This review presents an overview of techniques for examining and manipulating cell surface oligosaccharides through genetic, enzymatic, and chemical strategies. Treatment of cells with the compounds abrogated mucin-type O-linked glycosylation but not N-linked glycosylation and also induced apoptosis. Fucosylated glycoproteins are involved in many cell-cell recognition events and are markers of embryonic and malignant tissue. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. Here, we report a novel mass spectrometry-based strategy for detection of N-glycosites in the yeast proteome. Site-selective protein modification based on covalent reactions of peptide tags and small molecules is a key capability for basic research as well as for the development of new therapeutic bioconjugates. The chemical reporter is then covalently modified in a highly selective fashion with an exogenously delivered probe. Carolyn Bertozzi is the Anne T. and Robert M. Bass Professor of Chemistry and Professor of Chemical & Systems Biology and Radiology (by courtesy) at Stanford University, the Baker Family Director at Sarafan ChEM-H, and an Investigator of the Howard Hughes Medical Institute. View details for DOI 10.1016/j.jprot.2016.04.009, View details for DOI 10.1021/acscentsci.6b00194, View details for PubMedCentralID PMC4965850, View details for DOI 10.1021/acscentsci.6b00167, View details for PubMedCentralID PMC4919776. View details for DOI 10.1021/acs.jproteome.6b01053. To determine whether PapA3 participates in PAT assembly, we expressed the protein heterologously and evaluated its acyltransferase activity in vitro. 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. Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. WebCarolyn Bertozzi is the Baker Family Director of Sarafan ChEM-H, the Anne T. and Robert M. Bass Professor of Chemistry, and a professor of chemical and systems biology and of A library of potential bisubstrate analogue inhibitors (1) targeting sulfotransferase enzymes was generated by the chemoselective ligation of the PAPS mimic 2 with a panel of 447 aldehydes. Here we studied the function of novel germline variants in CSF3R at amino acid N610. A combination of PZA and the {Au(PPh3)}-thiol polymeric species appears to lead to enhanced efficacy of 1 against M. tuberculosis. Here we applied the bioorthogonal chemical reporter technique for the molecular imaging of mucin-type O-glycans in live C. elegans. Identification of new components of the RipC-FtsEX cell separation pathway of Corynebacterineae. View details for DOI 10.1074/jbc.M313103200, View details for Web of Science ID 000222445300003. Zhou, X., Rodriguez-Rivera, F. P., Lim, H., Bell, J. C., Bernhardt, T. G., Bertozzi, C. R., Theriot, J. Ketones within the glycoconjugates on ManLev-treated cells were then reacted with synthetic aminooxy and hydrazide-functionalized carbohydrates. We were able to bypass the salvage pathway by using an azide-functionalized analogue of GDP-fucose. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). For N-glycopeptides, HCD and sceHCD generate similar numbers of identifications, although sceHCD generally provides higher quality spectra. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. 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. 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). View details for DOI 10.1016/j.jmb.2006.08.080, View details for Web of Science ID 000242160600003, View details for PubMedCentralID PMC1769331. View details for Web of Science ID 000223369800037. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. Finally, we showed in a mouse model of infection that the loss of cyp128 exhibits a hypervirulent phenotype similar to that in previous studies of the stf3 mutant. Marcaurelle, L. A., Pratt, M. R., Bertozzi, C. R. A new approach to mineralization of biocompatible hydrogel scaffolds: An efficient process toward 3-dimensional bonelike composites, Synthesis of a bisubstrate analogue targeting estrogen sulfotransferase. Both reactive partners are abiotic and chemically orthogonal to native cellular components. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. View details for DOI 10.1021/jacs.9b04695, View details for Web of Science ID 000484082700023. Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. Most clinically approved biomarkers of cancer are glycoproteins, and those residing on the cell surface are of particular interest in biotherapeutics. View details for DOI 10.1021/jacs.6b12541, View details for PubMedCentralID PMC5345120. These studies establish Cu-free click chemistry as a bioorthogonal reaction that can be executed in the physiologically relevant context of a mouse. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). Post-translational modifications (PTMs) on proteins often function to regulate signaling cascades, with the activation of T cells during an adaptive immune response being a classic example. View details for Web of Science ID 000314794400031, View details for PubMedCentralID PMC3583381. 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. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. She grew up in Lexington, Massachusetts with two sisters, one of which is on the mathematics faculty at the University of California, Los Angeles (UCLA). View details for DOI 10.1021/jacs.8b03074, View details for Web of Science ID 000435525500001, View details for DOI 10.3389/fmicb.2018.01117, View details for Web of Science ID 000433326300001. View details for Web of Science ID 000082757300015. 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. Biosynthesis of L-selectin ligands: Sulfation of sialyl Lewis x-related oligosaccharides by a family of GlcNAc-6-sulfotransferases. The conjugation proceeds under mild conditions with excellent ligation efficiencies and in a stereoselective manner, providing glycopolymers with pendant glycans accommodated mostly in their cyclic beta-glycosidic form. These synthetic targets incorporate a selectively protected serine residue at the reducing terminus, providing a functional handle for further conjugation. Grunwell, J. R., Rath, V. L., Rasmussen, J., Cabrilo, Z., Bertozzi, C. R. Discovery of sulfated metabolites in mycobacteria with a genetic and mass spectrometric approach. After completing postdoctoral work at UCSF in the field of cellular immunology, she joined the UC Berkeley faculty in 1996. This type of enzyme catalyzes the initial step of mucin-type O-glycosylation, that is, the transfer of GalNAc in O-glycosidic linkage to serine and threonine residues in polypeptides. One of the most fundamental queries is the extent to which the combinations of DNA-, RNA- and PTM-level variations explode the complexity of the human proteome. Bertozzi, C. R., Chang, C. J., Davis, B. G., Olvera de la Cruz, M., Tirrell, D. A., Zhao, D. Integrins Form an Expanding Diffusional Barrier that Coordinates Phagocytosis. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. View details for Web of Science ID 000260193100005, View details for PubMedCentralID PMC2680727. 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. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. However, current bioaerosol sampling approaches have reported low detection yields in sputum-positive TB cases. A., Bertozzi, C. R. Site-specific chemical protein conjugation using genetically encoded aldehyde tags. Grossman, H. L., Myers, W. R., Vreeland, V. J., Bruehl, R., Alper, M. D., Bertozzi, C. R., Clarke, J. Modular assembly of glycoproteins: Towards the synthesis of GlyCAM-1 by using expressed protein ligation, A strategy for functional proteomic analysis of glycosidase activity from cell lysates, Biomimetic engineering of carbon nanotubes by using cell surface mucin mimics. Here, we describe the computation-guided rational design of a cysteine- and lysine-containing 11-residue peptide sequence that reacts with 2-cyanobenzothiazole (CBT) derivatives. They are difficult to study because of the complex interplay of 20 distinct glycosyltransferase isoenzymes that initiate this form of glycosylation, the polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts). 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. Her father, William Bertozzi, was a physics professor at MIT. Thus, subtle structural differences in PSA resulting from the incorporation of SiaProp residues do not alter the antiadhesive properties of polysialylated NCAM. In cells, GlcNAc6ST-1 exists as a dimer; two cysteine residues within the stem and transmembrane domain were found to be critical for dimerization. Nuclear translocation of Gal-1, in turn, was regulated by discrete cell-surface glycans restricted to the front of the mammary end buds. Using this approach quantities of homogeneous material were obtained for structural and functional analysis. In an emerging strategy, glycans are imaged by metabolic labeling with chemical reporters and subsequent ligation to fluorescent probes. The Staudinger ligation takes advantage of the electrophilicity of the azide; however, the azide can also participate in cycloaddition reactions. WebBertozzi is trying to drag sugars into the light. This work provides a method to study the biosynthesis of fucosylated glycans in vivo. A., Bertozzi, C. R. Investigating cellular metabolism of synthetic azidosugars with the Staudinger ligation. View details for DOI 10.1016/j.cub.2020.09.082. We report a method for selective labeling, affinity enrichment, and identification of cell-surface glycoproteins. A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). View details for DOI 10.1021/jacs.0c07700. View details for Web of Science ID 000182959600044. Metabolic labeling with GalNAz followed by Staudinger ligation provides a means for proteomic analysis of this posttranslational modification and for identifying O-linked glycoprotein fingerprints associated with disease. Our results indicate that, unlike UDP-GlcNAc 2-epimerase, which promotes biosynthesis of sialic acid, GlcNAc 2-epimerase can serve a catabolic role, diverting metabolic flux away from the sialic acid pathway. Sogi, K. M., Holsclaw, C. M., Fragiadakis, G. K., Nomura, D. K., Leary, J. 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 mucin granule lumen consists of a matrix meshwork embedded in a highly selective fashion with an exogenously probe., although sceHCD generally provides higher quality spectra applied the bioorthogonal chemical reporter is then covalently in., providing a functional handle for further conjugation glycome in living organisms describe the computation-guided rational design a! Orthogonal to native cellular components father, William Bertozzi, C. M., Fragiadakis, G. K. Nomura... 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