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)


2019-20 Courses

Stanford Advisees


All Publications

  • 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


    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-+
  • 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


    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-+


    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 inávivo by eliciting granuloma vascularization and suggest potential approaches for host-directed therapies.

    View details for PubMedID 30308157

  • 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


    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

  • 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


    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

  • 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


    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

  • 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

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


    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 inávivo 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 inávivo.

    View details for DOI 10.1016/j.chembiol.2015.07.009

    View details for PubMedID 26256477

    View details for PubMedCentralID PMC4567249

  • 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


    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-?


    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

  • 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-?


    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


    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


    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


    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


    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 aáfunctional 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

  • 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)


    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 ┼. 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

  • 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


    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

  • 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


    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

  • 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


    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


    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


    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

  • 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)


    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

  • 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


    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

  • 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


    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


    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

  • 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


    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


    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


    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

  • 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


    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

  • 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


    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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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-?


    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


    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

  • 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


    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


    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

  • Copper-Free Click Chemistry: Bioorthogonal Reagents for Tagging Azides ALDRICHIMICA ACTA Baskin, J. M., Bertozzi, C. R. 2010; 43 (1): 15-23
  • In Vivo Imaging of Caenorhabditis elegans Glycans ACS CHEMICAL BIOLOGY Laughlin, S. T., Bertozzi, C. R. 2009; 4 (12): 1068-1072


    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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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


    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


    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


    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

  • 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

  • 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


    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


    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


    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

  • 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


    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

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


    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


    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


    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


    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-GuÚrin (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

  • 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


    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

  • 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


    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


    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

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


    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

  • 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


    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


    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

  • 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


    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

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


    [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

  • 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

  • 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


    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

  • 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


    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

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


    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

  • 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


    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

  • Synthetic glycopeptides and glycoproteins as tools for biology CHEMICAL SOCIETY REVIEWS Pratt, M. R., Bertozzi, C. R. 2005; 34 (1): 58-68


    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

  • 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


    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

  • 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


    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


    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

  • 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


    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

  • 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

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


    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

  • 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


    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


    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


    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 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


    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

  • 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
  • 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
  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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

  • 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


    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


    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

  • 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


    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


    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

  • 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


    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

  • 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


    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
  • 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


    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

  • 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


    [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

  • 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

  • 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

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