function, or dissociate from proteins after internalization. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA) to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

    View details for DOI 10.1038/nbt1355

    View details for Web of Science ID 000251457800039

    View details for PubMedID 18059260

    View details for PubMedCentralID PMC2654346

  • Molecular orientation of membrane-anchored mucin glycoprotein mimics JOURNAL OF PHYSICAL CHEMISTRY B Parthasarathy, R., Rabuka, D., Bertozzi, C. R., Groves, J. T. 2007; 111 (42): 12133-12135

    Abstract

    Mucin glycoproteins contribute to a wide range of cell-surface phenomena. Their dense glycosylation is believed to confer structural rigidity as well as molecular extension beyond the glycocalyx, crucial to interaction with the cellular environment. However, controlled investigations of the relationships between glycosylation, rigidity, and extension of membrane-bound mucins or similar macromolecules are lacking, largely because of the absence of tractable experimental models. We have therefore made use of recently developed synthetic mucin mimetics, in which the core alpha-GalNAc monosaccharides of natural mucins are conjugated to a lipidated polymer backbone and anchored to fluid, solid-supported lipid membranes, and fluorescence interference contrast microscopy, an optical technique that provides nanometer-scale topographic information about objects near a reflective interface, to measure the orientation of the mucin mimics relative to the membrane plane. Data from two independent probes, fluorophores conjugated directly to the polymer backbone and fluorescent proteins bound to the sugar groups, unexpectedly show that the mucin mimic molecules lie flat along the membrane. Rigidity and core glycosylation are therefore insufficient to ensure molecular projection outward from a membrane surface.

    View details for DOI 10.1021/jp072136q

    View details for Web of Science ID 000250260500015

    View details for PubMedID 17915910

  • Copper-free click chemistry for dynamic in vivo imaging PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Baskin, J. M., Prescher, J. A., Laughlin, S. T., Agard, N. J., Chang, P. V., Miller, I. A., Lo, A., Codelli, J. A., Bertozzi, C. R. 2007; 104 (43): 16793-16797

    Abstract

    Dynamic imaging of proteins in live cells is routinely performed by using genetically encoded reporters, an approach that cannot be extended to other classes of biomolecules such as glycans and lipids. Here, we report a Cu-free variant of click chemistry that can label these biomolecules rapidly and selectively in living systems, overcoming the intrinsic toxicity of the canonical Cu-catalyzed reaction. The critical reagent, a substituted cyclooctyne, possesses ring strain and electron-withdrawing fluorine substituents that together promote the [3 + 2] dipolar cycloaddition with azides installed metabolically into biomolecules. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics.

    View details for Web of Science ID 000250487600015

    View details for PubMedID 17942682

    View details for PubMedCentralID PMC2040404

  • Synthesis of mono- and dideoxygenated alpha,alpha-trehalose analogs CARBOHYDRATE RESEARCH Lin, F. L., van Halbeek, H., Bertozzi, C. R. 2007; 342 (14): 2014-2030

    Abstract

    In this work, we describe the synthesis and NMR characterization of four mono- and four dideoxygenated analogs of alpha,alpha-D-trehalose. The symmetrical (2,2'-, 3,3'-, 4,4'- and 6,6'-) dideoxy analogs were obtained via selective protection and subsequent radical deoxygenation of the desired hydroxyl group set. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions. Complete assignment of all (1)H and (13)C resonances in the spectra of these deoxytrehaloses was achieved through the extensive use of 2D [(1)H,(1)H] and [(1)H,(13)C] correlation NMR experiments. The synthesis of these trehalose analogs sets the stage for future biochemical and NMR-based studies to probe the substrate interactions of trehalose with the recently identified mycobacterial sulfotransferase Stf0.

    View details for DOI 10.1016/j.carres.2007.05.009

    View details for Web of Science ID 000249191300005

    View details for PubMedID 17559818

    View details for PubMedCentralID PMC2072868

  • Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Paulick, M. G., Wise, A. R., Forstner, M. B., Groves, J. T., Bertozzi, C. R. 2007; 129 (37): 11543-11550

    Abstract

    Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphatidylinositol (GPI) anchor is a posttranslational modification that anchors the modified proteins in the outer leaflet of the plasma membrane. GPI-anchored proteins play vital roles in signal transduction, the vertebrate immune response, and the pathobiology of trypanosomal parasites. While many GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. We synthesized a series of GPI-protein analogues bearing modified anchor structures that were designed to dissect the contribution of various glycan components to the GPI-protein's membrane behavior. These anchor analogues were similar in length to native GPI anchors and included mimics of the native structure's three domains. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). These modified GFPs were incorporated in supported lipid bilayers, and their mobilities were analyzed using fluorescence correlation spectroscopy. The data from these experiments suggest that the GPI anchor is more than a simple membrane-anchoring device; it also may prevent transient interactions between the attached protein and the underlying lipid bilayer, thereby permitting rapid diffusion in the bilayer. The ability to generate chemically defined analogues of GPI-anchored proteins is an important step toward elucidating the molecular functions of this interesting post-translational modification.

    View details for DOI 10.1021/ja073271j

    View details for Web of Science ID 000249464900052

    View details for PubMedID 17715922

  • A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Leavy, T. M., Bertozzi, C. R. 2007; 17 (14): 3851-3854

    Abstract

    O-GlcNAc transferase (OGT) catalyzes the addition of N-acetylglucosamine (O-GlcNAc) onto a diverse array of intracellular proteins. Although hundreds of proteins are known to be modified by O-GlcNAc, a strict amino acid consensus sequence for OGT has not been identified. In this study, we describe the development of a high-throughput assay for OGT and use it to profile the specificity of the enzyme among a panel of peptide substrates.

    View details for DOI 10.1016/j.bmcl.2007.05.008

    View details for Web of Science ID 000248074600008

    View details for PubMedID 17531489

    View details for PubMedCentralID PMC3225185

  • Imaging cell surface glycans with bioorthogonal chemical reporters JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chang, P. V., Prescher, J. A., Hangauer, M. J., Bertozzi, C. R. 2007; 129 (27): 8400-?

    View details for DOI 10.1021/ja070238o

    View details for Web of Science ID 000247759400001

    View details for PubMedID 17579403

    View details for PubMedCentralID PMC2535820

  • PapA1and PapA2 are acyltransferases essential for the biosynthesis of the Mycobacterium tuberculosis virulence factor Sulfolipid-1 PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kumar, P., Schelle, M. W., Jain, M., Lin, F. L., Petzold, C. J., Leavell, M. D., Leary, J. A., Cox, J. S., Bertozzi, C. R. 2007; 104 (27): 11221-11226

    Abstract

    Mycobacterium tuberculosis produces numerous exotic lipids that have been implicated as virulence determinants. One such glycolipid, Sulfolipid-1 (SL-1), consists of a trehalose-2-sulfate (T2S) core acylated with four lipid moieties. A diacylated intermediate in SL-1 biosynthesis, SL(1278), has been shown to activate the adaptive immune response in human patients. Although several proteins involved in SL-1 biosynthesis have been identified, the enzymes that acylate the T2S core to form SL(1278) and SL-1, and the biosynthetic order of these acylation reactions, are unknown. Here we demonstrate that PapA2 and PapA1 are responsible for the sequential acylation of T2S to form SL(1278) and are essential for SL-1 biosynthesis. In vitro, recombinant PapA2 converts T2S to 2'-palmitoyl T2S, and PapA1 further elaborates this newly identified SL-1 intermediate to an analog of SL(1278). Disruption of papA2 and papA1 in M. tuberculosis confirmed their essential role in SL-1 biosynthesis and their order of action. Finally, the Delta papA2 and Delta papA1 mutants were screened for virulence defects in a mouse model of infection. The loss of SL-1 (and SL(1278)) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection.

    View details for DOI 10.1073/pnas.0611649104

    View details for Web of Science ID 000247900000020

    View details for PubMedID 17592143

    View details for PubMedCentralID PMC2040880

  • Introducing genetically encoded aldehydes into proteins NATURE CHEMICAL BIOLOGY Carrico, I. S., Carlson, B. L., Bertozzi, C. R. 2007; 3 (6): 321-322

    Abstract

    Methods for introducing bioorthogonal functionalities into proteins have become central to protein engineering efforts. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. This genetically encoded 'aldehyde tag' is no larger than a His(6) tag and can be exploited for numerous protein labeling applications.

    View details for DOI 10.1038/nchembio878

    View details for Web of Science ID 000246816400009

    View details for PubMedID 17450134

  • A cell nanoinjector based on carbon nanotubes PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Chen, X., Kis, A., Zettl, A., Bertozzi, C. R. 2007; 104 (20): 8218-8222

    Abstract

    Technologies for introducing molecules into living cells are vital for probing the physical properties and biochemical interactions that govern the cell's behavior. Here, we report the development of a nanoscale cell injection system (termed the nanoinjector) that uses carbon nanotubes to deliver cargo into cells. A single multiwalled carbon nanotube attached to an atomic force microscope (AFM) tip was functionalized with cargo via a disulfide-based linker. Penetration of cell membranes with this "nanoneedle" was controlled by the AFM. The following reductive cleavage of the disulfide bonds within the cell's interior resulted in the release of cargo inside the cells, after which the nanoneedle was retracted by AFM control. The capability of the nanoinjector was demonstrated by injection of protein-coated quantum dots into live human cells. Single-particle tracking was used to characterize the diffusion dynamics of injected quantum dots in the cytosol. This technique causes no discernible membrane or cell damage, and can deliver a discrete number of molecules to the cell's interior without the requirement of a carrier solvent.

    View details for DOI 10.1073/pnas.0700567104

    View details for Web of Science ID 000246599900007

    View details for PubMedID 17485677

    View details for PubMedCentralID PMC1895932

  • Hierarchical assembly of model cell surfaces: Synthesis of mucin mimetic polymers and their display on supported bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rabuka, D., Parthasarathy, R., Lee, G. S., Chen, X., Groves, J. T., Bertozzi, C. R. 2007; 129 (17): 5462-5471

    Abstract

    Molecular level analysis of cell-surface phenomena could benefit from model systems comprising structurally defined components. Here we present the first step toward bottom-up assembly of model cell surfaces-the synthesis of mucin mimetics and their incorporation into artificial membranes. Natural mucins are densely glycosylated O-linked glycoproteins that serve numerous functions on cell surfaces. Their large size and extensive glycosylation makes the synthesis of these biopolymers impractical. We designed synthetically tractable glycosylated polymers that possess rodlike extended conformations similar to natural mucins. The glycosylated polymers were end-functionalized with lipid groups and embedded into supported lipid bilayers where they interact with protein receptors in a structure-dependent manner. Furthermore, their dynamic behavior in synthetic membranes mirrored that of natural biomolecules. This system provides a unique framework with which to study the behavior of mucin-like macromolecules in a controlled, cell surface-mimetic environment.

    View details for DOI 10.1021/ja067819i

    View details for Web of Science ID 000245946400051

    View details for PubMedID 17425309

    View details for PubMedCentralID PMC2535821

  • Vaccine efficacy of an attenuated but persistent Mycobacterium tuberculosis cysH mutant JOURNAL OF MEDICAL MICROBIOLOGY Senaratne, R. H., Mougous, J. D., Reader, J. R., Williams, S. J., Zhang, T., Bertozzi, C. R., Riley, L. W. 2007; 56 (4): 454-458

    Abstract

    The emergence of drug-resistant Mycobacterium tuberculosis strains and the widespread occurrence of AIDS demand newer and more efficient control of tuberculosis. The protective efficacy of the current Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine is highly variable. Therefore, development of an effective new vaccine has gained momentum in recent years. Recently, several M. tuberculosis mutants were tested as potential vaccine candidates in the mouse model of tuberculosis. However, only some of these mutants were able to generate protection equivalent to that of BCG in mice. This study reports the vaccine potential of an attenuated 5'-adenosine phosphosulfate reductase mutant (DeltacysH) of M. tuberculosis. Immunization of mice with either BCG or DeltacysH followed by infection with the virulent M. tuberculosis Erdman strain demonstrated that DeltacysH can generate protection equivalent to that of the BCG vaccine.

    View details for DOI 10.1099/jmm.0.46983-0

    View details for Web of Science ID 000245795600002

    View details for PubMedID 17374883

  • Lipidomics reveals control of Mycobacterium tuberculosis virulence lipids via metabolic coupling PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Jain, M., Petzold, C. J., Schelle, M. W., Leavell, M. D., Mougous, J. D., Bertozzi, C. R., Leary, J. A., Cox, J. S. 2007; 104 (12): 5133-5138

    Abstract

    Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Using a mass spectrometric approach to simultaneously monitor hundreds of lipids, we discovered that the size and abundance of two lipid virulence factors, phthiocerol dimycocerosate (PDIM) and sulfolipid-1 (SL-1), are controlled by the availability of a common precursor, methyl malonyl CoA (MMCoA). Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. Furthermore, perturbation of MMCoA metabolism attenuated pathogen replication in mice. Importantly, we detected increased PDIM synthesis in bacteria growing within host tissues and in bacteria grown in culture on odd-chain fatty acids. Because M. tuberculosis catabolizes host lipids to grow during infection, we propose that growth of M. tuberculosis on fatty acids in vivo leads to increased flux of MMCoA through lipid biosynthetic pathways, resulting in increased virulence lipid synthesis. Our results suggest that the shift to host lipid catabolism during infection allows for increased virulence lipid anabolism by the bacterium.

    View details for DOI 10.1073/pnas.0610634104

    View details for Web of Science ID 000245256700066

    View details for PubMedID 17360366

    View details for PubMedCentralID PMC1829275

  • Noncovalent complexes of APS reductase from M-tuberculosis: Delineating a mechanistic model using ESI-FTICR MS JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY Gao, H., Leary, J., Carroll, K. S., Bertozzi, C. R., Chen, H. 2007; 18 (2): 167-178

    Abstract

    ESI-FTICR MS was utilized to characterize a 4Fe-4S containing protein Mycobacterium tuberculosis APS reductase. This enzyme catalyzes the reduction of APS to sulfite and AMP with reducing equivalents from the protein cofactor, thioredoxin. Under nondenaturing conditions, a distribution of the apoprotein, a 2Fe-2S intermediate, and the 4Fe-4S holoprotein were observed. Accurate mass measurements indicated an oxidation state of +2 for the 4Fe-4S cluster, with no disulfide bond in the holoenzyme. Gas-phase stability of the 4Fe-4S cluster was investigated using both in-source and collision induced dissociation, which provided information regarding the relative gas-phase binding strength of iron towards protein ligands and inorganic sulfides. Noncovalent complexes of the holoprotein with several ligands, including APS, thioredoxin, and AMP, were also investigated. Calculated values of dissociation constants for the complexes indicate that AMP binds with a higher affinity to the enzyme intermediate than to the free enzyme. The implications of the binary and ternary complexes observed by gas-phase noncovalent interactions in the mechanism of APS reduction are discussed.

    View details for DOI 10.1016/j.jasms.2006.08.010

    View details for Web of Science ID 000244109300001

    View details for PubMedID 17023175

    View details for PubMedCentralID PMC2755055

  • Self-assembled cellular microarrays patterned using DNA barcodes LAB ON A CHIP Douglas, E. S., Chandra, R. A., Bertozzi, C. R., Mathies, R. A., Francis, M. B. 2007; 7 (11): 1442-1448

    Abstract

    The successful integration of living cells into synthetic devices requires precise control over cell patterning. Here we describe a versatile platform that can accomplish this goal through DNA hybridization. Living cells functionalized with exogenous cell-surface DNA strands bind to cognate sequences of DNA printed on glass slides. Attachment via these "cell-adhesion barcodes" is rapid and specific, with close-packed arrays of cells forming within minutes. The biophysical properties of the system are characterized, and the technique is used to form complex cellular patterns with single-cell line widths and self-assembled cellular microarrays. Key advantages of DNA-directed cell binding include the ability to immobilize both adherent and non-adherent cells, to capture cells selectively from a mixed population, to tune the binding properties of the cells, and to reuse substrates prepared with widely available DNA printing technologies.

    View details for DOI 10.1039/b708666k

    View details for Web of Science ID 000250428200015

    View details for PubMedID 17960269

  • Metabolic labeling of glycans with azido sugars and subsequent glycan-profiling and visualization via Staudinger ligation NATURE PROTOCOLS Laughlin, S. T., Bertozzi, C. R. 2007; 2 (11): 2930-2944

    Abstract

    Metabolic labeling of glycans with a bioorthogonal chemical reporter such as the azide enables their visualization in cells and organisms as well as the enrichment of specific glycoprotein types for proteomic analysis. This process involves two steps. Azido sugars are fed to cells or organisms and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged with imaging probes or epitope tags, either ex vivo or in vivo, using an azide-specific reaction. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Applications to the visualization of cellular glycans and enrichment of glycoproteins for proteomic analysis are described. The synthesis of the azido sugars (ManNAz, GalNAz, GlcNAz or 6AzFuc) or detection reagents (phosphine-FLAG or phosphine-FLAG-His6) can be completed in approximately 1 week. A cell metabolic labeling experiment can be completed in approximately 4 d.

    View details for DOI 10.1038/nprot.2007.422

    View details for Web of Science ID 000253140000033

    View details for PubMedID 18007630

  • Synthetic glycobiology: exploits in the Golgi compartment CURRENT OPINION IN CHEMICAL BIOLOGY Czlapinski, J. L., Bertozzi, C. R. 2006; 10 (6): 645-651

    Abstract

    The challenge of engineering glycosylation has been confronted by synthetic chemists, biochemists and cell biologists, each with the primary goal of optimizing glycoconjugates for therapeutic applications. In nature, glycans are constructed by glycosyltransferases that are organized in an assembly line in the endoplasmic reticulum and Golgi compartment. Recent insights into the domain architecture, localization and regulation of glycosyltransferases have provided a platform for engineering their position within the secretory pathway and access to substrates. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. These efforts epitomize the emerging field of 'synthetic glycobiology'.

    View details for DOI 10.1016/j.cbpa.2006.10.009

    View details for Web of Science ID 000242919700018

    View details for PubMedID 17056297

  • Using phage display to select antibodies recognizing post-translational modifications independently of sequence context MOLECULAR & CELLULAR PROTEOMICS Kehoe, J. W., Velappan, N., Walbolt, M., Rasmussen, J., King, D., Lou, J., Knopp, K., Pavlik, P., Marks, J. D., Bertozzi, C. R., Bradbury, A. R. 2006; 5 (12): 2350-2363

    Abstract

    Many cellular activities are controlled by post-translational modifications, the study of which is hampered by the lack of specific reagents due in large part to their ubiquitous and non-immunogenic nature. Although antibodies against specifically modified sequences are relatively easy to obtain, it is extremely difficult to derive reagents recognizing post-translational modifications independently of the sequence context surrounding the modification. In this study, we examined the possibility of selecting such antibodies from large phage antibody libraries using sulfotyrosine as a test case. Sulfotyrosine is a post-translational modification important in many extracellular protein-protein interactions, including human immunodeficiency virus infection. After screening almost 8000 selected clones, we were able to isolate a single specific single chain Fv using two different selection strategies, one of which included elution with tyrosine sulfate. This antibody was able to recognize sulfotyrosine independently of its sequence context in test peptides and a number of different natural proteins. Antibody reactivity was lost by antigen treatment with sulfatase or preincubation with soluble tyrosine sulfate, indicating its specificity. The isolation of this antibody signals the potential of phage antibody libraries in the derivation of reagents specific for post-translational modifications, although the extensive screening required indicates that such antibodies are extremely rare.

    View details for DOI 10.1074/mcp.M600314-MCP200

    View details for Web of Science ID 000242852000012

    View details for PubMedID 16971384

  • Substrate recognition, protein dynamics, and iron-sulfur cluster in Pseudomonas aeruginosa adenosine 5 '-phosphosulfate reductase JOURNAL OF MOLECULAR BIOLOGY Chartron, J., Carroll, K. S., Shiau, C., Gao, H., Leary, J. A., Bertozzi, C. R., Stout, C. D. 2006; 364 (2): 152-169

    Abstract

    APS reductase catalyzes the first committed step of reductive sulfate assimilation in pathogenic bacteria, including Mycobacterium tuberculosis, and is a promising target for drug development. We report the 2.7 A resolution crystal structure of Pseudomonas aeruginosa APS reductase in the thiosulfonate intermediate form of the catalytic cycle and with substrate bound. The structure, high-resolution Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry, and quantitative kinetic analysis, establish that the two chemically discrete steps of the overall reaction take place at distinct sites on the enzyme, mediated via conformational flexibility of the C-terminal 18 residues. The results address the mechanism by which sulfonucleotide reductases protect the covalent but labile enzyme-intermediate before release of sulfite by the protein cofactor thioredoxin. P. aeruginosa APS reductase contains an [4Fe-4S] cluster that is essential for catalysis. The structure reveals an unusual mode of cluster coordination by tandem cysteine residues and suggests how this arrangement might facilitate conformational change and cluster interaction with the substrate. Assimilatory 3'-phosphoadenosine 5'-phosphosulfate (PAPS) reductases are evolutionarily related, homologous enzymes that catalyze the same overall reaction, but do so in the absence of an [Fe-S] cluster. The APS reductase structure reveals adaptive use of a phosphate-binding loop for recognition of the APS O3' hydroxyl group, or the PAPS 3'-phosphate group.

    View details for DOI 10.1016/j.jmb.2006.08.080

    View details for Web of Science ID 000242160600003

    View details for PubMedID 17010373

    View details for PubMedCentralID PMC1769331

  • Sulfate metabolism in mycobacteria CHEMBIOCHEM Schelle, M. W., Bertozzi, C. R. 2006; 7 (10): 1516-1524

    Abstract

    Pathogenic bacteria have developed numerous mechanisms to survive inside a hostile host environment. The human pathogen Mycobacterium tuberculosis (M. tb) is thought to control the human immune response with diverse biomolecules, including a variety of exotic lipids. One prevalent M. tb-specific sulfated metabolite, termed sulfolipid-1 (SL-1), has been correlated with virulence though its specific biological function is not known. Recent advances in our understanding of SL-1 biosynthesis will help elucidate the role of this curious metabolite in M. tb infection. Furthermore, the study of SL-1 has led to questions regarding the significance of sulfation in mycobacteria. Examples of sulfated metabolites as mediators of interactions between bacteria and plants suggest that sulfation is a key modulator of extracellular signaling between prokaryotes and eukaryotes. The discovery of novel sulfated metabolites in M. tb and related mycobacteria strengthens this hypothesis. Finally, mechanistic and structural data from sulfate-assimilation enzymes have revealed how M. tb controls the flux of sulfate in the cell. Mutants with defects in sulfate assimilation indicate that the fate of sulfur in M. tb is a critical survival determinant for the bacteria during infection and suggest novel targets for tuberculosis drug therapy.

    View details for DOI 10.1002/cbic.200600224

    View details for Web of Science ID 000241392400005

    View details for PubMedID 16933356

  • A chemical reporter strategy to probe glycoprotein fucosylation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rabuka, D., Hubbard, S. C., Laughlin, S. T., Argade, S. P., Bertozzi, C. R. 2006; 128 (37): 12078-12079

    Abstract

    Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Here we report a method for rapid profiling of fucosylated glycoproteins from human cells using 6-azido fucose as a metabolic label.

    View details for DOI 10.1021/ja064619y

    View details for Web of Science ID 000240465200023

    View details for PubMedID 16967952

    View details for PubMedCentralID PMC3233198

  • Chemical technologies for probing glycans CELL Prescher, J. A., Bertozzi, C. R. 2006; 126 (5): 851-854

    Abstract

    Glycans are central to many biological processes, but efforts to define their functions at the molecular level have been frustrated by a lack of suitable technologies. Here we highlight chemical tools that are beginning to address this need.

    View details for DOI 10.1016/j.cell.2006.08.017

    View details for Web of Science ID 000240675000013

    View details for PubMedID 16959565

  • A role for sulfation-desulfation in the uptake of bisphenol A into breast tumor cells CHEMISTRY & BIOLOGY Stowell, C. L., Barvian, K. K., Young, P. C., Bigsby, R. M., Verdugo, D. E., Bertozzi, C. R., Widlanski, T. S. 2006; 13 (8): 891-897

    Abstract

    Bisphenol A (BPA) is a widely used plasticizer whose estrogenic properties may impact hormone-responsive disorders and fetal development. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. This may be a result of BPA sulfation/desulfation providing a pathway for selective uptake into hormone-responsive cells. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Treatment of MCF-7 cells with BPAS leads to desulfation and uptake of BPA. No BPAS is found inside the cells. These findings suggest a mechanism for the selective uptake of BPA into cells expressing estrone sulfatase. Therefore, sulfation may increase the estrogenic potential of xenobiotics.

    View details for DOI 10.1016/j.chembiol.2006.06.016

    View details for Web of Science ID 000240329800013

    View details for PubMedID 16931338

  • Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Link, A. J., Vink, M. K., Agard, N. J., Prescher, J. A., Bertozzi, C. R., Tirrell, D. A. 2006; 103 (27): 10180-10185

    Abstract

    The incorporation of noncanonical amino acids into recombinant proteins in Escherichia coli can be facilitated by the introduction of new aminoacyl-tRNA synthetase activity into the expression host. We describe here a screening procedure for the identification of new aminoacyl-tRNA synthetase activity based on the cell surface display of noncanonical amino acids. Screening of a saturation mutagenesis library of the E. coli methionyl-tRNA synthetase (MetRS) led to the discovery of three MetRS mutants capable of incorporating the long-chain amino acid azidonorleucine into recombinant proteins with modest efficiency. The Leu-13 --> Gly (L13G) mutation is found in each of the three MetRS mutants, and the MetRS variant containing this single mutation is highly efficient in producing recombinant proteins that contain azidonorleucine.

    View details for DOI 10.1073/pnas.0601167103

    View details for Web of Science ID 000239069400007

    View details for PubMedID 16801548

    View details for PubMedCentralID PMC1502431

  • Interfacing carbon nanotubes with living cells JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, X., Tam, U. C., Czlapinski, J. L., Lee, G. S., Rabuka, D., Zettl, A., Bertozzi, C. R. 2006; 128 (19): 6292-6293

    Abstract

    We developed a polymer coating for carbon nanotubes (CNTs) that mimics the mucin glycoprotein coating of mammalian cells. CNTs coated with these mucin mimic polymers have two novel properties: they can bind to carbohydrate receptors, providing a means for biomimetic interactions with cell surfaces, and, importantly, they are rendered nontoxic to cells.

    View details for DOI 10.1021/ja060276s

    View details for Web of Science ID 000237590400012

    View details for PubMedID 16683774

  • Probing mucin-type O-linked glycosylation in living animals PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Dube, D. H., Prescher, J. A., Quang, C. N., Bertozzi, C. R. 2006; 103 (13): 4819-4824

    Abstract

    Changes in O-linked protein glycosylation are known to correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. Here, we report a technique for rapid profiling of O-linked glycoproteins in living animals by metabolic labeling with N-azidoacetylgalactosamine (GalNAz) followed by Staudinger ligation with phosphine probes. After injection of mice with a peracetylated form of GalNAz, azide-labeled glycoproteins were observed in a variety of tissues, including liver, kidney, and heart, in serum, and on isolated splenocytes. B cell glycoproteins were robustly labeled with GalNAz but T cell glycoproteins were not, suggesting fundamental differences in glycosylation machinery or metabolism. Furthermore, GalNAz-labeled B cells could be selectively targeted with a phosphine probe by Staudinger ligation within the living animal. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of this posttranslational modification and for identifying O-linked glycoprotein fingerprints associated with disease.

    View details for DOI 10.1073/pnas.0506855103

    View details for Web of Science ID 000236472500008

    View details for PubMedID 16549800

    View details for PubMedCentralID PMC1405625

  • A sulfated metabolite produced by stf3 negatively regulates the virulence of Mycobacterium tuberculosis PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mougous, J. D., Senaratne, R. H., Petzold, C. J., Jain, M., Lee, D. H., Schelle, M. W., Leavell, M. D., Cox, J. S., Leary, J. A., Riley, L. W., Bertozzi, C. R. 2006; 103 (11): 4258-4263

    Abstract

    Sulfated molecules have been shown to modulate isotypic interactions between cells of metazoans and heterotypic interactions between bacterial pathogens or symbionts and their eukaryotic host cells. Mycobacterium tuberculosis, the causative agent of tuberculosis, produces sulfated molecules that have eluded functional characterization for decades. We demonstrate here that a previously uncharacterized sulfated molecule, termed S881, is localized to the outer envelope of M. tuberculosis and negatively regulates the virulence of the organism in two mouse infection models. Furthermore, we show that the biosynthesis of S881 relies on the universal sulfate donor 3'-phosphoadenosine-5'-phosphosulfate and a previously uncharacterized sulfotransferase, stf3. These findings extend the known functions of sulfated molecules as general modulators of cell-cell interactions to include those between a bacterium and a human host.

    View details for DOI 10.1073/pnas.0510861103

    View details for Web of Science ID 000236429300059

    View details for PubMedID 16537518

    View details for PubMedCentralID PMC1449680

  • 5 '-Adenosinephosphosulphate reductase (CysH) protects Mycobacterium tuberculosis against free radicals during chronic infection phase in mice MOLECULAR MICROBIOLOGY Senaratne, R. H., De Silva, A. D., Williams, S. J., Mougous, J. D., Reader, J. R., Zhang, T. J., Chan, S., Sidders, B., Lee, D. H., Chan, J., Bertozzi, C. R., Riley, L. W. 2006; 59 (6): 1744-1753

    Abstract

    A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). Here we report that 5'-adenosinephosphosulphate reductase (CysH), an enzyme essential for the production of reduced-sulphur-containing metabolites, is critical for Mycobacterium tuberculosis (Mtb) survival in chronic infection phase in mice. Disruption of cysH rendered Mtb auxotrophic for cysteine and methionine, and attenuated virulence in BALB/c and C57BL/6 immunocompetent mice. The mutant and wild-type Mtb replicated similarly during the acute phase of infection, but the mutant showed reduced viability during the persistent phase of the infection. The cysH mutant caused disease and death after 4-7 weeks of infection in four different groups of mice - Rag1(-/-), NOS2(-/-), gp91phox(-/-) NOS2(-/-) and gp91phox(-/-) mice given aminoguanidine [to suppress the effects of nitric oxide synthase 2 (NOS2)]- indicating minimal metabolic effect on the cysH mutant survival in these mice. The cysH mutant was also susceptible to peroxynitrite and hydrogen peroxide in vitro. These results show that CysH is important for Mtb protection during the chronic infection phase, and that resistance to nitrosative and oxidative stress may be the mechanism of this protection. Thus, this metabolic gene of an intracellular pathogen could have a secondary role in protection against the host immune response. Finally the lack of an endogenous human orthologue of cysH and its possible role in defence against adaptive immunity renders CysH an attractive enzyme for further studies as a target for therapeutics active against CTBI.

    View details for DOI 10.1111/j.1365-2958.2006.05075.x

    View details for Web of Science ID 000235842600009

    View details for PubMedID 16553880

  • Mucin granule intraluminal organization in living mucous/goblet cells - Roles of protein post-translational modifications and secretion JOURNAL OF BIOLOGICAL CHEMISTRY Perez-Vilar, J., Mabolo, R., McVaugh, C. T., Bertozzi, C. R., Boucher, R. C. 2006; 281 (8): 4844-4855

    Abstract

    Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Secretory products can both diffuse, although very slowly, through the meshwork pores and interact noncovalently with the matrix. Using a green fluorescent protein-mucin fusion protein (SHGFP-MUC5AC/CK) as a FRAP (fluorescence recovery after photobleaching) probe, we have assessed in living mucous cells the relative importance of different protein post-translational modifications on the intragranular organization. Long term inhibition of mucin-type O-glycosylation, sialylation, or sulfation altered SHGFP-MUC5AC/CK characteristic diffusion time (t(1/2)), whereas all but sulfation diminished its mobile fraction. Reduction of protein disulfide bonds with tris(hydroxypropyl)phosphine resulted in virtually complete immobilization of the SHGFP-MUC5AC/CK intragranular pool. However, when activity of the vacuolar H+-ATPase was also inhibited, disulfide reduction decreased SHGFP-MUC5AC/CK t((1/2)) while diminishing its intraluminal concentration. Similar FRAP profiles were observed in granules that remained in the cells after the addition of a mucin secretagogue. Taken together these results suggest that: (a) the relative content of O-glycans and intragranular anionic groups is crucial for protein diffusion through the intragranular meshwork; (b) protein-protein, rather than carbohydrate-mediated, interactions are responsible for binding of SHGFP-MUC5AC/CK to the immobile fraction, although the degree of matrix O-glycosylation and sialylation affects such interactions; (c) intragranular organization does not depend on covalent multimerization of mucins or the presence of native disulfide bonds in the intragranular mucin/proteins, but rather on specific protein-mediated interactions that are important during the early stages of mucin matrix condensation; (d) alterations of the intragranular matrix precede granule discharge, which can be partial and, accordingly, does not necessarily involve the disappearance of the granule.

    View details for DOI 10.1074/jbc.M510520200

    View details for Web of Science ID 000235426200035

    View details for PubMedID 16377632

  • Molecular basis for G protein control of the prokaryotic ATP sulfurylase MOLECULAR CELL Mougous, J. D., Lee, D. H., Hubbard, S. C., Schelle, M. W., Vocadlo, D. J., Berger, J. M., Bertozzi, C. R. 2006; 21 (1): 109-122

    Abstract

    Sulfate assimilation is a critical component of both primary and secondary metabolism. An essential step in this pathway is the activation of sulfate through adenylation by the enzyme ATP sulfurylase (ATPS), forming adenosine 5'-phosphosulfate (APS). Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. To discover the molecular basis of this unusual role for a G protein, we biochemically characterized and solved the X-ray crystal structure of a complex between Pseudomonas syringae ATPS (CysD) and its associated regulatory G protein (CysN). The structure of CysN*D shows the two proteins in tight association; however, the nucleotides bound to each subunit are spatially segregated. We provide evidence that conserved switch motifs in the G domain of CysN allosterically mediate interactions between the nucleotide binding sites. This structure suggests a molecular mechanism by which conserved G domain architecture is used to energetically link GTP turnover to the production of an essential metabolite.

    View details for DOI 10.1016/j.molcel.2005.10.034

    View details for Web of Science ID 000234764700011

    View details for PubMedID 16387658

  • An alpha-formylglycine building block for Fmoc-based solid-phase peptide synthesis ORGANIC LETTERS Rush, J., Bertozzi, C. R. 2006; 8 (1): 131-134

    Abstract

    [reaction: see text] Nearly all known sulfatases share a common active site modification that is required for their activity: conversion of cysteine to alpha-formylglycine. We report the synthesis of an alpha-formylglycine building block suitable for Fmoc-based solid-phase peptide synthesis. The building block was incorporated into a synthetic peptide derived from the active site of a Mycobacterium tuberculosis sulfatase.

    View details for DOI 10.1021/o1052623t

    View details for Web of Science ID 000234391600034

    View details for PubMedID 16381585

    View details for PubMedCentralID PMC2527029

  • A comparative study of bioorthogonal reactions with azides ACS CHEMICAL BIOLOGY Agard, N. J., Baskin, J. M., Prescher, J. A., Lo, A., Bertozzi, C. R. 2006; 1 (10): 644-648

    Abstract

    Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chemical reporter. Once introduced into target biomolecules, either metabolically or through chemical modification, the azide can be tagged with probes using one of three highly selective reactions: the Staudinger ligation, the Cu(I)-catalyzed azide-alkyne cycloaddition, or the strain-promoted [3 + 2] cycloaddition. Here, we compared these chemistries in the context of various biological applications, including labeling of biomolecules in complex lysates and on live cell surfaces. The Cu(I)-catalyzed reaction was found to be most efficient for detecting azides in protein samples but was not compatible with live cells due to the toxicity of the reagents. Both the Staudinger ligation and the strain-promoted [3 + 2] cycloaddition using optimized cyclooctynes were effective for tagging azides on live cells. The best reagent for this application was dependent upon the specific structure of the azide. These results provide a guide for biologists in choosing a suitable ligation chemistry.

    View details for DOI 10.1021/cb6003228

    View details for Web of Science ID 000243895200017

    View details for PubMedID 17175580

  • Metabolic labeling of glycans with azido sugars for visualization and glycoproteomics GLYCOBIOLOGY Laughlin, S. T., Agard, N. J., Baskin, J. M., Carrico, I. S., Chang, P. V., Ganguli, A. S., Hangauer, M. J., Lo, A., Prescher, J. A., Bertozzi, C. R. 2006; 415: 230-250

    Abstract

    The staggering complexity of glycans renders their analysis extraordinarily difficult, particularly in living systems. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. This technology involves metabolic labeling of glycans with a specifically reactive, abiotic functional group, the azide. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. The azido sugars are then covalently tagged, either ex vivo or in vivo, using one of two azide-specific chemistries: the Staudinger ligation, or the strain-promoted [3+2] cycloaddition. These reactions can be used to tag glycans with imaging probes or epitope tags, thus enabling the visualization or enrichment of glycoconjugates. Applications to noninvasive imaging and glycoproteomic analyses are discussed.

    View details for DOI 10.1016/S0076-6879(06)15015-6

    View details for Web of Science ID 000242168500015

    View details for PubMedID 17116478

  • Programmable cell adhesion encoded by DNA hybridization ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Chandra, R. A., Douglas, E. S., Mathies, R. A., Bertozzi, C. R., Francis, M. B. 2006; 45 (6): 896-901

    View details for DOI 10.1002/ANIE.200502421

    View details for Web of Science ID 000235246600005

    View details for PubMedID 16370010

  • Investigation of the iron-sulfur cluster in Mycobacterium tuberculosis APS reductase: Implications for substrate binding and catalysis BIOCHEMISTRY Carroll, K. S., Gao, H., Chen, H. Y., Leary, J. A., Bertozzi, C. R. 2005; 44 (44): 14647-14657

    Abstract

    The sulfur assimilation pathway is a key metabolic system in prokaryotes that is required for production of cysteine and cofactors such as coenzyme A. In the first step of the pathway, APS reductase catalyzes the reduction of adenosine 5'-phosphosulfate (APS) to adenosine 5'-phosphate (AMP) and sulfite with reducing equivalents from the protein cofactor, thioredoxin. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Of the sequence motifs that are associated with 4Fe-4S centers, the cysteine dyad is atypical and has generated discussion with respect to coordination as well as the cluster's larger functional significance. Herein, we have used biochemical, spectroscopic, and mass spectrometry analysis to investigate the iron-sulfur cluster and its role in the mechanism of Mycobacterium tuberculosis APS reductase. Site-directed mutagenesis of any cysteine residue within the conserved motif led to a loss of cluster with a concomitant loss in catalytic activity, while secondary structure was preserved. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. Taken together, these results demonstrate that the active site functionally communicates with the iron-sulfur cluster and also suggest a functional significance for the cysteine dyad in promoting site differentiation within the 4Fe-4S cluster.

    View details for DOI 10.1021/bi051344a

    View details for Web of Science ID 000233068900026

    View details for PubMedID 16262264

  • Biomimetic bonelike composites and novel bioactive glass coatings ADVANCED ENGINEERING MATERIALS Tomsia, A. P., Saiz, E., Song, J., Bertozzi, C. R. 2005; 7 (11): 999-1004
  • Synthesis of lipidated green fluorescent protein and its incorporation in supported lipid bilayers JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Grogan, M. J., Kaizuka, Y., Conrad, R. M., Groves, J. T., Bertozzi, C. R. 2005; 127 (41): 14383-14387

    Abstract

    Herein we report a semisynthetic method of producing membrane-anchored proteins. Ligation of synthetic lipids with designed anchor structures to proteins was performed using native chemical ligation (NCL) of a C-terminal peptide thioester and an N-terminal cysteine lipid. This strategy mimics the natural glycosylphosphatidylinositol (GPI) linkage found in many natural membrane-associated proteins; however, the synthetic method utilizes simple lipid anchors without glycans. Synthetically lipidated recombinant green fluorescent protein (GFP) was shown to be stably anchored to the membrane, and its lateral fluidity was quantitatively characterized by direct fluorescence imaging in supported membranes. Circumventing the steps of purification from native cell membranes, this methodology facilitates the reconstitution of membrane-associated proteins.

    View details for DOI 10.1021/ja052407f

    View details for Web of Science ID 000232605600062

    View details for PubMedID 16218633

  • The chemistry and biology of mucin-type O-linked glycosylation BIOORGANIC & MEDICINAL CHEMISTRY Hang, H. C., Bertozzi, C. R. 2005; 13 (17): 5021-5034

    Abstract

    Mucin-type O-linked glycosylation is a fundamental post-translational modification that is involved in a variety of important biological processes. However, the lack of chemical tools to study mucin-type O-linked glycosylation has hindered our molecular understanding of O-linked glycans in many biological contexts. The review discusses the significance of mucin-type O-linked glycosylation initiated by the polypeptide N-acetylgalactosaminyltransferases in biology and development of chemical tools to study these enzymes and their substrates.

    View details for DOI 10.1016/j.bmc.2005.04.085

    View details for Web of Science ID 000231341900006

    View details for PubMedID 16005634

  • Compositional profiling of heparin/heparan sulfate using mass spectrometry: assay for specificity of a novel extracellular human endosulfatase GLYCOBIOLOGY Saad, O. M., Ebel, H., Uchimura, K., ROSEN, S. D., Bertozzi, C. R., Leary, J. A. 2005; 15 (8): 818-826

    Abstract

    An important class of carbohydrates studied within the field of glycobiology, heparin and heparan sulfate (HS) have been implicated in a diverse array of biological functions. Changes in their sulfation pattern and domain organization have been associated with different pathological situations such as viral infectivity, tumor growth, and metastasis. To obtain structural information about these biomolecules, and the modifications they may undergo during different stages of cell growth and development, a mass spectrometry-based method was developed and used to obtain unambiguous structural information on the glycosaminoglycans (GAGs) that comprise heparin/HS. The method was applied to assay for the heparin substrate specificity of a newly discovered human extracellular endosulfatase, HSulf-2, which has been implicated in tumorigenesis. This new protocol incorporates 12 known heparin disaccharides, including three sets of isomers. A unique response factor (R) is determined for each disaccharide, whereas a multiplexed and data processing method is incorporated for faster data acquisition and quantification purposes. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues.

    View details for DOI 10.1093/glycob/cwi064

    View details for Web of Science ID 000230346400006

    View details for PubMedID 15843596

  • A conserved mechanism for sulfonucleotide reduction PLOS BIOLOGY Carroll, K. S., Gao, H., Chen, H. Y., Stout, C. D., Leary, J. A., Bertozzi, C. R. 2005; 3 (8): 1418-1435

    Abstract

    Sulfonucleotide reductases are a diverse family of enzymes that catalyze the first committed step of reductive sulfur assimilation. In this reaction, activated sulfate in the context of adenosine-5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) is converted to sulfite with reducing equivalents from thioredoxin. The sulfite generated in this reaction is utilized in bacteria and plants for the eventual production of essential biomolecules such as cysteine and coenzyme A. Humans do not possess a homologous metabolic pathway, and thus, these enzymes represent attractive targets for therapeutic intervention. Here we studied the mechanism of sulfonucleotide reduction by APS reductase from the human pathogen Mycobacterium tuberculosis, using a combination of mass spectrometry and biochemical approaches. The results support the hypothesis of a two-step mechanism in which the sulfonucleotide first undergoes rapid nucleophilic attack to form an enzyme-thiosulfonate (E-Cys-S-SO(3-)) intermediate. Sulfite is then released in a thioredoxin-dependent manner. Other sulfonucleotide reductases from structurally divergent subclasses appear to use the same mechanism, suggesting that this family of enzymes has evolved from a common ancestor.

    View details for DOI 10.1371/journal.pbio.0030250

    View details for Web of Science ID 000231243800014

    View details for PubMedID 16008502

    View details for PubMedCentralID PMC1175818

  • Glycans in cancer and inflammation. Potential for therapeutics and diagnostics NATURE REVIEWS DRUG DISCOVERY Dube, D. H., Bertozzi, C. R. 2005; 4 (6): 477-488

    Abstract

    Changes in glycosylation are often a hallmark of disease states. For example, cancer cells frequently display glycans at different levels or with fundamentally different structures than those observed on normal cells. This phenomenon was first described in the early 1970s, but the molecular details underlying such transformations were poorly understood. In the past decade advances in genomics, proteomics and mass spectrometry have enabled the association of specific glycan structures with disease states. In some cases, the functional significance of disease-associated changes in glycosylation has been revealed. This review highlights changes in glycosylation associated with cancer and chronic inflammation and new therapeutic and diagnostic strategies that are based on the underlying glycobiology.

    View details for DOI 10.1038/nrd1751

    View details for Web of Science ID 000229578100018

    View details for PubMedID 15931257

  • Kinetic measurements and mechanism determination of Stf0 sulfotransferase using mass spectrometry ANALYTICAL BIOCHEMISTRY Pi, N., Hoang, M. B., Gao, H., Mougous, J. D., Bertozzi, C. R., Leary, J. A. 2005; 341 (1): 94-104

    Abstract

    Mycobacterial carbohydrate sulfotransferase Stf0 catalyzes the sulfuryl group transfer from 3'-phosphoadenosine-5'-phosphosulfate (PAPS) to trehalose. The sulfation of trehalose is required for the biosynthesis of sulfolipid-1, the most abundant sulfated metabolite found in Mycobacterium tuberculosis. In this paper, an efficient enzyme kinetics assay for Stf0 using electrospray ionization (ESI) mass spectrometry is presented. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. In addition, Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry was employed to detect the noncovalent complexes, the Stf0-PAPS and Stf0-trehalose binary complexes, and a Stf0-3'-phosphoadenosine 5'-phosphate-trehalose ternary complex. The results from our study strongly suggest a rapid equilibrium random sequential Bi-Bi mechanism for Stf0 with formation of a ternary complex intermediate. In this mechanism, PAPS and trehalose bind and their products are released in random fashion. To our knowledge, this is the first detailed mechanistic data reported for Stf0, which further demonstrates the power of mass spectrometry in elucidating the reaction pathway and catalytic mechanism of promising enzymatic systems.

    View details for DOI 10.1016/j.ab.2005.02.004

    View details for Web of Science ID 000229122600012

    View details for PubMedID 15866533

  • Chemistry in living systems NATURE CHEMICAL BIOLOGY Prescher, J. A., Bertozzi, C. R. 2005; 1 (1): 13-21

    Abstract

    Dissecting complex cellular processes requires the ability to track biomolecules as they function within their native habitat. Although genetically encoded tags such as GFP are widely used to monitor discrete proteins, they can cause significant perturbations to a protein's structure and have no direct extension to other classes of biomolecules such as glycans, lipids, nucleic acids and secondary metabolites. In recent years, an alternative tool for tagging biomolecules has emerged from the chemical biology community--the bioorthogonal chemical reporter. In a prototypical experiment, a unique chemical motif, often as small as a single functional group, is incorporated into the target biomolecule using the cell's own biosynthetic machinery. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. This review highlights the development of bioorthogonal chemical reporters and reactions and their application in living systems.

    View details for DOI 10.1038/nchembio0605-13

    View details for Web of Science ID 000232621100006

    View details for PubMedID 16407987

  • Mineralization of synthetic polymer scaffolds: A bottom-up approach for the development of artificial bone JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Song, J., Malathong, V., Bertozzi, C. R. 2005; 127 (10): 3366-3372

    Abstract

    The controlled integration of organic and inorganic components confers natural bone with superior mechanical properties. Bone biogenesis is thought to occur by templated mineralization of hard apatite crystals by an elastic protein scaffold, a process we sought to emulate with synthetic biomimetic hydrogel polymers. Cross-linked polymethacrylamide and polymethacrylate hydrogels were functionalized with mineral-binding ligands and used to template the formation of hydroxyapatite. Strong adhesion between the organic and inorganic materials was achieved for hydrogels functionalized with either carboxylate or hydroxy ligands. The mineral-nucleating potential of hydroxyl groups identified here broadens the design parameters for synthetic bonelike composites and suggests a potential role for hydroxylated collagen proteins in bone mineralization.

    View details for DOI 10.1021/ja043776z

    View details for Web of Science ID 000227627800041

    View details for PubMedID 15755154

  • Mechanistic investigation of the Staudinger ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lin, F. L., Hoyt, H. M., van Halbeek, H., Bergman, R. G., Bertozzi, C. R. 2005; 127 (8): 2686-2695

    Abstract

    The Staudinger ligation of azides and phosphines has found widespread use in the field of chemical biology, but the mechanism of the transformation has not been characterized in detail. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The Staudinger ligation with alkyl azides was second-order overall and proceeded more rapidly in polar, protic solvents. Hammett analyses demonstrated that electron-donating substituents on the phosphine accelerate the overall reaction. The electronic and steric properties of the ester had no significant impact on the overall rate but did affect product ratios. Finally, the structure of an intermediate that accumulates under anhydrous conditions was identified. These findings establish a platform for optimizing the Staudinger ligation for expanded use in biological applications.

    View details for DOI 10.1021/ja044461m

    View details for Web of Science ID 000227308100055

    View details for PubMedID 15725026

  • Functional hydrogel-biomineral composites inspired by natural bone Symposium on Polymer Biocatalysis and Biomaterials held at the 2003 ACS National Meeting Song, J., Bertozzi, C. R. AMER CHEMICAL SOC. 2005: 96?106
  • Synthetic glycopeptides and glycoproteins as tools for biology CHEMICAL SOCIETY REVIEWS Pratt, M. R., Bertozzi, C. R. 2005; 34 (1): 58-68

    Abstract

    Investigations into the roles of protein glycosylation have revealed functions such as modulating protein structure and localization, cell-cell recognition, and signaling in multicellular systems. However, detailed studies of these events are hampered by the heterogeneous nature of biosynthetic glycoproteins that typically exist in numerous glycoforms. Research into protein glycosylation, therefore, has benefited from homogeneous, structurally-defined glycoproteins obtained by chemical synthesis. This tutorial review focuses on recent applications of homogeneous synthetic glycopeptides and glycoproteins for studies of structure and function. In addition, the future of synthetic glycopeptides and glycoproteins as therapeutics is discussed.

    View details for DOI 10.1039/b400593g

    View details for Web of Science ID 000226522400020

    View details for PubMedID 15643490

  • Azido sialic acids can modulate cell-surface interactions CHEMBIOCHEM Luchansky, S. J., Bertozzi, C. R. 2004; 5 (12): 1706-1709

    View details for DOI 10.1002/cbic.200400148

    View details for Web of Science ID 000225672400013

    View details for PubMedID 15568180

  • A small-molecule switch for Golgi sulfotransferases PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA de Graffenried, C. L., Laughlin, S. T., Kohler, J. J., Bertozzi, C. R. 2004; 101 (48): 16715-16720

    Abstract

    The study of glycan function is a major frontier in biology that could benefit from small molecules capable of perturbing carbohydrate structures on cells. The widespread role of sulfotransferases in modulating glycan function makes them prime targets for small-molecule modulators. Here, we report a system for conditional activation of Golgi-resident sulfotransferases using a chemical inducer of dimerization. Our approach capitalizes on two features shared by these enzymes: their requirement of Golgi localization for activity on cellular substrates and the modularity of their catalytic and localization domains. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Both the activity and specificity of the inducible enzymes were indistinguishable from their WT counterparts. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. The approach provides a means for studying sulfate-dependent processes in cellular systems and, potentially, in vivo.

    View details for DOI 10.1073/pnas.0403681101

    View details for Web of Science ID 000225508400004

    View details for PubMedID 15548609

    View details for PubMedCentralID PMC534710

  • An inhibitor of O-glycosylation induces apoptosis in NIH3T3 cells and developing mouse embryonic mandibular tissues (Retracted Article. See vol 283, pg 4460, 2008) JOURNAL OF BIOLOGICAL CHEMISTRY Tian, E., Ten Hagen, K. G., SHUM, L., Hang, H. C., Imbert, Y., Young, W. W., Bertozzi, C. R., Tabak, L. A. 2004; 279 (48): 50382-50390
  • A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of blomolecules in living systems JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Agard, N. J., Prescher, J. A., Bertozzi, C. R. 2004; 126 (46): 15046-15047

    Abstract

    Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. Two notable examples are the Staudinger ligation of azides and phosphines and the Cu(I)-catalyzed [3 + 2] cycloaddition of azides and alkynes ("click chemistry"). The Staudinger ligation has sufficient biocompatibility for performance in living animals but suffers from phosphine oxidation and synthetic challenges. Click chemistry obviates the requirement of phosphines, but the Cu(I) catalyst is toxic to cells, thereby precluding in vivo applications. Here we present a strain-promoted [3 + 2] cycloaddition between cyclooctynes and azides that proceeds under physiological conditions without the need for a catalyst. The utility of the reaction was demonstrated by selective modification of biomolecules in vitro and on living cells, with no apparent toxicity.

    View details for DOI 10.1021/ja0449981

    View details for Web of Science ID 000225233600024

    View details for PubMedID 15547999

  • Global gene expression of cells attached to a tissue engineering scaffold BIOMATERIALS Klapperich, C. M., Bertozzi, C. R. 2004; 25 (25): 5631-5641

    Abstract

    A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Little is known about how cells respond on a molecular level to tissue engineering scaffold materials. In this work we used oligonucleotide microarrays to interrogate gene expression profiles associated with cell-biomaterial interactions. We seeded collagen-glycosaminoglycan meshes, a widely used tissue engineering scaffold material, with human IMR-90 fibroblasts and compared transcript levels with control cells grown on tissue culture polystyrene. Genes involved in cell signaling, extracellular matrix remodeling, inflammation, angiogenesis and hypoxia were all activated in cells on the collagen-GAG mesh. Understanding the impact of a scaffold on attached cells will facilitate the design of improved tissue engineering materials.

    View details for DOI 10.1016/j.biomaterials.2004.01.025

    View details for Web of Science ID 000222040300008

    View details for PubMedID 15159079

  • Directing flux in glycan biosynthetic pathways with a small molecule switch CHEMBIOCHEM Kohler, J. J., Czlapinski, J. L., Laughlin, S. T., Schelle, M. W., de Graffenried, C. L., Bertozzi, C. R. 2004; 5 (10): 1455-1458

    View details for DOI 10.1002/cbic.200400156

    View details for Web of Science ID 000224453500014

    View details for PubMedID 15457531

  • Metabolic functionalization of recombinant glycoproteins BIOCHEMISTRY Luchansky, S. J., Argade, S., Hayes, B. K., Bertozzi, C. R. 2004; 43 (38): 12358-12366

    Abstract

    Glycoproteins are essential for cellular communication and are the most rapidly growing class of therapeutic agents. Chemical modification of glycoproteins has been employed to improve their in vivo efficacy or to label them for detection. Methods for the controlled derivatization of glycoproteins are presently limited by the repertoire of natural amino acid side chain and carbohydrate functionalities. Here, we use metabolic oligosaccharide engineering to introduce a bioorthogonal functional group, the azide, into cellular and recombinant glycoproteins for subsequent chemical elaboration via Staudinger ligation. As most therapeutic glycoproteins are sialylated and require this saccharide for optimal pharmacokinetics, we targeted sialic acid as a host for azides using N-azidoacetylmannosamine (ManNAz) as a biosynthetic precursor. Metabolic conversion of ManNAz to N-azidoacetylsialic acid (SiaNAz) within membrane-bound and secreted glycoproteins was quantified in a variety of cell types. SiaNAz was found to comprise between 4% and 41% of total sialosides, depending on the system. Metabolic labeling of recombinant interferon-beta and GlyCAM-Ig was achieved, demonstrating the utility of the method for functionalizing N-linked and O-linked glycoproteins of therapeutic interest. More generally, the generation of recombinant glycoproteins containing chemical handles within their glycans provides a means for studying their behavior and for improving their in vivo efficacy.

    View details for DOI 10.1021/bi049274f

    View details for Web of Science ID 000224032900044

    View details for PubMedID 15379575

  • The stem region of the sulfotransferase GlcNAc6ST-1 is a determinant of substrate specificity JOURNAL OF BIOLOGICAL CHEMISTRY de Graffenried, C. L., Bertozzi, C. R. 2004; 279 (38): 40035-40043

    Abstract

    The GlcNAc-6-sulfotransferases are a family of Golgi-resident enzymes that modulate glycan function. Two members of this family, GlcNAc6ST-1 and -2, collaborate in the biosynthesis of ligands for the leukocyte adhesion molecule L-selectin. Although their biochemical properties are similar in vitro, the enzymes have distinct glycoprotein substrate preferences in vivo. The sulfotransferases share similar overall architecture with the exception of an extended stem region in GlcNAc6ST-1 that is absent in GlcNAc6ST-2. In this study we probed the importance of the stem region with respect to substrate preference, localization, and oligomerization. Analysis of truncation mutants demonstrated that perturbation of the stem region of GlcNAc6ST-1 affects the cellular substrate preference of the enzyme without altering its retention within the Golgi. A chimeric enzyme comprising the stem region of GlcNAc6ST-1 inserted between the catalytic and transmembrane domains of GlcNAc6ST-2 had the same substrate preference as native GlcNAc6ST-1. In cells, GlcNAc6ST-1 exists as a dimer; two cysteine residues within the stem and transmembrane domain were found to be critical for dimerization. However, disruption of the dimer by mutagenesis did not affect either localization or substrate preference. Collectively, these results indicate that the stem region of GlcNAc6ST-1 influences substrate specificity, independent of its role in dimerization or Golgi retention.

    View details for DOI 10.1074/jbc.M405709200

    View details for Web of Science ID 000223791500101

    View details for PubMedID 15220337

  • Chemical tools for the study of polysialic acid 4th International Conference on Sialobiology Samuel, J., Bertozzi, C. R. GAKUSHIN PUBL CO. 2004: 305?18
  • Chemical remodelling of cell surfaces in living animals NATURE Prescher, J. A., Dube, D. H., Bertozzi, C. R. 2004; 430 (7002): 873-877

    Abstract

    Cell surfaces are endowed with biological functionality designed to mediate extracellular communication. The cell-surface repertoire can be expanded to include abiotic functionality through the biosynthetic introduction of unnatural sugars into cellular glycans, a process termed metabolic oligosaccharide engineering. This technique has been exploited in fundamental studies of glycan-dependent cell-cell and virus-cell interactions and also provides an avenue for the chemical remodelling of living cells. Unique chemical functional groups can be delivered to cell-surface glycans by metabolism of the corresponding unnatural precursor sugars. These functional groups can then undergo covalent reaction with exogenous agents bearing complementary functionality. The exquisite chemical selectivity required of this process is supplied by the Staudinger ligation of azides and phosphines, a reaction that has been performed on cultured cells without detriment to their physiology. Here we demonstrate that the Staudinger ligation can be executed in living animals, enabling the chemical modification of cells within their native environment. The ability to tag cell-surface glycans in vivo may enable therapeutic targeting and non-invasive imaging of changes in glycosylation during disease progression.

    View details for DOI 10.1038/nature02791

    View details for Web of Science ID 000223369800037

    View details for PubMedID 15318217

  • Identification, function and structure of the mycobacterial sulfotransferase that initiates sulfolipid-1 biosynthesis NATURE STRUCTURAL & MOLECULAR BIOLOGY Mougous, J. D., Petzold, C. J., Senaratne, R. H., Lee, D. H., Akey, D. L., Lin, F. L., Munchel, S. E., Pratt, M. R., Riley, L. W., Leary, J. A., Berger, J. M., Bertozzi, C. R. 2004; 11 (8): 721-729

    Abstract

    Sulfolipid-1 (SL-1) is an abundant sulfated glycolipid and potential virulence factor found in Mycobacterium tuberculosis. SL-1 consists of a trehalose-2-sulfate (T2S) disaccharide elaborated with four lipids. We identified and characterized a conserved mycobacterial sulfotransferase, Stf0, which generates the T2S moiety of SL-1. Biochemical studies demonstrated that the enzyme requires unmodified trehalose as substrate and is sensitive to small structural perturbations of the disaccharide. Disruption of stf0 in Mycobacterium smegmatis and M. tuberculosis resulted in the loss of T2S and SL-1 formation, respectively. The structure of Stf0 at a resolution of 2.6 A reveals the molecular basis of trehalose recognition and a unique dimer configuration that encloses the substrate into a bipartite active site. These data provide strong evidence that Stf0 carries out the first committed step in the biosynthesis of SL-1 and establish a system for probing the role of SL-1 in M. tuberculosis infection.

    View details for DOI 10.1038/nsmb802

    View details for Web of Science ID 000222930600016

    View details for PubMedID 15258569

  • The roles of enzyme localisation and complex formation in glycan assembly within the Golgi apparatus CURRENT OPINION IN CELL BIOLOGY de Graffenried, C. L., Bertozzi, C. R. 2004; 16 (4): 356-363

    Abstract

    Cell surface glycans govern numerous cell-cell interactions are therefore key determinants of multicellular biology. They originate from biosynthetic pathways comprising an assembly line of glycosyltransferases within the Golgi compartment. Although the mechanisms of Golgi enzyme localisation are still under debate, the distribution of these enzymes among the Golgi cisternae can dictate the overall structures produced by the cell. Fine-tuning of glycan biosynthetic pathways is further accomplished by specific associations among glycosyltransferases. Together, localisation and association govern the assembly of complex glycans and thereby regulate interactions at the cell surface.

    View details for DOI 10.1016/j.ceb.2004.06.007

    View details for Web of Science ID 000223130800003

    View details for PubMedID 15261667

  • Functional self-assembling bolaamphiphilic polydiacetylenes as colorimetric sensor scaffolds JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Song, J., Cisar, J. S., Bertozzi, C. R. 2004; 126 (27): 8459-8465

    Abstract

    Conjugated polymers capable of responding to external stimuli by changes in optical, electrical, or electrochemical properties can be used for the construction of direct sensing devices. Polydiacetylene-based systems are attractive for sensing applications due to their colorimetric response to changes in the local environment. Here we present the design, preparation, and characterization of self-assembling functional bolaamphiphilic polydiacetylenes (BPDAs) inspired by nature's strategy for membrane stabilization. We show that by placing polar headgroups on both ends of the diacetylene lipids in a transmembranic fashion and by altering the chemical nature of the polar surface residues, the conjugated polymers can be engineered to display a range of radiation-, thermal-, and pH-induced colorimetric responses. We observed dramatic nanoscopic morphological transformations accompanying charge-induced chromatic transitions, suggesting that both side-chain disordering and main-chain rearrangement play important roles in altering the effective conjugation lengths of the poly(ene-yne). These results establish the foundation for further development of BPDA-based colorimetric sensors.

    View details for DOI 10.1021/ja039825+

    View details for Web of Science ID 000222612600032

    View details for PubMedID 15238003

  • Trehalose is required for growth of Mycobacterium smegmatis JOURNAL OF BIOLOGICAL CHEMISTRY Woodruff, P. J., Carlson, B. L., Siridechadilok, B., Pratt, M. R., Senaratne, R. H., Mougous, J. D., Riley, L. W., Williams, S. J., Bertozzi, C. R. 2004; 279 (28): 28835-28843

    Abstract

    Mycobacteria contain high levels of the disaccharide trehalose in free form as well as within various immunologically relevant glycolipids such as cord factor and sulfolipid-1. By contrast, most bacteria use trehalose solely as a general osmoprotectant or thermoprotectant. Mycobacterium tuberculosis and Mycobacterium smegmatis possess three pathways for the synthesis of trehalose. Most bacteria possess only one trehalose biosynthesis pathway and do not elaborate the disaccharide into more complex metabolites, suggesting a distinct role for trehalose in mycobacteria. We disabled key enzymes required for each of the three pathways in M. smegmatis by allelic replacement. The resulting trehalose biosynthesis mutant was unable to proliferate and enter stationary phase unless supplemented with trehalose. At elevated temperatures, however, the mutant was unable to proliferate even in the presence of trehalose. Genetic complementation experiments showed that each of the three pathways was able to recover the mutant in the absence of trehalose, even at elevated temperatures. From a panel of trehalose analogs, only those with the native alpha,alpha-(1,1) anomeric stereochemistry rescued the mutant, whereas alternate stereoisomers and general osmo- and thermoprotectants were inactive. These findings suggest a dual role for trehalose as both a thermoprotectant and a precursor of critical cell wall metabolites.

    View details for DOI 10.1074/jbc.M313103200

    View details for Web of Science ID 000222445300003

    View details for PubMedID 15102847

  • Syntheses of 6-sulfo sialyl Lewis X glycans corresponding to the L-selectin ligand "Sulfoadhesin" ORGANIC LETTERS Pratt, M. R., Bertozzi, C. R. 2004; 6 (14): 2345-2348

    Abstract

    [structure: see text] Divergent syntheses of sulfated sialyl Lewis X oligosaccharides corresponding to the core 1 and core 6 branches of the L-selectin ligand are reported. These synthetic targets incorporate a selectively protected serine residue at the reducing terminus, providing a functional handle for further conjugation.

    View details for DOI 10.1021/ol0493195

    View details for Web of Science ID 000222420300014

    View details for PubMedID 15228275

  • Deconvoluting the functions of polypeptide N-alpha-acetylgalactosaminyltransferase family members by glycopeptide substrate profiling CHEMISTRY & BIOLOGY Pratt, M. R., Hang, H. C., Ten Hagen, K. G., Rarick, J., Gerken, T. A., Tabak, L. A., Bertozzi, C. R. 2004; 11 (7): 1009-1016

    Abstract

    The polypeptide N-alpha-acetylgalactosaminyltransferases (ppGalNAcTs) play a key role in mucin-type O-linked glycan biosynthesis by installing the initial GalNAc residue on the protein scaffold. The preferred substrates and functions of the >20 isoforms in mammals are not well understood. However, current data suggest that glycosylated mucin domains are created by the successive, often hierarchical, action of several specific ppGalNAcTs. Herein we analyzed the glycopeptide substrate preferences of several ppGalNAcT family members using a library screening approach. A 56-member glycopeptide library designed to reflect a diversity of glycan clustering was assayed for substrate activity with ppGalNAcT isoforms using an azido-ELISA. The data suggest that the ppGalNAcTs can be classified into at least four types, which working together, are able to produce densely glycosylated mucin glycoproteins.

    View details for DOI 10.1016/j.chembiol.2004.05.009

    View details for Web of Science ID 000222987300018

    View details for PubMedID 15271359

  • Expanding the diversity of unnatural cell-surface sialic acids CHEMBIOCHEM Luchansky, S. J., Goon, S., Bertozzi, C. R. 2004; 5 (3): 371-374

    View details for DOI 10.1002/cbic.200300789

    View details for Web of Science ID 000220196800018

    View details for PubMedID 14997530

  • Small molecule inhibitors of mucin-type O-linked glycosylation from a uridine-based library CHEMISTRY & BIOLOGY Hang, H. C., Yu, C., Ten Hagen, K. G., Tian, E., Winans, K. A., Tabak, L. A., Bertozzi, C. R. 2004; 11 (3): 337-345

    Abstract

    The polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs, also abbreviated ppGaNTases) initiate mucin-type O-linked glycosylation and therefore play pivotal roles in cell-cell communication and protection of tissues. In order to develop new tools for studying mucin-type O-linked glycosylation, we screened a 1338 member uridine-based library to identify small molecule inhibitors of ppGalNAcTs. Using a high-throughput enzyme-linked lectin assay (ELLA), two inhibitors of murine ppGalNAcT-1 (K(I) approximately 8 microM) were identified that also inhibit several other members of the family. The compounds did not inhibit other mammalian glycosyltransferases or nucleotide sugar utilizing enzymes, suggesting selectivity for the ppGalNAcTs. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. These uridine analogs represent the first generation of chemical tools to study the functions of mucin-type O-linked glycosylation.

    View details for DOI 10.1016/j.chembiol.2004.02.023

    View details for Web of Science ID 000220502900010

    View details for PubMedID 15123263

  • Probing glycosyltransferase activities with the Staudinger ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hang, H. C., Yu, C., Pratt, M. R., Bertozzi, C. R. 2004; 126 (1): 6-7

    Abstract

    The development of rapid screening methods for probing glycosyltransferase activities is essential for advancing the field of glycobiology. While assays for specific glycosyltransferases exist, there is no generalizable method that can be applied across the enzyme superfamily. Herein we describe a novel glycosyltransferase assay that exploits their unnatural substrate tolerance and the unique chemical reactivity of the azide. We applied this "azido-ELISA" to the family of polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transfer N-azidoacetylgalactosamine (GalNAz) from the unnatural nucleotide sugar donor UDP-GalNAz. The azide was detected and quantified by Staudinger ligation with a phosphine probe in a microtiter plate format. This approach should be applicable to any glycosyltransferase or group-transfer enzyme that tolerates unnatural azido substrates.

    View details for DOI 10.1021/ja037692m

    View details for Web of Science ID 000187945400003

    View details for PubMedID 14709032

  • Detection of bacteria in suspension by using a superconducting quantum interference device PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Grossman, H. L., Myers, W. R., Vreeland, V. J., Bruehl, R., Alper, M. D., Bertozzi, C. R., Clarke, J. 2004; 101 (1): 129-134

    Abstract

    We demonstrate a technique for detecting magnetically labeled Listeria monocytogenes and for measuring the binding rate between antibody-linked magnetic particles and bacteria. This sensitive assay quantifies specific bacteria in a sample without the need to immobilize them or wash away unbound magnetic particles. In the measurement, we add 50-nm-diameter superparamagnetic magnetite particles, coated with antibodies, to an aqueous sample containing L. monocytogenes. We apply a pulsed magnetic field to align the magnetic dipole moments and use a high-transition temperature superconducting quantum interference device, an extremely sensitive detector of magnetic flux, to measure the magnetic relaxation signal when the field is turned off. Unbound particles randomize direction by Brownian rotation too quickly to be detected. In contrast, particles bound to L. monocytogenes are effectively immobilized and relax in about 1 s by rotation of the internal dipole moment. This Néel relaxation process is detected by the superconducting quantum interference device. The measurements indicate a detection limit of (5.6 +/- 1.1) x 10(6) L. monocytogenes in our sample volume of 20 microl. If the sample volume were reduced to 1 nl, we estimate that the detection limit could be improved to 230 +/- 40 L. monocytogenes cells. Time-resolved measurements yield the binding rate between the particles and bacteria.

    View details for DOI 10.1073/pnas.0307128101

    View details for Web of Science ID 000187937200026

    View details for PubMedID 14688406

    View details for PubMedCentralID PMC314150

  • Functional glass slides for in vitro evaluation of interactions between osteosarcoma TE85 cells and mineral-binding ligands JOURNAL OF MATERIALS CHEMISTRY Song, J., Chen, J., Klapperich, C. M., Eng, V., Bertozzi, C. R. 2004; 14 (17): 2643-2648

    View details for DOI 10.1039/b408597n

    View details for Web of Science ID 000223490500010

  • Modular assembly of glycoproteins: Towards the synthesis of GlyCAM-1 by using expressed protein ligation ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Macmillan, D., Bertozzi, C. R. 2004; 43 (11): 1355-1359

    View details for DOI 10.1002/anie.200352673

    View details for Web of Science ID 000220266000010

    View details for PubMedID 15368405

  • A strategy for functional proteomic analysis of glycosidase activity from cell lysates ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Vocadlo, D. J., Bertozzi, C. R. 2004; 43 (40): 5338-5342

    View details for DOI 10.1002/anie.200454235

    View details for Web of Science ID 000224592400009

    View details for PubMedID 15468183

  • Biomimetic engineering of carbon nanotubes by using cell surface mucin mimics ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Chen, X., Lee, G. S., Zettl, A., Bertozzi, C. R. 2004; 43 (45): 6111-6116

    View details for DOI 10.1002/anie.200460620

    View details for Web of Science ID 000225445200005

    View details for PubMedID 15549753

  • A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Hang, H. C., Yu, C., Kato, D. L., Bertozzi, C. R. 2003; 100 (25): 14846-14851

    Abstract

    Mucin-type O-linked glycoproteins are involved in a variety of biological interactions in higher eukaryotes. The biosynthesis of these glycoproteins is initiated by a family of polypeptide N-acetyl-alpha-galactosaminyltransferases (ppGalNAcTs) that modify proteins in the secretory pathway. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. Herein we present a method for labeling mucin-type O-linked glycoproteins with a unique chemical tag, the azide, which permits their selective covalent modification from complex cell lysates. From a panel of synthetic derivatives, we identified an azido GalNAc analog (N-azidoacetylgalactosamine, GalNAz) that is metabolized by numerous cell types and installed on mucin-type O-linked glycoproteins by the ppGalNAcTs. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. The approach was validated by labeling a recombinant glycoprotein that is known to possess O-linked glycans with GalNAz. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. The ability to label mucin-type O-linked glycoproteins with chemical tags should facilitate their identification by proteomic strategies.

    View details for DOI 10.1073/pnas.2335201100

    View details for Web of Science ID 000187227200045

    View details for PubMedID 14657396

    View details for PubMedCentralID PMC299823

  • Regulating cell surface glycosylation by small molecule control of enzyme localization CHEMISTRY & BIOLOGY Kohler, J. J., Bertozzi, C. R. 2003; 10 (12): 1303-1311

    Abstract

    Cell surface carbohydrates mediate interactions between the cell and its environment. Glycosyltransferases responsible for synthesis of cell surface oligosaccharides are therefore essential administrators of cellular communication. These enzymes often comprise large families. Redundancy of related family members and embryonic lethality both complicate genetic methods for deconvoluting functions of glycosyltransferases. We report a chemical method in which the activity of an individual glycosyltransferase is controlled by a small molecule. The approach exploits the requirement of Golgi localization, a common feature of glycosyltransferase superfamily members. In our approach, the glycosyltransferase is separated into two domains, one that determines localization and one responsible for catalysis. Control of enzyme activity is achieved using a small molecule to regulate association of the two domains. We used this method to regulate production of sialyl Lewis x by alpha1,3-fucosyltransferase VII in living cells.

    View details for DOI 10.1016/j.chembiol.2003.11.018

    View details for Web of Science ID 000187633800020

    View details for PubMedID 14700637

  • Golgi localization of carbohydrate sulfotransferases is a determinant of L-selectin ligand biosynthesis JOURNAL OF BIOLOGICAL CHEMISTRY de Graffenried, C. L., Bertozzi, C. R. 2003; 278 (41): 40282-40295

    Abstract

    Sulfation of endothelial glycoproteins by the sulfotransferase GlcNAc6ST-2 is a regulatory modification that promotes binding of the leukocyte adhesion molecule L-selectin. GlcNAc6ST-2 is a member of a family of related enzymes that act on similar carbohydrate substrates in vitro but discrete glycoproteins in vivo. We demonstrate that GlcNAc6ST-1, -2, and -3 have distinct Golgi distributions, with GlcNAc6ST-1 confined to the trans-Golgi network, GlcNAc6ST-3 confined to the early secretory pathway, and GlcNAc6ST-2 distributed throughout the Golgi. Their localization was correlated with preferred activity on either N-linked or O-linked glycoproteins. A chimera comprising the localization domain of GlcNAc6ST-1 fused to the catalytic domain of GlcNAc6ST-2 was confined to the trans-Golgi network and adopted the substrate preference of GlcNAc6ST-1. We propose a model in which Golgi enzyme localization and competition orchestrate the biosynthesis of L-selectin ligands.

    View details for DOI 10.1074/jbc.M304928200

    View details for Web of Science ID 000185713800121

    View details for PubMedID 12855678

  • cDNA cloning and expression of UDP-N-acetyl-D-galactosamine : polypeptide N-acetylgalactosaminyltransferase T1 from Toxoplasma gondii MOLECULAR AND BIOCHEMICAL PARASITOLOGY Wojczyk, B. S., Stwora-Wojczyk, M. M., Hagen, F. K., Striepen, B., Hang, H. C., Bertozzi, C. R., Roos, D. S., Spitalnik, S. L. 2003; 131 (2): 93-107

    Abstract

    We report the cloning, expression, and characterization of the first UDP-GalNAc:polypetide N-acetylgalactosaminyltransferase (ppGalNAc-T) from the human disease-causing parasite, Toxoplasma gondii. This enzyme is also the first characterized ppGalNAc-T of protozoan origin. This type of enzyme catalyzes the initial step of mucin-type O-glycosylation, that is, the transfer of GalNAc in O-glycosidic linkage to serine and threonine residues in polypeptides. We used polymerase chain reaction amplification with degenerate primers and hybridization screening of a T. gondii cDNA library to identify this enzyme. The resulting 84-kDa type II membrane protein contains a 49-amino acid N-terminal cytoplasmic domain, a 22-amino acid hydrophobic transmembrane domain, and a 680-amino acid C-terminal lumenal domain. Conceptual translation of the cDNA sequence reveals a relatively long (i.e. 135 amino acids) stem region and the presence of several important sequence motifs. The latter include a glycosyltransferase 1 (GT1) motif containing a DXH sequence, a Gal/GalNAc-T motif, and a region homologous to ricin lectin. Northern blot analysis identified a single 5.5-kb ppGalNAc-T transcript. Comparison of the cDNA and genomic DNA sequences reveals that this transferase is encoded by 10 exons in a 10 kb region. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited transferase activity in vitro. The identification of this enzyme in T. gondii demonstrates that this human parasite has its own enzymatic machinery for the O-glycosylation of toxoplasmal proteins.

    View details for DOI 10.1016/S0166-6851(03)00196-8

    View details for Web of Science ID 000185793400002

    View details for PubMedID 14511808

  • Metabolic oligosaccharide engineering as a tool for glycobiology CURRENT OPINION IN CHEMICAL BIOLOGY Dube, D. H., Bertozzi, C. R. 2003; 7 (5): 616-625

    Abstract

    Oligosaccharides transact information exchange at the cell surface and modulate the activities and distribution of proteins within cells. Recently, the ability to modify monosaccharide structures within cellular glycans through metabolic processes has offered a new avenue for biological studies. The technique of metabolic oligosaccharide engineering has been used to disrupt glycan biosynthesis, chemically modify cell surfaces, probe metabolic flux inside cells, and to identify specific glycoprotein subtypes from the proteome.

    View details for DOI 10.1016/j.cbpa.2003.08.006

    View details for Web of Science ID 000186448100015

    View details for PubMedID 14580567

  • Formation of 1,1-alpha,alpha-glycosidic bonds by intramolecular aglycone delivery. A convergent synthesis of trehalose ORGANIC LETTERS Pratt, M. R., Leigh, C. D., Bertozzi, C. R. 2003; 5 (18): 3185-3188

    Abstract

    [reaction: see text] We report a new synthesis of trehalose analogs that involves the use of intramolecular aglycone delivery for stereoselective formation of the 1,1-alpha,alpha-glycosidic bond. The glycosylation reaction afforded the desired isomer exclusively and in good yield.

    View details for DOI 10.1021/ol034836t

    View details for Web of Science ID 000185051300005

    View details for PubMedID 12943383

  • A chemical approach for identifying O-GlcNAc-modified proteins in cells PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Vocadlo, D. J., Hang, H. C., Kim, E. J., Hanover, J. A., Bertozzi, C. R. 2003; 100 (16): 9116-9121

    Abstract

    The glycosylation of serine and threonine residues with a single GlcNAc moiety is a dynamic posttranslational modification of many nuclear and cytoplasmic proteins. We describe a chemical strategy directed toward identifying O-GlcNAc-modified proteins from living cells or proteins modified in vitro. We demonstrate, in vitro, that each enzyme in the hexosamine salvage pathway, and the enzymes that affect this dynamic modification (UDP-GlcNAc:polypeptidtyltransferase and O-GlcNAcase), tolerate analogues of their natural substrates in which the N-acyl side chain has been modified to bear a bio-orthogonal azide moiety. Accordingly, treatment of cells with N-azidoacetylglucosamine results in the metabolic incorporation of the azido sugar into nuclear and cytoplasmic proteins. These O-azidoacetylglucosamine-modified proteins can be covalently derivatized with various biochemical probes at the site of protein glycosylation by using the Staudinger ligation. The approach was validated by metabolic labeling of nuclear pore protein p62, which is known to be posttranslationally modified with O-GlcNAc. This strategy will prove useful for both the identification of O-GlcNAc-modified proteins and the elucidation of the specific residues that bear this saccharide.

    View details for DOI 10.1073/pnas.1632821100

    View details for Web of Science ID 000184620000006

    View details for PubMedID 12874386

    View details for PubMedCentralID PMC171382

  • Chemoselective ligation applied to the synthesis of a biantennary N-linked glycoform of CD52 JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Pratt, M. R., Bertozzi, C. R. 2003; 125 (20): 6149-6159

    Abstract

    We report here a strategy for the synthesis of N-linked glycopeptide analogues that replace the glycosidic linkages extending from the core pentasaccharide with thioethers amenable to construction by chemoselective ligation. The key building block, a pentasaccharide-Asn analogue containing two thiol residues, was incorporated into CD52 by 9-fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. An undecasaccharide mimetic was then readily generated by alkylation of this glycopeptide with an N-bromoacetamido trisaccharide. The rapid assembly of a complex type N-linked glycopeptide mimetic was accomplished using this technique.

    View details for DOI 10.1021/ja029346v

    View details for Web of Science ID 000182959600044

    View details for PubMedID 12785846

  • MmpL8 is required for sulfolipid-1 biosynthesis and Mycobacterium tuberculosis virulence PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Converse, S. E., Mougous, J. D., Leavell, M. D., Leary, J. A., Bertozzi, C. R., Cox, J. S. 2003; 100 (10): 6121-6126

    Abstract

    Mycobacterium tuberculosis, the causative agent of human tuberculosis, is unique among bacterial pathogens in that it displays a wide array of complex lipids and lipoglycans on its cell surface. One of the more remarkable lipids is a sulfated glycolipid, termed sulfolipid-1 (SL-1), which is thought to mediate specific host-pathogen interactions during infection. However, a direct role for SL-1 in M. tuberculosis virulence has not been established. Here we show that MmpL8, a member of a large family of predicted lipid transporters in M. tuberculosis, is required for SL-1 production. The accumulation of an SL-1 precursor, termed SL(1278), in mmpL8 mutant cells indicates that MmpL8 is necessary for an intermediate step in the SL-1 biosynthesis pathway. We use a novel fractionation procedure to demonstrate that SL-1 is present on the cell surface, whereas SL(1278) is found exclusively in more internal layers. Importantly, we show that mmpL8 mutants are attenuated for growth in a mouse model of tuberculosis. However, SL-1 per se is not required for establishing infection as pks2 mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Thus, we hypothesize that either MmpL8 transports molecules in addition to SL-1 that mediate host-pathogen interactions or the accumulation of SL(1278) in mmpL8 mutant cells interferes with other pathways required for growth during the early stages of infection.

    View details for DOI 10.1073/pnas.1030024100

    View details for Web of Science ID 000182939400099

    View details for PubMedID 12724526

    View details for PubMedCentralID PMC156336

  • A fluorogenic dye activated by the Staudinger ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lemieux, G. A., de Graffenried, C. L., Bertozzi, C. R. 2003; 125 (16): 4708-4709

    Abstract

    Specific labeling of biomolecules with biochemical and biophysical probes is a central element of proteomics research. Here we describe a coumarin-phosphine dye that undergoes activation of coumarin fluorescence upon Staudinger ligation with azides. Since azides can be metabolically incorporated into cellular proteins and oligosaccharides, this dye may be a useful tool for profiling proteins and their posttranslational modifications.

    View details for DOI 10.1021/ja029013y

    View details for Web of Science ID 000182331800019

    View details for PubMedID 12696879

  • Metabolic incorporation of unnatural sialic acids into Haemophilus ducreyi lipooligosaccharides PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Goon, S., Schilling, B., Tullius, M. V., Gibson, B. W., Bertozzi, C. R. 2003; 100 (6): 3089-3094

    Abstract

    The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. Here we report that H. ducreyi can use unnatural sialic acids bearing elongated N-acyl groups from three to seven carbon atoms in length, resulting in outer membrane presentation of unnatural sialyl-LOS. The unnatural variant comprises approximately 90% of cell surface sialosides when exogenous substrates are added to the media at micromolar concentrations, despite the availability of natural sialic acid in the growth media. Although they represent the majority of cell surface sialosides, analogs with longer N-acyl groups diminish the overall level of LOS sialylation, culminating in complete inhibition of LOS sialylation by N-octanoyl sialic acid. Thus, sialylation of H. ducreyi LOS can be modulated with respect to the structure of the terminal sialic acid residue and the extent to which the LOS acceptor is modified by supplying the bacteria with various sialic acid analogs.

    View details for DOI 10.1073/pnas.0437851100

    View details for Web of Science ID 000181675200023

    View details for PubMedID 12615992

    View details for PubMedCentralID PMC152251

  • GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism JOURNAL OF BIOLOGICAL CHEMISTRY Luchansky, S. J., Yarema, K. J., Takahashi, S., Bertozzi, C. R. 2003; 278 (10): 8035-8042

    Abstract

    Sialic acid is a major determinant of carbohydrate-receptor interactions in many systems pertinent to human health and disease. N-Acetylmannosamine (ManNAc) is the first committed intermediate in the sialic acid biosynthetic pathway; thus, the mechanisms that control intracellular ManNAc levels are important regulators of sialic acid production. UDP-GlcNAc 2-epimerase and GlcNAc 2-epimerase are two enzymes capable of generating ManNAc from UDP-GlcNAc and GlcNAc, respectively. Whereas the former enzyme has been shown to direct metabolic flux toward sialic acid in vivo, the function of the latter enzyme is unclear. Here we study the effects of GlcNAc 2-epimerase expression on sialic acid production in cells. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway.

    View details for DOI 10.1074/jbc.M212127200

    View details for Web of Science ID 000181466800038

    View details for PubMedID 12499362

  • Synthesis of thioether-linked analogues of the 2,3-sialyl-TF and MECA-79 antigens: Mucin-type glycopeptides associated with cancer and inflammation CHEMBIOCHEM Marcaurelle, L. A., Pratt, M. R., Bertozzi, C. R. 2003; 4 (2-3): 224-228

    View details for Web of Science ID 000181517000014

    View details for PubMedID 12616638

  • A new approach to mineralization of biocompatible hydrogel scaffolds: An efficient process toward 3-dimensional bonelike composites JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Song, J., Saiz, E., Bertozzi, C. R. 2003; 125 (5): 1236-1243

    Abstract

    As a first step toward the design and fabrication of biomimetic bonelike composite materials, we have developed a template-driven nucleation and mineral growth process for the high-affinity integration of hydroxyapatite with a poly(2-hydroxyethyl methacrylate) (pHEMA) hydrogel scaffold. A mineralization technique was developed that exposes carboxylate groups on the surface of cross-linked pHEMA, promoting high-affinity nucleation and growth of calcium phosphate on the surface, along with extensive calcification of the hydrogel interior. Robust surface mineral layers a few microns thick were obtained. The same mineralization technique, when applied to a hydrogel that is less prone to surface hydrolysis, led to distinctly different mineralization patterns, in terms of both the extent of mineralization and the crystallinity of the apatite grown on the hydrogel surface. This template-driven mineralization technique provides an efficient approach toward bonelike composites with high mineral-hydrogel interfacial adhesion strength.

    View details for DOI 10.1021/ja028559h

    View details for Web of Science ID 000180713000046

    View details for PubMedID 12553825

  • Synthesis of a bisubstrate analogue targeting estrogen sulfotransferase JOURNAL OF ORGANIC CHEMISTRY Armstrong, J. I., Verdugo, D. E., Bertozzi, C. R. 2003; 68 (1): 170-173

    Abstract

    Sulfotransferases catalyze the transfer of a sulfuryl group from the eukaryotic sulfate donor 3'-phosphoadenosine 5'-phosphosulfate to an acceptor biomolecule. Sulfotransferases have been linked with several disease states, prompting our investigation of specific sulfotransferase inhibitors. Presented herein is the synthesis and evaluation of a bisubstrate analogue designed to inhibit estrogen sulfotransferase. The synthesis utilizes a novel, orthogonally protected 3'-phosphoadenosine 5'-phosphate (PAP) derivative allowing the selective functionalization of the 5'-phosphate with a sulfate acceptor mimic. Kinetic studies revealed significant inhibitory activity and provide guidance for improved inhibitor design.

    View details for DOI 10.1021/jo0260443

    View details for Web of Science ID 000180258800025

    View details for PubMedID 12515476

  • Drug targeting Mycobacterium tuberculosis cell wall synthesis: Development of a microtiter plate-based screen for UDP-galactopyranose mutase and identification of an inhibitor from a uridine-based library ANTIMICROBIAL AGENTS AND CHEMOTHERAPY Scherman, M. S., Winans, K. A., Stern, R. J., Jones, V., Bertozzi, C. R., McNeil, M. R. 2003; 47 (1): 378-382

    Abstract

    A microtiter plate assay for UDP-galactopyranose mutase, an essential cell wall biosynthetic enzyme of Mycobacterium tuberculosis, was developed. The assay is based on the release of tritiated formaldehyde from UDP-galactofuranose but not UDP-galactopyranose by periodate and was used to identify a uridine-based enzyme inhibitor from a chemical library.

    View details for DOI 10.1128/AAC.47.1.378-382.2003

    View details for Web of Science ID 000180149600058

    View details for PubMedID 12499218

    View details for PubMedCentralID PMC148999

  • Preparation of pHEMA-CP composites with high interfacial adhesion via template-driven mineralization International Workshop on Interfaces: Ceramic and Metal Interfaces: Control at the Atomic Level Song, J., Saiz, E., Bertozzi, C. R. ELSEVIER SCI LTD. 2003: 2905?19
  • Hydrogel polymers from alkylthio acrylates for biomedical applications Symposium on Polymer Gels Mukkamala, R., Kushner, A. M., Bertozzi, C. R. AMER CHEMICAL SOC. 2003: 163?174
  • Constructing azide-labeled cell surfaces using polysaccharide biosynthetic pathways RECOGNITION OF CARBOHYDRATES IN BIOLOGICAL SYSTEMS PT A: GENERAL PROCEDURES Luchansky, S. J., Hang, H. C., Saxon, E., Grunwell, J. R., Danielle, C. Y., Dube, D. H., Bertozzi, C. R. 2003; 362: 249-272

    View details for Web of Science ID 000185102400018

    View details for PubMedID 12968369

  • Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases BIOCHEMISTRY Grunwell, J. R., Rath, V. L., Rasmussen, J., Cabrilo, Z., Bertozzi, C. R. 2002; 41 (52): 15590-15600

    Abstract

    The installation of sulfate groups on the carbohydrate residues of glycoproteins, glycolipids, and glycosaminoglycans is a critical posttranslational modification that occurs in all higher eukaryotes. The Gal/GalNAc/GlcNAc-6-O-sulfotransferases (GSTs) are a recently discovered family of carbohydrate sulfotransferases that share significant sequence homology at the amino acid level and mediate a number of different biological processes such as leukocyte adhesion at sites of chronic inflammation. Structural and mechanistic studies of this family of sulfotransferases have been hindered by the lack of a productive recombinant protein expression system. We developed a baculovirus expression system for five of the seven cloned GSTs and determined their kinetic parameters using both thin-layer chromatography and a recently developed polymer dot-blot assay. We used these tools to perform the first site-directed mutagenesis study of a member of this sulfotransferase family, GST2. Using sequence alignments with other carbohydrate and cytosolic sulfotransferases, we selected residues within the putative binding regions for 3'-phosphoadenosine 5'-phosphosulfate (PAPS) and the carbohydrate substrate for mutagenesis. Kinetic analysis of the mutants identified residues that are essential for catalytic activity. These results should facilitate mechanistic studies and the development of small molecule inhibitors of this enzyme family to ameliorate chronic inflammatory diseases.

    View details for DOI 10.1021/bi0269557

    View details for Web of Science ID 000180171800015

    View details for PubMedID 12501187

  • Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Mougous, J. D., Leavell, M. D., Senaratne, R. H., Leigh, C. D., Williams, S. J., Riley, L. W., Leary, J. A., Bertozzi, C. R. 2002; 99 (26): 17037-17042

    Abstract

    The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A step in this direction will be the development of tools to identify metabolites that share common structural features. The importance of sulfated molecules in cell-cell communication motivated us to develop a rapid two-step method for identifying these metabolites in microorganisms, particularly in pathogenic mycobacteria. Sulfurcontaining molecules were initially identified by mass spectral analysis of cell extracts from bacteria labeled metabolically with a stable sulfur isotope (34SO 4 2-). To differentiate sulfated from reduced-sulfur-containing molecules, we employed a mutant lacking the reductive branch of the sulfate assimilation pathway. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. The method was applied to the discovery of several new sulfated molecules in Mycobacterium tuberculosis and Mycobacterium smegmatis. Because a sulfur auxotrophic strain is the only requirement of the approach, many microorganisms can be studied in this manner. Such genetic engineering in combination with stable isotopic labeling can be applied to various metabolic pathways and their products.

    View details for DOI 10.1073/pnas.252514899

    View details for Web of Science ID 000180101600095

    View details for PubMedID 12482950

    View details for PubMedCentralID PMC139265

  • Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Saxon, E., Luchansky, S. J., Hang, H. C., Yu, C., Lee, S. C., Bertozzi, C. R. 2002; 124 (50): 14893-14902

    Abstract

    The structure of sialic acid on living cells can be modulated by metabolism of unnatural biosynthetic precursors. Here we investigate the conversion of a panel of azide-functionalized mannosamine and glucosamine derivatives into cell-surface sialosides. A key tool in this study is the Staudinger ligation, a highly selective reaction between modified triarylphosphines and azides that produces an amide-linked product. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. The reaction provided a means to label the glycoconjugate-bound azidosugars with biochemical probes. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. These two strategies provide a means to selectively modify cell-surface glycans with exogenous probes.

    View details for DOI 10.1021/ja027748x

    View details for Web of Science ID 000179817000032

    View details for PubMedID 12475330

  • Carbohydrate sulfotransferases of the GalNAc/Gal/GlcNAc6ST family BIOCHEMISTRY Grunwell, J. R., Bertozzi, C. R. 2002; 41 (44): 13117-13126

    View details for DOI 10.1021/bi020507h

    View details for Web of Science ID 000178916000001

    View details for PubMedID 12403612

  • Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay BIOCHEMISTRY Pi, N., Armstrong, J. I., Bertozzi, C. R., Leary, J. A. 2002; 41 (44): 13283-13288

    Abstract

    A novel and efficient enzyme kinetics assay using electrospray ionization mass spectrometry was developed and applied to the bacterial carbohydrate sulfotransferase (NodST). NodST catalyzes the sulfuryl group transfer from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to chitobiose, generating 3'-phosphoadenosine 5'-phosphate (PAP) and chitobiose-6-OSO(3)(-) as products. Traditional spectrophotometric assays are not applicable to the NodST system since no shift in absorption accompanies sulfuryl group transfer. Alternative assays have employed thin-layer chromatography, but this procedure is time-consuming and requires radioactive materials. The ESI-MS assay presented herein requires no chromophoric substrate or product, and the analysis time is very short. The ESI-MS assay is used to determine NodST kinetic parameters, including K(M), V(max), and K(i) (for PAP). In addition, the mode of inhibition for PAP was rapidly determined. The results were in excellent agreement with those obtained from previous assays, verifying the accuracy and reliability of the ESI-MS assay. This unique technique is currently being used to investigate the enzymatic mechanism of NodST and to identify sulfotransferase inhibitors.

    View details for DOI 10.1021/bi020457g

    View details for Web of Science ID 000178916000019

    View details for PubMedID 12403630

  • 5 '-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria JOURNAL OF BIOLOGICAL CHEMISTRY Williams, S. J., Senaratne, R. H., Mougous, J. D., Riley, L. W., Bertozzi, C. R. 2002; 277 (36): 32606-32615

    Abstract

    Bacterial sulfate assimilation pathways provide for activation of inorganic sulfur for the biosynthesis of cysteine and methionine, through either adenosine 5'-phosphosulfate (APS) or 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as intermediates. PAPS is also the substrate for sulfotransferases that produce sulfolipids, putative virulence factors, in Mycobacterium tuberculosis such as SL-1. In this report, genetic complementation using Escherichia coli mutant strains deficient in APS kinase and PAPS reductase was used to define the M. tuberculosis and Mycobacterium smegmatis CysH enzymes as APS reductases. Consequently, the sulfate assimilation pathway of M. tuberculosis proceeds from sulfate through APS, which is acted on by APS reductase in the first committed step toward cysteine and methionine. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. Deletion of CysH from M. smegmatis afforded a cysteine and methionine auxotroph consistent with a metabolic branch point centered on APS. In addition, we have redefined the substrate specificity of the B. subtilis CysH, formerly designated a PAPS reductase, as an APS reductase, based on its ability to complement a mutant E. coli strain deficient in APS kinase. Together, these studies show that two conserved sequence motifs, CCXXRKXXPL and SXGCXXCT, found in the C termini of all APS reductases, but not in PAPS reductases, may be used to predict the substrate specificity of these enzymes. A functional domain of the M. tuberculosis CysC protein was cloned and expressed in E. coli, confirming the ability of this organism to make PAPS. The expression of recombinant M. tuberculosis APS kinase provides a means for the discovery of inhibitors of this enzyme and thus of the biosynthesis of SL-1.

    View details for DOI 10.1074/jbc.M204613200

    View details for Web of Science ID 000177859000029

    View details for PubMedID 12072441

  • A 96-well dot-blot assay for carbohydrate sulfotransferases ANALYTICAL BIOCHEMISTRY Verdugo, D. E., Bertozzi, C. R. 2002; 307 (2): 330-336

    Abstract

    Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). The assay proceeds by transfer of 35S-labeled sulfate from [35S]-3(')-phosphoadenosine-5(')-phosphosulfate (PAPS) to the free amino groups of de-N-sulfated heparin (NDST-1), or the 6-hydroxyl groups of N-acetylglucosamine residues linked to a polyacrylamide scaffold (HEC-GlcNAc-6-ST). The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product "dots" for subsequent quantification. This assay allows for direct product detection on the membrane, obviating excessive washing and elution steps endemic to other assays. The assay was validated by measuring K(M) values for PAPS and K(I) values for PAP, the product of sulfuryl transfer. The assay method should be useful for inhibitor screens for both enzymes. In addition, the general assay architecture should be readily applicable to high-throughput screens of other carbohydrate sulfotransferases.

    View details for Web of Science ID 000177962200018

    View details for PubMedID 12202251

  • Sulfotransferases and sulfatases in mycobacteria CHEMISTRY & BIOLOGY Mougous, J. D., Green, R. E., Williams, S. J., Brenner, S. E., Bertozzi, C. R. 2002; 9 (7): 767-776

    Abstract

    Analysis of the genomes of M. tuberculosis, M. leprae, M. smegmatis, and M. avium has revealed a large family of genes homologous to known sulfotransferases. Despite reports detailing a suite of sulfated glycolipids in many mycobacteria, a corresponding family of sulfotransferase genes remains uncharacterized. Here, a sequence-based analysis of newly discovered mycobacterial sulfotransferase genes, named stf1-stf10, is presented. Interestingly, two sulfotransferase genes are highly similar to mammalian sulfotransferases, increasing the list of mycobacterial eukaryotic-like protein families. The sulfotransferases join an equally complex family of mycobacterial sulfatases: a large family of sulfatase genes has been found in all of the mycobacterial genomes examined. As sulfated molecules are common mediators of cell-cell interactions, the sulfotransferases and sulfatases may be involved in regulating host-pathogen interactions.

    View details for Web of Science ID 000177297600001

    View details for PubMedID 12144918

  • Recent advances in the chemical synthesis of mucin-like glycoproteins GLYCOBIOLOGY Marcaurelle, L. A., Bertozzi, C. R. 2002; 12 (6): 69R-77R

    Abstract

    This purpose of this mini review is to familiarizereaders with the tools currently available for the synthesis of mucin-typeglycoproteins. The article will highlight recent approaches to thesynthesis of glycopeptide fragments bearing complex O-linkedglycans, as well as new strategies for the generation of full-lengthglycoproteins.

    View details for Web of Science ID 000177054900001

    View details for PubMedID 12107076

  • Conjugation of DNA to silanized colloidal semiconductor nanocrystalline quantum dots CHEMISTRY OF MATERIALS Parak, W. J., Gerion, D., Zanchet, D., Woerz, A. S., Pellegrino, T., Micheel, C., Williams, S. C., Seitz, M., Bruehl, R. E., Bryant, Z., Bustamante, C., Bertozzi, C. R., Alivisatos, A. P. 2002; 14 (5): 2113-2119

    View details for DOI 10.1021/cm0107878

    View details for Web of Science ID 000175790100030

  • Stereloselective synthesis of myo-inositol via ring-closing metathesis: A building block for glycosylphosphatidylinositol (GPI) anchor synthesis ORGANIC LETTERS Conrad, R. M., Grogan, M. J., Bertozzi, C. R. 2002; 4 (8): 1359-1361

    Abstract

    Here we report a concise stereoselective synthesis of myo-inositol via ring-closing metathesis. A readily available bis-Weinreb amide of D-tartrate served as a key intermediate. [reaction: see text]

    View details for DOI 10.1021/ol025680k

    View details for Web of Science ID 000174997600030

    View details for PubMedID 11950362

  • Chemical approaches to the investigation of cellular systems BIOORGANIC & MEDICINAL CHEMISTRY Cook, B. N., Bertozzi, C. R. 2002; 10 (4): 829-840

    Abstract

    Biochemistry in the context of a living cell or organism is complicated by many variables such as supramolecular organization, cytoplasmic viscosity, and substrate heterogeneity. While these variables are easily excluded or avoided in reconstituted systems, they must be dealt with in cellular environments. New developments have allowed researchers to begin probing the inner workings of the cell to gain new insight into cell function and metabolism. Advances in cellular imaging and in small molecule-controlled gene expression, signal transduction and cell surface modification are discussed in this review. These techniques have permitted the study of molecular components within the context of living cells.

    View details for Web of Science ID 000174151500001

    View details for PubMedID 11836089

  • Differential effects of unnatural sialic acids on the polysialylation of the neural cell adhesion molecule and neuronal behavior JOURNAL OF BIOLOGICAL CHEMISTRY Charter, N. W., Mahal, L. K., Koshland, D. E., Bertozzi, C. R. 2002; 277 (11): 9255-9261

    Abstract

    In this study we have examined how unnatural sialic acids can alter polysialic acid expression and influence the adhesive properties of the neural cell adhesion molecule (NCAM). Unnatural sialic acids are generated by metabolic conversion of synthetic N-acyl mannosamines and are typically incorporated into cell-surface glycoconjugates. However, N-butanoylmannosamine and N-pentanoylmannosamine are effective inhibitors of polysialic acid (PSA) synthesis in stably transfected HeLa cells expressing NCAM and the polysialyltransferase STX. These cells were used as substrates to examine the effect of inhibiting PSA synthesis on the development of neurons derived from the chick dorsal root ganglion. N-butanoylmannosamine blocked polysialylation of NCAM and significantly reduced neurite outgrowth comparable with enzymatic removal of PSA by endoneuraminidases. As a result, neurite outgrowth was similar to that observed for non-polysialylated NCAM. In contrast, previous studies have shown that N-propanoyl sialic acid (SiaProp), generated from N-propanoylmannosamine, is readily accepted by polysialyltransferases and permits the extension of poly(SiaProp) on NCAM. Despite being immunologically distinct, poly(SiaProp) can promote neurite outgrowth similarly to natural polysialic acid. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM.

    View details for DOI 10.1074/jbc.M111619200

    View details for Web of Science ID 000174400600071

    View details for PubMedID 11786551

  • Tyrosylprotein sulfotransferase inhibitors generated by combinatorial target-guided ligand assembly BIOORGANIC & MEDICINAL CHEMISTRY LETTERS Kehoe, J. W., Maly, D. J., Verdugo, D. E., Armstrong, J. I., Cook, B. N., Ouyang, Y. B., Moore, K. L., Ellman, J. A., Bertozzi, C. R. 2002; 12 (3): 329-332

    Abstract

    Tyrosylprotein sulfotransferases (TPSTs) catalyze the sulfation of tyrosine residues within secreted and membrane-bound proteins. The modification modulates protein-protein interactions in the extracellular environment. Here we use combinatorial target-guided ligand assembly to discover the first known inhibitors of human TPST-2.

    View details for Web of Science ID 000174145500016

    View details for PubMedID 11814789

  • Incorporation of azides into recombinant proteins for chemoselective modification by the Staudinger ligation PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Kiick, K. L., Saxon, E., Tirrell, D. A., Bertozzi, C. R. 2002; 99 (1): 19-24

    Abstract

    The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. One method to achieve this involves the utilization of a non-natural amino acid by the cell's native translational apparatus. Here we demonstrate that a methionine surrogate, azidohomoalanine, is activated by the methionyl-tRNA synthetase of Escherichia coli and replaces methionine in proteins expressed in methionine-depleted bacterial cultures. We further show that proteins containing azidohomoalanine can be selectively modified in the presence of other cellular proteins by means of Staudinger ligation with triarylphosphine reagents. Incorporation of azide-functionalized amino acids into proteins in vivo provides opportunities for protein modification under native conditions and selective labeling of proteins in the intracellular environment.

    View details for Web of Science ID 000173233300006

    View details for PubMedID 11752401

    View details for PubMedCentralID PMC117506

  • Homogeneous glycopeptides and glycoproteins for biological investigation ANNUAL REVIEW OF BIOCHEMISTRY Grogan, M. J., Pratt, M. R., Marcaurelle, L. A., Bertozzi, C. R. 2002; 71: 593-634

    Abstract

    Protein glycosylation is widely recognized as a modulator of protein structure, localization, and cell-cell recognition in multicellular systems. Glycoproteins are typically expressed as mixtures of glycoforms, their oligosaccharides being generated by a template-independent biosynthetic process. Investigation of their function has been greatly assisted by sources of homogeneous material. This review summarizes current efforts to obtain homogeneous glycopeptide and glycoprotein materials by a variety of methods that draw from the techniques of recombinant expression, chemical synthesis, enzymatic transformation, and chemoselective ligation. Some of these techniques remove obstacles to glycoprotein synthesis by installing nonnative linkages and other modifications for facilitated assembly. The end purpose of the described approaches is the production of glycosylated materials for experiments relevant to the biological investigation of glycoproteins, although the strategies presented apply to other posttranslational modifications as well.

    View details for DOI 10.1146/annurev.biochem.71.110601.135334

    View details for Web of Science ID 000177352600021

    View details for PubMedID 12045107

  • An inhibitor of the human UDP-GlcNAc 4-epimerase identified from a uridine-based library: A strategy to inhibit O-linked glycosylation CHEMISTRY & BIOLOGY Winans, K. A., Bertozzi, C. R. 2002; 9 (1): 113-129

    Abstract

    The biological study of O-linked glycosylation is particularly problematic, as chemical tools to control this modification are lacking. An inhibitor of the UDP-GlcNAc 4-epimerase that synthesizes UDP-GalNAc, the donor initiating O-linked glycosylation, would be a powerful reagent for reversibly inhibiting O-linked glycosylation. We synthesized a 1338 member library of uridine analogs directed to the epimerase by virtue of substrate mimicry. Screening of the library identified an inhibitor with a K(i) value of 11 microM. Tests against related enzymes confirmed the compound's specificity for the UDP-GlcNAc 4-epimerase. Inhibitors of a key step of O-linked glycan biosynthesis can be discovered from a directed library screen. Progeny thereof may be powerful tools for controlling O-linked glycosylation in cells.

    View details for Web of Science ID 000173696100012

    View details for PubMedID 11841944

  • Kinetic parameters for small-molecule drug delivery by covalent cell surface targeting BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS Nauman, D. A., Bertozzi, C. R. 2001; 1568 (2): 147-154

    Abstract

    Human cells incubated with N-levulinoylmannosamine (ManLev) process this unnatural metabolic precursor into N-levulinoyl sialic acid (SiaLev), which is incorporated into cell surface glycoconjugates. A key feature of SiaLev is the presence of a ketone group that can be exploited in chemoselective ligation reactions to deliver small-molecule probes to the cell surface. A mathematical model was developed and tested experimentally to evaluate the prospects of using cell surface ketones as targets for covalent small-molecule drug delivery. We quantified the absolute number of ketone groups displayed on cell surfaces as a function of the concentration of ManLev in the medium. The apparent rate constants for the hydrolysis and disappearance of the cell surface conjugates were determined, as well as the apparent rate constant for the formation of covalent bonds with cell surface ketones. These values and the mathematical model confirm that chemoselective reactions on the cell surface can deliver to cells similar numbers of molecules as antibodies. Thus, cell surface ketones are a potential vehicle for a metabolically controlled small-molecule drug delivery system.

    View details for Web of Science ID 000173078400005

    View details for PubMedID 11750762

  • Synthesis of oxime-linked mucin mimics containing the tumor-related T-N and sialyl T-N antigens ORGANIC LETTERS Marcaurelle, L. A., SHIN, Y. S., Goon, S., Bertozzi, C. R. 2001; 3 (23): 3691-3694

    Abstract

    [reaction--see text] The synthesis of oxime-linked mucin mimics was accomplished via the incorporation of multiple ketone residues into a peptide followed by reaction with aminooxy sugars corresponding to the tumor-related T(N) and sialyl T(N) (ST(N)) antigens.

    View details for DOI 10.1021/ol0166247

    View details for Web of Science ID 000172181700022

    View details for PubMedID 11700114

  • Surface molecular recognition PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA Sampson, N. S., Mrksich, M., Bertozzi, C. R. 2001; 98 (23): 12870-12871

    Abstract

    The spatial display of cellular ligands and receptors is important for cell adhesion and communication. Two approaches that emphasize developing selective methods to dissect, modify, and control receptor-ligand interactions at the cellular interface are discussed.

    View details for Web of Science ID 000172076800007

    View details for PubMedID 11687628

    View details for PubMedCentralID PMC60788

  • Substrate specificity of the sialic acid biosynthetic pathway BIOCHEMISTRY Jacobs, C. L., Goon, S., Yarema, K. J., Hinderlich, S., Hang, H. C., Chai, D. H., Bertozzi, C. R. 2001; 40 (43): 12864-12874

    Abstract

    Unnatural analogues of sialic acid can be delivered to mammalian cell surfaces through the metabolic transformation of unnatural N-acetylmannosamine (ManNAc) derivatives. In previous studies, mannosamine analogues bearing simple N-acyl groups up to five carbon atoms in length were recognized as substrates by the biosynthetic machinery and transformed into cell surface sialoglycoconjugates [Keppler, O. T., et al. (2001) Glycobiology 11, 11R-18R]. Such structural alterations to cell surface glycans can be used to probe carbohydrate-dependent phenomena. This report describes our investigation into the extent of tolerance of the pathway toward additional structural alterations of the N-acyl substituent of ManNAc. A panel of analogues with ketone-containing N-acyl groups that varied in the length or steric bulk was chemically synthesized and tested for metabolic conversion to cell surface glycans. We found that extension of the N-acyl chain to six, seven, or eight carbon atoms dramatically reduced utilization by the biosynthetic machinery. Likewise, branching from the linear chain reduced metabolic conversion. Quantitation of metabolic intermediates suggested that cellular metabolism is limited by the phosphorylation of the N-acylmannosamines by ManNAc 6-kinase in the first step of the pathway. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Identification of ManNAc 6-kinase as a bottleneck for unnatural sialic acid biosynthesis provides a target for expanding the metabolic promiscuity of mammalian cells.

    View details for Web of Science ID 000171962100014

    View details for PubMedID 11669623

  • Biosynthesis of sialylated lipooligosaccharides in Haemophilis ducreyi is dependent on exogenous sialic acid and not mannosamine. Incorporation studies using N-acylmannosamine analogues, N-glycolylneuraminic acid, and C-13-labeled N-acetylneuraminic acid BIOCHEMISTRY Schilling, B., Goon, S., Samuels, N. M., Gaucher, S. P., Leary, J. A., Bertozzi, C. R., Gibson, B. W. 2001; 40 (42): 12666-12677

    Abstract

    Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Incorporation of sialosides into LOS was assessed by matrix-assisted laser desorption and electrospray ionization mass spectrometry. A Fourier transform ion cyclotron resonance mass spectrometer provided accurate mass measurements, and a quadrupole time-of-flight instrument was used to obtain characteristic fragment ions and partial carbohydrate sequences. Exogenously supplied N-acetylmannosamine analogues were not converted to LOS-associated sialosides at a detectable level. In contrast, exogenous (13)C-labeled N-acetylneuraminic acid ([(13)C]NeuAc) and N-glycolylneuraminic acid (NeuGc) were efficiently incorporated into LOS in a dose-dependent fashion. Moreover, approximately 1.3 microM total exogenous sialic acid was sufficient to obtain about 50% of the maximum production of sialic acid-containing glycoforms observed under in vitro growth conditions. Together, these data suggest that the expressed levels of sialylated LOS glycoforms observed in H. ducreyi are in large part controlled by the exogenous concentrations of sialic acid and at levels one might expect in vivo. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used.

    View details for Web of Science ID 000171801100023

    View details for PubMedID 11601991

  • A small-molecule modulator of poly-alpha 2,8-sialic acid expression on cultured neurons and tumor cells SCIENCE Mahal, L. K., Charter, N. W., Angata, K., Fukuda, M., Koshland, D. E., Bertozzi, C. R. 2001; 294 (5541): 380-382

    Abstract

    Poly-alpha2,8-sialic acid (PSA) has been implicated in numerous normal and pathological processes, including development, neuronal plasticity, and tumor metastasis. We report that cell surface PSA expression can be reversibly inhibited by a small molecule, N-butanoylmannosamine (ManBut). Inhibition occurs through a metabolic mechanism in which ManBut is converted to unnatural sialic acid derivatives that effectively act as chain terminators during cellular PSA biosynthesis. N-Propanoylmannosamine (ManProp), which differs from ManBut by a single methylene group, did not inhibit PSA biosynthesis. Modulation of PSA expression by chemical means has a role complementary to genetic and biochemical approaches in the study of complex PSA-mediated events.

    View details for Web of Science ID 000171601400045

    View details for PubMedID 11598302

  • Chemoselective approaches to glycoprotein assembly ACCOUNTS OF CHEMICAL RESEARCH Hang, H. C., Bertozzi, C. R. 2001; 34 (9): 727-736

    Abstract

    Oligosaccharides on proteins and lipids play central roles in human health and disease. The molecular analysis of glycoconjugate function has benefited tremendously from new methods for their chemical synthesis, which provides homogeneous material not attainable from biosynthetic systems. Still, glycoconjugate synthesis requires the manipulation of multiple stereocenters and protecting groups and remains the domain of a few expert laboratories around the world. This Account summarizes chemoselective approaches for assembling homogeneous glycoconjugates that attempt to reduce the barriers to their synthesis. The objective of these methods is to make glycoconjugate synthesis accessible to a broader community, thereby accelerating progress in glycobiology.

    View details for DOI 10.1021/ar9901570

    View details for Web of Science ID 000171204900005

    View details for PubMedID 11560472

  • A library approach to the generation of bisubstrate analogue sulfotransferase inhibitors ORGANIC LETTERS Armstrong, J. I., Ge, X., Verdugo, D. E., Winans, K. A., Leary, J. A., Bertozzi, C. R. 2001; 3 (17): 2657-2660

    Abstract

    [reaction: see text]. A library of potential bisubstrate analogue inhibitors (1) targeting sulfotransferase enzymes was generated by the chemoselective ligation of the PAPS mimic 2 with a panel of 447 aldehydes. Preliminary screening has identified compounds that inhibit estrogen sulfotransferase (EST), an enzyme relevant to breast cancer.

    View details for Web of Science ID 000170600300010

    View details for PubMedID 11506602

  • Metabolic selection of glycosylation defects in human cells NATURE BIOTECHNOLOGY Yarema, K. J., Goon, S., Bertozzi, C. R. 2001; 19 (6): 553-558

    Abstract

    Changes in glycosylation are often associated with disease progression, but the genetic and metabolic basis of these events is rarely understood in detail at a molecular level. We describe a metabolism-based approach to the selection of mutants in glycoconjugate biosynthesis that provides insight into regulatory mechanisms for oligosaccharide expression and metabolic flux. Unnatural intermediates are used to challenge a specific pathway, and cell surface expression of their metabolic products provides a readout of flux in that pathway and a basis for selecting genetic mutants. The approach was applied to the sialic acid metabolic pathway in human cells, yielding novel mutants with phenotypes related to the inborn metabolic defect sialuria and metastatic tumor cells.

    View details for Web of Science ID 000169081700029

    View details for PubMedID 11385460

  • Polymerized liposome assemblies: Bifunctional macromolecular selectin inhibitors mimicking physiological selectin ligands BIOCHEMISTRY Bruehl, R. E., Dasgupta, F., Katsumoto, T. R., Tan, J. H., Bertozzi, C. R., Spevak, W., Ahn, D. J., ROSEN, S. D., Nagy, J. O. 2001; 40 (20): 5964-5974

    Abstract

    Monomeric sialyl Lewis(X) (sLe(x)) and sLe(x)-like oligosaccharides are minimal structures capable of supporting selectin binding in vitro. However, their weak binding interactions do not correlate with the high-affinity binding interactions witnessed in vivo. The polyvalent display of carbohydrate groups found on cell surface glycoprotein structures may contribute to the enhanced binding strength of selectin-mediated adhesion. Detailed biochemical analyses of physiological selectin ligands have revealed a complicated composition of molecules that bind to the selectins in vivo and suggest that there are other requirements for tight binding beyond simple carbohydrate multimerization. In an effort to mimic the high-affinity binding, polyvalent scaffolds that contain multicomponent displays of selectin-binding ligands have been synthesized. Here, we demonstrate that the presentation of additional anionic functional groups in the form of sulfate esters, on a polymerized liposome surface containing a multimeric array of sLe(x)-like oligosaccharides, generates a highly potent, bifunctional macromolecular assembly. This assembly inhibits L-, E-, and P-selectin binding to GlyCAM-1, a physiological ligand better than sLe(x)-like liposomes without additional anionic charge. These multivalent arrays are 4 orders of magnitude better than the monovalent carbohydrate. Liposomes displaying 3'-sulfo Lewis(X)-like oligosaccharides, on the other hand, show slight loss of binding with introduction of additional anionic functional groups for E- and P-selectin and negligible change for L-selectin. The ability to rapidly and systematically vary the composition of these assemblies is a distinguishing feature of this methodology and may be applied to the study of other systems where composite binding determinants are important for high-affinity binding.

    View details for Web of Science ID 000168932900013

    View details for PubMedID 11352731

  • Biosynthesis of L-selectin ligands: Sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases BIOCHEMISTRY Bowman, K. G., Cook, B. N., de Graffenried, C. L., Bertozzi, C. R. 2001; 40 (18): 5382-5391

    Abstract

    The leukocyte adhesion molecule L-selectin mediates lymphocyte homing to secondary lymphoid organs and to certain sites of inflammation. The cognate ligands for L-selectin possess the unusual sulfated tetrasaccharide epitope 6-sulfo sialyl Lewis x (Siaalpha2-->3Galbeta1-->4[Fucalpha1-->3][SO(3)-->6]GlcNAc). Sulfation of GlcNAc within sialyl Lewis x is a crucial modification for L-selectin binding, and thus, the underlying sulfotransferase may be a key modulator of lymphocyte trafficking. Four recently discovered GlcNAc-6-sulfotransferases are the first candidate contributors to the biosynthesis of 6-sulfo sLex in the context of L-selectin ligands. Here we report the in vitro activity of the four GlcNAc-6-sulfotransferases on a panel of synthetic oligosaccharide substrates that comprise structural motifs derived from sialyl Lewis x. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. On the basis of these results, we propose possible pathways for 6-sulfo sialyl Lewis x biosynthesis and suggest that sulfation may be an early committed step.

    View details for DOI 10.1021/bi001750o

    View details for Web of Science ID 000168490400007

    View details for PubMedID 11331001

  • The evolving academic research environment CHEMICAL & ENGINEERING NEWS Bertozzi, C. R. 2001; 79 (13): 271-271
  • Chemical glycobiology SCIENCE Bertozzi, C. R., Kiessling, L. L. 2001; 291 (5512): 2357-2364

    Abstract

    Chemical tools have proven indispensable for studies in glycobiology. Synthetic oligosaccharides and glycoconjugates provide materials for correlating structure with function. Synthetic mimics of the complex assemblies found on cell surfaces can modulate cellular interactions and are under development as therapeutic agents. Small molecule inhibitors of carbohydrate biosynthetic and processing enzymes can block the assembly of specific oligosaccharide structures. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. Carbohydrate biosynthetic pathways are often amenable to interception with synthetic unnatural substrates. Such metabolic interference can block the expression of oligosaccharides or alter the structures of the sugars presented on cells. Collectively, these chemical approaches are contributing great insight into the myriad biological functions of oligosaccharides.

    View details for Web of Science ID 000167618700043

    View details for PubMedID 11269316

  • Chemical synthesis of lymphotactin: A glycosylated chemokine with a C-terminal mucin-like domain CHEMISTRY-A EUROPEAN JOURNAL Marcaurelle, L. A., Mizoue, L. S., Wilken, J., Oldham, L., KENT, S. B., Handel, T. M., Bertozzi, C. R. 2001; 7 (5): 1129-1132

    Abstract

    The synthesis of a 93-residue chemokine, lymphotactin, containing eight sites of O-linked glycosylation, was achieved using the technique of native chemical ligation. A single GalNAc residue was incorporated at each glycosylation site using standard Fmoc-chemistry to achieve the first total synthesis of a mucin-type glycoprotein. Using this approach quantities of homogeneous material were obtained for structural and functional analysis.

    View details for Web of Science ID 000167417700020

    View details for PubMedID 11303872

  • Modulating cell surface immunoreactivity by metabolic induction of unnatural carbohydrate antigens CHEMISTRY & BIOLOGY Lemieux, G. A., Bertozzi, C. R. 2001; 8 (3): 265-275

    Abstract

    Sialic acid is a component of many tumor-associated oligosaccharide antigens. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. We explored whether unnatural cell surface sialosides produced by metabolism can act as neo-antigens and modulate the immunogenicity of cells.Immunization of rabbits with synthetic conjugates of an unnatural sialic acid bound to keyhole limpet hemocyanin produced significant titers of antibodies that were specific for the structurally altered sialic acid. The antibodies recognized cells that were fed the unnatural biosynthetic precursor, and were capable of directing complement-mediated lysis.Structural alteration of sialic acids replaces a tolerized self-antigen with an antigenic determinant. Incorporation of unnatural sialosides into cell surface glycoconjugates through biosynthetic means can alter the immunoreactivity of cells, providing new possibilities for tumor immunotherapy.

    View details for Web of Science ID 000168383900005

    View details for PubMedID 11306351

  • Chemoselective elaboration of O-linked glycopeptide mimetics by alkylation of 3-ThioGalNAc JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Marcaurelle, L. A., Bertozzi, C. R. 2001; 123 (8): 1587-1595

    Abstract

    A critical branch point in mucin-type oligosaccharides is the beta 1-->3 glycosidic linkage to the core alpha-N-acetylgalactosamine (GalNAc) residue. We report here a strategy for the synthesis of O-linked glycopeptide analogues that replaces this linkage with a thioether amenable to construction by chemoselective ligation. The key building block was a 2-azido-3-thiogalactose-Thr analogue that was incorporated into a peptide by fluorenylmethoxycarbonyl (Fmoc)-based solid-phase peptide synthesis. Higher order oligosaccharides were readily generated by alkylation of the corresponding 3-thioGalNAc with N-bromoacetamido sugars. The rapid assembly of "core 1"and "core 3" O-linked glycopeptide mimetics was accomplished in this fashion.

    View details for DOI 10.1021/ja003713q

    View details for Web of Science ID 000167162100006

    View details for PubMedID 11456757

  • Ketone isosteres of 2-N-acetamidosugars as substrates for metabolic cell surface engineering JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Hang, H. C., Bertozzi, C. R. 2001; 123 (6): 1242-1243

    View details for Web of Science ID 000166873000031

    View details for PubMedID 11456684

  • Characterizing glycosylation pathways GENOME BIOLOGY Yarema, K. J., Bertozzi, C. R. 2001; 2 (5)

    Abstract

    Numerous factors that influence cell-surface carbohydrate composition remain to be elucidated. The combination of novel biochemical and metabolism-based approaches with emerging genomic methods promises to accelerate efforts to understand glycosylation.

    View details for Web of Science ID 000207584000003

    View details for PubMedID 11387039

    View details for PubMedCentralID PMC138933

  • Chemical and biological strategies for engineering cell surface glycosylation ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY Saxon, E., Bertozzi, C. R. 2001; 17: 1-23

    Abstract

    Oligosaccharides play a crucial role in many of the recognition, signaling, and adhesion events that take place at the surface of cells. Abnormalities in the synthesis or presentation of these carbohydrates can lead to misfolded and inactive proteins, as well as to several debilitating disease states. However, their diverse structures, which are the key to their function, have hampered studies by biologists and chemists alike. This review presents an overview of techniques for examining and manipulating cell surface oligosaccharides through genetic, enzymatic, and chemical strategies.

    View details for Web of Science ID 000172448800001

    View details for PubMedID 11687482

  • Sulfation of N-acetylglucosamine by chondroitin 6-sulfotransferase 2 (GST-5) JOURNAL OF BIOLOGICAL CHEMISTRY Bhakta, S., Bartes, A., Bowman, K. G., Kao, W. M., Polsky, I., Lee, J. K., Cook, B. N., Bruehl, R. E., ROSEN, S. D., Bertozzi, C. R., Hemmerich, S. 2000; 275 (51): 40226-40234

    Abstract

    Based on sequence homology with a previously cloned human GlcNAc 6-O-sulfotransferase, we have identified an open reading frame (ORF) encoding a novel member of the Gal/GalNAc/GlcNAc 6-O-sulfotransferase (GST) family termed GST-5 on the human X chromosome (band Xp11). GST-5 has recently been characterized as a novel GalNAc 6-O-sulfotransferase termed chondroitin 6-sulfotransferase-2 (Kitagawa, H., Fujita, M., Itio, N., and Sugahara K. (2000) J. Biol. Chem. 275, 21075-21080). We have coexpressed a human GST-5 cDNA with a GlyCAM-1/IgG fusion protein in COS-7 cells and observed four-fold enhanced [(35)S]sulfate incorporation into this mucin acceptor. All mucin-associated [(35)S]sulfate was incorporated as GlcNAc-6-sulfate or Galbeta1-->4GlcNAc-6-sulfate. GST-5 was also expressed in soluble epitope-tagged form and found to catalyze 6-O-sulfation of GlcNAc residues in synthetic acceptor structures. In particular, GST-5 was found to catalyze 6-O-sulfation of beta-benzyl GlcNAc but not alpha- or beta-benzyl GalNAc. In the mouse genome we have found a homologous ORF that predicts a novel murine GlcNAc 6-O-sulfotransferase with 88% identity to the human enzyme. This gene was mapped to mouse chromosome X at band XA3.1-3.2. GST-5 is the newest member of an emerging family of carbohydrate 6-O-sulfotransferases that includes chondroitin 6-sulfotransferase (GST-0), keratan-sulfate galactose 6-O-sulfotransferase (GST-1), the ubiquitously expressed GlcNAc 6-O-sulfotransferase (GST-2), high endothelial cell GlcNAc 6-O-sulfotransferase (GST-3), and intestinal GlcNAc 6-O-sulfotransferase (GST-4).

    View details for Web of Science ID 000166039500060

    View details for PubMedID 10956661

  • New directions in glycoprotein engineering TETRAHEDRON MACMILLAN, D., Bertozzi, C. R. 2000; 56 (48): 9515-9525
  • Minimal sulfated carbohydrates for recognition by L-selectin and the MECA-79 antibody JOURNAL OF BIOLOGICAL CHEMISTRY Bruehl, R. E., Bertozzi, C. R., ROSEN, S. D. 2000; 275 (42): 32642-32648

    Abstract

    Sulfated forms of sialyl-Le(X) containing Gal-6-SO(4) or GlcNAc-6-SO(4) have been implicated as potential recognition determinants on high endothelial venule ligands for L-selectin. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. Using a panel of sulfated lactose (Galbeta1-->4Glc) neoglycolipids as substrates in direct binding assays, we found that 6',6-disulfolactose was the preferred structure for L-selectin, although significant binding to 6'- and 6-sulfolactose was observed as well. Binding was EDTA-sensitive and blocked by L-selectin-specific monoclonal antibodies. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Instead, MECA-79 bound preferentially to 6-sulfolactose. The difference in preferred substrates between L-selectin and MECA-79 may explain the variable activity of MECA-79 as an inhibitor of lymphocyte adhesion to high endothelial venules in lymphoid organs. Our results suggest that both Gal-6-SO(4) and GlcNAc-6-SO(4) may contribute to L-selectin recognition, either as components of sulfosialyl-Le(X) capping groups or in internal structures. By contrast, only GlcNAc-6-SO(4) appears to contribute to MECA-79 binding.

    View details for Web of Science ID 000090003800038

    View details for PubMedID 10938267

  • Biosynthetic incorporation of unnatural sialic acids into polysialic acid on neural cells GLYCOBIOLOGY Charter, N. W., Mahal, L. K., Koshland, D. E., Bertozzi, C. R. 2000; 10 (10): 1049-1056

    Abstract

    In this study we demonstrate that polysialyltransferases are capable of accepting unnatural substrates in terminally differentiated human neurons. Polysialyltransferases catalyze the glycosylation of the neural cell adhesion molecule (NCAM) with polysialic acid (PSA). The unnatural sialic acid analog, N-levulinoyl sialic acid (SiaLev), was incorporated into cell surface glycoconjugates including PSA by the incubation of cultured neurons with the metabolic precursor N-levulinoylmannosamine (ManLev). The ketone group within the levulinoyl side chain of SiaLev was then used as a chemical handle for detection using a biotin probe. The incorporation of SiaLev residues into PSA was demonstrated by protection from sialidases that can cleave natural sialic acids but not those bearing unnatural N-acyl groups. The presence of SiaLev groups on the neuronal cell surface did not impede neurite outgrowth or significantly affect the distribution of PSA on neuronal compartments. Since PSA is important in neural plasticity and development, this mechanism for modulating PSA structure might be useful for functional studies.

    View details for Web of Science ID 000089664300011

    View details for PubMedID 11030751

  • Differential carbohydrate recognition of two GlcNAc-6-sulfotransferases with possible roles in L-selectin ligand biosynthesis JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Cook, B. N., Bhakta, S., Biegel, T., Bowman, K. G., Armstrong, J. I., Hemmerich, S., Bertozzi, C. R. 2000; 122 (36): 8612-8622
  • A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds ORGANIC LETTERS Saxon, E., Armstrong, J. I., Bertozzi, C. R. 2000; 2 (14): 2141-2143

    Abstract

    [reaction: see text] Here we report a novel modification of our previously reported "Staudinger ligation" that generates an amide bond from an azide and a specifically functionalized phosphine. This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biological chemistry.

    View details for Web of Science ID 000088039800042

    View details for PubMedID 10891251

  • Cell surface engineering by a modified Staudinger reaction SCIENCE Saxon, E., Bertozzi, C. R. 2000; 287 (5460): 2007-2010

    Abstract

    Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. A ligation modeled after the Staudinger reaction forms an amide bond by coupling of an azide and a specifically engineered triarylphosphine. Both reactive partners are abiotic and chemically orthogonal to native cellular components. Azides installed within cell surface glycoconjugates by metabolism of a synthetic azidosugar were reacted with a biotinylated triarylphosphine to produce stable cell-surface adducts. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions.

    View details for Web of Science ID 000085902800059

    View details for PubMedID 10720325

  • Tyrosine sulfation: a modulator of extracellular protein-protein interactions CHEMISTRY & BIOLOGY Kehoe, J. W., Bertozzi, C. R. 2000; 7 (3): R57-R61

    Abstract

    Tyrosine sulfation is a post-translational modification of many secreted and membrane-bound proteins. Its biological roles have been unclear. Recent work has implicated tyrosine sulfate as a determinant of protein-protein interactions involved in leukocyte adhesion, hemostasis and chemokine signaling.

    View details for Web of Science ID 000085780300001

    View details for PubMedID 10712936

  • Discovery of carbohydrate sulfotransferase inhibitors from a kinase-directed library ANGEWANDTE CHEMIE-INTERNATIONAL EDITION Armstrong, J. I., Portley, A. R., Chang, Y. T., Nierengarten, D. M., Cook, B. N., Bowman, K. G., Bishop, A., Gray, N. S., Shokat, K. M., Schultz, P. G., Bertozzi, C. R. 2000; 39 (7): 1303-?
  • Metabolic labeling of glycoproteins with chemical tags through unnatural sialic acid biosynthesis APPLICATIONS OF CHIMERIC GENES AND HYBRID PROTEINS PT B Jacobs, C. L., Yarema, K. J., Mahal, L. K., Nauman, D. A., Charters, N. W., Bertozzi, C. R. 2000; 327: 260-275

    View details for Web of Science ID 000165500500020

    View details for PubMedID 11044989

  • Fmoc-based synthesis of peptide-(alpha)thioesters: Application to the total chemical synthesis of a glycoprotein by native chemical ligation JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Shin, Y., Winans, K. A., Backes, B. J., KENT, S. B., Ellman, J. A., Bertozzi, C. R. 1999; 121 (50): 11684-11689
  • A chemically synthesized version of the insect antibacterial glycopeptide, diptericin, disrupts bacterial membrane integrity BIOCHEMISTRY Winans, K. A., King, D. S., Rao, V. R., Bertozzi, C. R. 1999; 38 (36): 11700-11710

    Abstract

    Insects protect themselves against bacterial infection by secreting a battery of antimicrobial peptides into the hemolymph. Despite recent progress, important mechanistic questions, such as the precise bacterial targets, the nature of any cooperation that occurs between peptides, and the purpose of multiple peptide isoforms, remain largely unanswered. We report herein the chemical synthesis and preliminary mechanistic investigation of diptericin, an 82 residue glycopeptide that contains regions similar to two different types of antibacterial peptides. A revised, highly practical synthesis of the precursor N(alpha)-Fmoc-Thr(Ac(3)-alpha-D-GalNAc) allowed us to produce sufficient quantities of the glycopeptide for mechanistic assays. The synthetic, full-length polypeptide proved to be active in growth inhibition assays with an IC(50) of approximately 250 nM, a concentration similar to that found in the insect hemolymph. Biological analysis of diptericin fragments indicated that the main determinant of antibacterial activity lay in the C-terminal region that is similar to the attacin peptides, although the N-terminal segment, related to the proline-rich family of antibacterial peptides, augmented that activity by 100-fold. In all assays, activity appeared glycosylation independent. Circular dichroism of unglycosylated diptericin indicated that the peptide lacked structure both in plain buffer and in the presence of liposomes. Diptericin increased the permeability of the outer and inner membranes of Escherichia coli D22 cells, suggesting possible mechanisms of action. The ability to access glycopeptides of this type through chemical synthesis will facilitate further mechanistic studies.

    View details for Web of Science ID 000082757300015

    View details for PubMedID 10512626

  • Engineering novel cell surface receptors for virus-mediated gene transfer JOURNAL OF BIOLOGICAL CHEMISTRY Lee, J. H., Baker, T. J., Mahal, L. K., Zabner, J., Bertozzi, C. R., WIEMER, D. F., Welsh, M. J. 1999; 274 (31): 21878-21884

    Abstract

    The absence of viral receptors is a major barrier to efficient gene transfer in many cells. To overcome this barrier, we developed an artificial receptor based on expression of a novel sugar. We fed cells an unnatural monosaccharide, a modified mannosamine that replaced the acetyl group with a levulinate group (ManLev). ManLev was metabolized and incorporated into cell-surface glycoconjugates. The synthetic sugar decorated the cell surface with a unique ketone group that served as a foundation on which we built an adenovirus receptor by covalently binding biotin hydrazide to the ketone. The artificial receptor enhanced adenoviral vector binding and gene transfer to cells that are relatively resistant to adenovirus infection. These data are the first to suggest the feasibility of a strategy that improves the efficiency of gene transfer by using the biosynthetic machinery of the cell to engineer novel sugars on the cell surface.

    View details for Web of Science ID 000081721100060

    View details for PubMedID 10419507

  • Exploiting differences in sialoside expression for selective targeting of MRI contrast reagents JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Lemieux, G. A., Yarema, K. J., Jacobs, C. L., Bertozzi, C. R. 1999; 121 (17): 4278-4279
  • New directions in the synthesis of glycopeptide mimetics CHEMISTRY-A EUROPEAN JOURNAL Marcaurelle, L. A., Bertozzi, C. R. 1999; 5 (5): 1384-1390
  • Probing the surface structural rearrangement of hydrogels by sum-frequency generation spectroscopy JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chen, Q., Zhang, D., Somorjai, G., Bertozzi, C. R. 1999; 121 (2): 446-447
  • Carbohydrate sulfotransferases: mediators of extracellular communication CHEMISTRY & BIOLOGY Bowman, K. G., Bertozzi, C. R. 1999; 6 (1): R9-R22

    Abstract

    Sulfated carbohydrates mediate diverse extracellular recognition events in both normal and pathological processes. The sulfotransferases that generate specific carbohydrate 'sulfoforms' have recently been recognized as key modulators of these processes and therefore represent potential therapeutic targets.

    View details for Web of Science ID 000082564500003

    View details for PubMedID 9889154

  • Chemoselective ligation reactions with proteins, oligosaccharides and cells TRENDS IN BIOTECHNOLOGY Lemieux, G. A., Bertozzi, C. R. 1998; 16 (12): 506-513

    Abstract

    Traditional chemical synthesis does not lend itself to the easy, rapid construction of even moderately sized biomolecules, because it requires elaborate protection schemes. Furthermore, many biological studies would be aided by the ability to assemble biomolecules under physiological conditions. These challenges have motivated the development of chemoselective ligation, the selective covalent coupling of mutually and uniquely reactive functional groups under mild, aqueous conditions. This technique has attracted significant attention recently for the synthesis of biological macromolecules of defined homogeneous composition, the design of self-assembling drugs and the chemical remodeling of cell surfaces.

    View details for Web of Science ID 000077466300003

    View details for PubMedID 9881482

  • Inner space exploration: the chemical biologist's guide to the cell CHEMISTRY & BIOLOGY Winans, K. A., Bertozzi, C. R. 1998; 5 (12): R313-R315

    View details for Web of Science ID 000077383600001

    View details for PubMedID 9862801

  • Metabolic delivery of ketone groups to sialic acid residues - Application to cell surface glycoform engineering JOURNAL OF BIOLOGICAL CHEMISTRY Yarema, K. J., Mahal, L. K., Bruehl, R. E., Rodriguez, E. C., Bertozzi, C. R. 1998; 273 (47): 31168-31179

    Abstract

    The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Using a recently described method (Mahal, L. K., Yarema, K. J., and Bertozzi, C. R. (1997) Science 276, 1125-1128), we delivered a uniquely reactive ketone group to endogenous cell surface sialic acid residues by treating cells with the ketone-bearing metabolic precursor N-levulinoylmannosamine (ManLev). The ketone undergoes highly selective condensation reactions with complementary nucleophiles such as aminooxy and hydrazide groups. The detailed quantitative parameters of ManLev metabolism in human and nonhuman-derived cell lines were determined to establish a foundation for the modification of cell surfaces with novel epitopes at defined cell-surface densities. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. The remodeled cells were endowed with novel lectin binding profiles as determined by flow cytometry analysis. The simplicity and generality of this method make it well suited for use in the study of carbohydrate-mediated cell surface interactions.

    View details for Web of Science ID 000077136900049

    View details for PubMedID 9813021

  • Synthesis of an oxime-linked neoglycopeptide with glycosylation-dependent activity similar to its native counterpart TETRAHEDRON LETTERS Marcaurelle, L. A., Rodriguez, E. C., Bertozzi, C. R. 1998; 39 (46): 8417-8420
  • Direct incorporation of unprotected ketone groups into peptides during solid-phase synthesis: Application to the one-step modification of peptides with two different biophysical probes for FRET TETRAHEDRON LETTERS Marcaurelle, L. A., Bertozzi, C. R. 1998; 39 (40): 7279-7282
  • Identification of an N-acetylglucosamine-6-O-sulfotransferase activity specific to lymphoid tissue: an enzyme with a possible role in lymphocyte homing CHEMISTRY & BIOLOGY Bowman, K. G., Hemmerich, S., Bhakta, S., SINGER, M. S., Bistrup, A., ROSEN, S. D., Bertozzi, C. R. 1998; 5 (8): 447-460

    Abstract

    The leukocyte adhesion molecule L-selection participates in the initial attachment of blood-borne lymphocytes to high endothelial venules (HEVs) during lymphocyte homing to secondary lymphoid organs, and contributes to leukocyte adhesion and extravasation in HEV-like vessels at sites of chronic inflammation. The L-selection ligands on lymph mode HEVs are mucin-like glycoproteins adorned with the unusual sulfated carbohydrate epitope, 6-sulfo sialyl Lewis x. Sulfation of this epitope on the N-acetylglucosamine (GlcNAc) residue confers high-avidity L-selection binding, and is thought to be restricted in the vasculature to sites of sustained lymphocyte recruitment. The GlcNAc-6-0 sulfotransferase that installs the sulfate ester may be a key modulator of lymphocyte recruitment to secondary lymphoid organs and sites of chronic inflammation and is therefore a potential target for anti-inflammatory therapy.A GlcNAc-6-0-sulfotransferase activity was identified within porcine lymph nodes and characterized using a rapid, sensitive, and quantitative assay. We synthesized two unnatural oligosaccharide substrates, GlcNAc beta 1-->6Gal alpha-R and Gal beta 1-->4GlcNAc beta 1-->6Gal alpha-R, that incorporate structural motifs from the native L-selection ligands into an unnatural C-glycosyl hydrocarbon scaffold. The sulfotransferase incorporated greater than tenfold more sulfate into the disaccharide than the trisaccharide, indicating a requirement for a terminal GlcNAc. Activity across tissues was highly restricted to the HEVs within peripheral lymph node.The restricted expression of the GlcNAc-6-0-sulfotransferase activity to lymph node HEVs strongly suggestions a role in the biosynthesis of L-selection ligands. In addition, similar sulfated epitopes are known to be expressed on HEV-like vessels of chronically inflamed tissues; indicating that this sulfotransferase may also contribute to inflammatory lymphocyte recruitment. We identified a concise disaccharide motif, GlcNAc beta 1-->6Gal alpha-R, that preserved both recognition and specificity determinants for the GlcNAc-6-0-sulfotransferase. The absence of activity on the trisaccharide Gal beta 1-->6Gal alpha-R indicates a requirement for a substrate with a terminal GlcNAc residue, suggesting that sulfation precedes further biosynthetic assembly of L-selection ligands.

    View details for Web of Science ID 000075388100009

    View details for PubMedID 9710564

  • Chemical approaches to glycobiology and emerging carbohydrate-based therapeutic agents CURRENT OPINION IN CHEMICAL BIOLOGY Yarema, K. J., Bertozzi, C. R. 1998; 2 (1): 49-61

    Abstract

    The contributions of cell surface oligosaccharides to critical biological processes such as leukocyte-endothelial cell adhesion, bacterial and viral infection, and immunological recognition of tumor cells and foreign tissue are now understood in significant molecular detail. These discoveries at the forefront of biological research have motivated the design of synthetic glycoconjugates as tools for the fundamental study of glycobiology and as candidates for future generations of therapeutic and pharmaceutical reagents. During the past two years, significant progress has been made in the design and synthesis of carbohydrate-based inhibitors of selectins, receptors involved in the attachment of leukocytes to endothelial cells at sites of inflammation. Monomeric and multivalent oligosaccharides that bind to bacterial and viral receptors have been shown to abrogate infection by agents such as Helicobacter pilori, influenza virus and HIV. The identification of certain cell surface oligosaccharides as potent antigens has prompted their use in tumor vaccines, and inspired new approaches to the management of tissue rejection subsequent to xenotransplantation. To better understand how cell surface oligosaccharides function within their native context, novel chemical approaches to modulating cell surface oligosaccharides structures are now being developed. These stratergies for cell surface 'glycoform remodeling' promise to facilitate the investigation of carbohydrate mediated cell-cell interactions.

    View details for Web of Science ID 000072701000007

    View details for PubMedID 9667919

  • A strategy for the chemoselective synthesis of O-linked glycopeptides with native sugar-peptide linkages JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Rodriguez, E. C., Winans, K. A., King, D. S., Bertozzi, C. R. 1997; 119 (41): 9905-9906
  • Engineered cell surfaces: Fertile ground for molecular landscaping CHEMISTRY & BIOLOGY Mahal, L. K., Bertozzi, C. R. 1997; 4 (6): 415-422

    Abstract

    The cell surface contains a wealth of information that determines how cells interact with their environment. Methods for directing the cell surface expression of novel protein-based and oligosaccharide-based epitopes are stimulating new directions in biotechnology and biomedical research.

    View details for Web of Science ID A1997XK27100002

    View details for PubMedID 9224572

  • Engineering chemical reactivity on cell surfaces through oligosaccharide biosynthesis SCIENCE Mahal, L. K., Yarema, K. J., Bertozzi, C. R. 1997; 276 (5315): 1125-1128

    Abstract

    Cell surface oligosaccharides can be engineered to display unusual functional groups for the selective chemical remodeling of cell surfaces. An unnatural derivative of N-acetyl-mannosamine, which has a ketone group, was converted to the corresponding sialic acid and incorporated into cell surface oligosaccharides metabolically, resulting in the cell surface display of ketone groups. The ketone group on the cell surface can then be covalently ligated under physiological conditions with molecules carrying a complementary reactive functional group such as the hydrazide. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The versatility of this technology was demonstrated by an example of selective drug delivery. Cells were decorated with biotin through selective conjugation to ketone groups, and selectively killed in the presence of a ricin A chain-avidin conjugate.

    View details for Web of Science ID A1997WZ22500048

    View details for PubMedID 9173543

  • An ELISA for selectins based on binding to a physiological ligand JOURNAL OF IMMUNOLOGICAL METHODS Bertozzi, C. R., SINGER, M. S., ROSEN, S. D. 1997; 203 (2): 157-165

    Abstract

    Members of the selectin family of adhesion receptors, consisting of L-, P- and E-selectin, mediate the initial interaction between leukocytes and endothelium during leukocyte trafficking from the blood into tissue sites. These receptors have attracted great attention in recent years due to their participation in a number of acute and chronic inflammatory diseases. We describe here a new ELISA that measures the binding between selectin-IgG chimeras and a physiological ligand for L-selectin and can be used to screen selectin inhibitors. The ligand used is a mucin-like glycoprotein known as GlyCAM-1, which is derived from high endothelial venules in secondary lymphoid organs. We demonstrate binding of all three selectins to GlyCAM-1 and demonstrate that the binding interactions satisfy a number of important criteria. The advantage of this ELISA over previous assays is that a macromolecular physiological ligand is employed, rather than a fortuitous or simplified carbohydrate ligand. Thus, the protein-carbohydrate interactions, as well as other interactions contributing to ligand recognition, can be investigated. The assay is suitable for high-throughout screening of compounds and may find use in the identification of selectin antagonists with anti-inflammatory potential.

    View details for Web of Science ID A1997WW72400006

    View details for PubMedID 9149809

  • L-selectin-carbohydrate interactions: Relevant modifications of the Lewis x trisaccharide BIOCHEMISTRY Sanders, W. J., Katsumoto, T. R., Bertozzi, C. R., ROSEN, S. D., Kiessling, L. L. 1996; 35 (47): 14862-14867

    Abstract

    Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. Specifically, three carbohydrate binding proteins termed selectins (E-, P-, and L-selectin) have been shown to be essential for leukocyte rolling along the vascular endothelium, the first step in the recruitment of leukocytes from the blood into inflammatory sites or into secondary lymphoid organs. Although this phenomenon is well-established, little is known about the molecular-level interactions on which it depends. All three selectins recognize sulfated and sialylated derivatives of the Lewis x [Le(x):Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and Lewis a [Le(a): Gal beta 1-->3(Fuc alpha 1-->4)GlcNAc] trisaccharide cores with affinities in the millimolar range, and it is believed that variants of these structures are the carbohydrate determinants of selectin recognition. Recently it was shown that the mucin GlyCAM-1, a secreted physiological ligand for L-selectin, is capped with sulfated derivatives of sialyl Lewis x [sLe(x): Sia alpha 2-->3Gal beta 1-->4(Fuc alpha 1-->3)GlcNAc] and that sulfation is required for the high-affinity interaction between GlyCAM-1 and L-selectin. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Our results suggest that 6-sulfo sLe(x) binds to L-selectin with higher affinity than does sLe(x) or 6'-sulfo sLe(x) and that sulfation of sLe(x) capping groups on GlyCAM-1 at the 6-position is important for L-selectin recognition.

    View details for Web of Science ID A1996VV23700024

    View details for PubMedID 8942649

  • Synthesis of beta-C-glycosides of N-acetylglucosamine via Keck allylation directed by neighboring phthalimide groups JOURNAL OF ORGANIC CHEMISTRY Roe, B. A., Boojamra, C. G., Griggs, J. L., Bertozzi, C. R. 1996; 61 (18): 6442-6445
  • Tin-mediated phosphorylation: Synthesis and selectin binding of a phospho Lewis a analog TETRAHEDRON LETTERS Manning, D. D., Bertozzi, C. R., ROSEN, S. D., Kiessling, L. L. 1996; 37 (12): 1953-1956
  • C-glycosyl aldehydes: Synthons for C-linked disaccharides JOURNAL OF ORGANIC CHEMISTRY Kobertz, W. R., Bertozzi, C. R., Bednarski, M. D. 1996; 61 (5): 1894-1897
  • Leukocyte adhesion - Two selectins converge on sulphate CURRENT BIOLOGY ROSEN, S. D., Bertozzi, C. R. 1996; 6 (3): 261-?
  • SULFATED DISACCHARIDE INHIBITORS OF L-SELECTIN - DERIVING STRUCTURAL LEADS FROM A PHYSIOLOGICAL SELECTIN LIGAND BIOCHEMISTRY Bertozzi, C. R., Fukuda, S., ROSEN, S. D. 1995; 34 (44): 14271-14278

    Abstract

    The selectins are a family of three adhesion molecules (L-, P-, and E-) that direct the interaction of circulating leukocytes with endothelial cells during the first step in recruitment to tissue sites. Their involvement in inflammatory disease makes the selectins attractive targets for anti-inflammatory therapy. The sialyl Lewis x tetrasaccharide binds weakly to all three selectins and has demonstrated anti-inflammatory activity in vivo. However, the synthetic difficulties inherent to sialylated and fucosylated oligosaccharides motivate the search for alternative antagonists. Here we demonstrate that information gained from the biochemical analysis of a physiological selectin ligand can provide new leads for small molecule design. Previous structural analysis of the oligosaccharide chains on GlyCAM-1, an endothelial-derived ligand for L-selectin, revealed two novel structures: 6'-sulfo sialyl Lewis x and 6-sulfo sialyl Lewis x. The sulfate esters on these structures are thought to be essential for high-affinity binding to L-selectin. By incorporating sulfate esters on the analogous positions of the disaccharide lactose, we generated a simple small molecule (lactose 6',6-disulfate) with greater inhibitory potency for L-selectin than sialyl Lewis x.

    View details for Web of Science ID A1995TD84500001

    View details for PubMedID 7578028

  • CRACKING THE CARBOHYDRATE CODE FOR SELECTIN RECOGNITION CHEMISTRY & BIOLOGY Bertozzi, C. R. 1995; 2 (11): 703-708

    Abstract

    Selectins are central in the inflammatory response; the discovery that they bind to carbohydrate ligands has galvanized carbohydrate chemists to search for inhibitors of the process. Recent progress in identifying and analyzing physiological selectin counter-receptors suggests new approaches to the design of ligands that bind to specific selectins.

    View details for Web of Science ID A1995TG85900001

    View details for PubMedID 9383476

  • THE SELECTINS AND THEIR LIGANDS CURRENT OPINION IN CELL BIOLOGY ROSEN, S. D., Bertozzi, C. R. 1994; 6 (5): 663-673

    Abstract

    The selectins are a family of carbohydrate-binding proteins, or lectins, that have stimulated tremendous interest because of their involvement in a wide array of interactions between leukocytes and endothelial cells. Highlights of recent progress include an extension of the list of instances of selectin participation in inflammatory diseases, further definition of selectin carbohydrate specificities, and identification of their carbohydrate-based ligands.

    View details for Web of Science ID A1994PH46500004

    View details for PubMedID 7530461

  • IDENTIFICATION OF THE SULFATED MONOSACCHARIDES OF GLYCAM-1, AN ENDOTHELIAL-DERIVED LIGAND FOR L-SELECTIN BIOCHEMISTRY Hemmerich, S., Bertozzi, C. R., Leffler, H., ROSEN, S. D. 1994; 33 (16): 4820-4829

    Abstract

    L-Selectin, a receptor bearing a C-type lectin domain, mediates the initial attachment of lymphocytes to high endothelial venules of lymph nodes. One of the endothelial-derived ligands for L-selectin is GlyCAM-1 (previously known as Sgp50), a mucin-like glycoprotein with sulfated, sialylated, and fucosylated O-linked oligosaccharide chains. Sialylation, sulfation, and fucosylation appear to be required for the avid interaction of this ligand with L-selectin, but the exact carbohydrate structures involved in recognition remain undefined. In this study, we examine the nature of the sulfate-modified carbohydrates of GlyCAM-1. GlyCAM-1 was metabolically labeled in lymph node organ culture with 35SO4 and a panel of tritiated carbohydrate precursors. Mild hydrolysis conditions were established that released sulfated oligosaccharides without cleavage of sulfate esters. Low molecular weight and singly charged fragments, obtained by a combination of gel filtration and anion-exchange chromatography, were analyzed. The structural identification of the fragments relied on the use of a variety of radiolabeled sugar precursors, further chemical and enzymatic hydrolysis, and high-pH anion-exchange chromatography analysis. Sulfated constituents of GlyCAM-1 were identified as Gal-6-SO4, GlcNAc-6-SO4, (SO4-6)Gal beta 1-->4GlcNAc, and Gal beta 1-->4(SO4-6)GlcNAc. In the accompanying paper [Hemmerich, S., & Rosen, S.D. (1994) Biochemistry 33, 4830-4835] evidence is presented that (SO4-6)Gal beta 1-->4GlcNAc forms the core of a sulfated sialyl Lewis x structure that may comprise a recognition determinant on GlyCAM-1.

    View details for Web of Science ID A1994NH63300010

    View details for PubMedID 7512827

  • FLUORESCENCE PROBES IN BIOCHEMISTRY - AN EXAMINATION OF THE NONFLUORESCENT BEHAVIOR OF DANSYLAMIDE BY PHOTOACOUSTIC CALORIMETRY ANALYTICAL BIOCHEMISTRY GRABOWSKI, J. J., Bertozzi, C. R., Jacobsen, J. R., Jain, A., Marzluff, E. M., Suh, A. Y. 1992; 207 (2): 214-226

    Abstract

    Photoacoustic calorimetry is shown to be a simple, precise, and accurate method for the quantification of the photophysics of a fluorescence probe, e.g., dansylamide, in a variety of solvents. This technique, which is described in detail, provides a direct measurement of the energy that is released nonradiatively following photostimulation, and can therefore be used to indirectly determine the amount of energy released via luminescent pathways. Photoacoustic calorimetry combined with established absorption and fluorescence methodologies provides a complete arsenal for characterizing the photophysical properties of many systems. Comparison of the photoacoustic signal for dansylamide versus standard compounds (ferrocene, tetraphenylethylene, 8-anilinonaphthalene-1-sulfonate, and/or 5,5'-dithiobis(2-nitrobenzoic acid) in 12 different solvents gave fh values (fraction of each absorbed 337.1-nm photon returned as heat) from a low of 0.530 in 1,4-dioxane to a high of 0.973 in water. The trend noted with solvent polarity is different and more revealing than that determined by the more classical approach of examining either the wavelength of the emission maximum or the fluorescence quantum yield.

    View details for Web of Science ID A1992KF46900003

    View details for PubMedID 1481973

  • A RECEPTOR-MEDIATED IMMUNE-RESPONSE USING SYNTHETIC GLYCOCONJUGATES JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bertozzi, C. R., Bednarski, M. D. 1992; 114 (14): 5543-5546
  • THE SYNTHESIS OF 2-AZIDO C-GLYCOSYL SUGARS TETRAHEDRON LETTERS Bertozzi, C. R., Bednarski, M. D. 1992; 33 (22): 3109-3112
  • ANTIBODY TARGETING TO BACTERIAL-CELLS USING RECEPTOR-SPECIFIC LIGANDS JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Bertozzi, C. R., Bednarski, M. D. 1992; 114 (6): 2242-2245
  • AN EFFICIENT METHOD FOR THE SYNTHESIS OF ALPHA-C-GLYCOSYL AND BETA-C-GLYCOSYL ALDEHYDES TETRAHEDRON LETTERS Kobertz, W. R., Bertozzi, C. R., Bednarski, M. D. 1992; 33 (6): 737-740
  • THE SYNTHESIS OF HETEROBIFUNCTIONAL LINKERS FOR THE CONJUGATION OF LIGANDS TO MOLECULAR PROBES JOURNAL OF ORGANIC CHEMISTRY Bertozzi, C. R., Bednarski, M. D. 1991; 56 (13): 4326-4329
  • COADSORPTION OF FERROCENE-TERMINATED AND UNSUBSTITUTED ALKANETHIOLS ON GOLD - ELECTROACTIVE SELF-ASSEMBLED MONOLAYERS JOURNAL OF THE AMERICAN CHEMICAL SOCIETY Chidsey, C. E., Bertozzi, C. R., PUTVINSKI, T. M., Mujsce, A. M. 1990; 112 (11): 4301-4306

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