MS of material collected on the tissue paper blots revealed 67 GalNAz-labeled proteins, including intracellular proteins. WebCarolyn Bertozzi (1966-ngin 10-ngiet 10-ngit ) he M-koet ke yit-chak fa-hok-k. View details for Web of Science ID 000227627800041. The loss of SL-1 (and SL(1278)) did not appear to affect bacterial replication or trafficking, suggesting that the functions of SL-1 are specific to human infection. Despite decades of accumulated knowledge about proteins and their post-translational modifications (PTMs), numerous questions remain regarding their molecular composition and biological function. [19] The discovery led to her field of research, glycobiology. Finally, we demonstrate that the cell-surface Staudinger ligation is compatible with hydrazone formation from metabolically introduced ketones. View details for DOI 10.1073/pnas.1608069113. Here, we compared these chemistries in the context of various biological applications, including labeling of biomolecules in complex lysates and on live cell surfaces. This enzyme catalyzes the reduction of APS to sulfite and AMP with reducing equivalents from the protein cofactor, thioredoxin. The efficacy of antimicrobial drugs against Mycobacterium tuberculosis, an intracellular bacterial pathogen, is generally first established by testing compounds against bacteria in axenic culture. In some cases, the functional significance of disease-associated changes in glycosylation has been revealed. 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. 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. Here we describe an efficient dot-blot assay for high-throughput screening of two enzymes, heparan sulfate N-deacetylase/N-sulfotransferase (NDST-1) and high-endothelial cell GlcNAc-6-sulfotransferase (HEC-GlcNAc-6-ST). Li phin vn-chng si to pan-lan-chhan , ng cho-tet yung phin-si fet-ch khok-chhng kh ke nui-yng. Her biography is available in 16 different languages on Wikipedia. With their native proteinaceous backbones and natural glycosidic linkages, these agonists are attractive for translational applications. The profile, which comprised 546 proteins, suggests diverse functions of the phagosome and potential connections to secretory processes, toll-like receptor signaling, and autophagy. We show that this probe faithfully recapitulates cellular fatty acid uptake and can be used in animal systems as a valuable tool to localize and quantitate in real time lipid fluxes such as intestinal fatty acid absorption and brown adipose tissue activation. View details for Web of Science ID 000355248100027, View details for DOI 10.1021/acscentsci.5b00185, View details for PubMedCentralID PMC4827500. Glycosylation is a prevalent, yet heterogeneous modification with a broad range of implications in molecular biology. These alkenyl compounds arise from a hydrogen transfer reaction from solvent to the unisolable intermediate, and we postulate that the reaction proceeds via a radical mechanism originating from the strained alkynyl bond that has unusually high radical character. 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 See vol 283, pg 4460, 2008). Thus, NGLY1 inhibition prevents Nrf1 activation and represents a new therapeutic approach for cancers that depend on proteasome homeostasis. Czlapinski, J. L., Schelle, M. W., Miller, L. W., Laughlin, S. T., Kohler, J. J., Cornish, V. W., Bertozzi, C. R. Structural Characterization of a Novel Sulfated Menaquinone produced by stf3 from Mycobacterium tuberculosis. Cell surface lipooligosaccharides (LOS) of H. ducreyi are thought to play important biological roles in host infection. New developments have allowed researchers to begin probing the inner workings of the cell to gain new insight into cell function and metabolism. Sulfomenaquinone (SMK) is a recently identified metabolite that is unique to the Mycobacterium tuberculosis (M. tuberculosis) complex and is shown to modulate its virulence. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Beatty, K. E., Williams, M., Carlson, B. L., Swarts, B. M., Warren, R. M., van Helden, P. D., Bertozzi, C. R. Synthesis and Reactivity of Dibenzoselenacycloheptynes. 2016 by John Wiley & Sons, Inc. View details for DOI 10.1002/9780470559277.ch150185, View details for PubMedCentralID PMC4809415. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Lin, F. L., Hoyt, H. M., van Halbeek, H., Bergman, R. G., Bertozzi, C. R. Synthetic glycopeptides and glycoproteins as tools for biology, Functional hydrogel-biomineral composites inspired by natural bone, Azido sialic acids can modulate cell-surface interactions, A small-molecule switch for Golgi sulfotransferases. The development of rapid screening methods for probing glycosyltransferase activities is essential for advancing the field of glycobiology. View details for DOI 10.1016/j.chembiol.2011.12.016, View details for Web of Science ID 000300917300009, View details for PubMedCentralID PMC3292763. Here, we systematically explore the advantages and disadvantages of conventional HCD, sceHCD, ETD, and EThcD for intact glycopeptide analysis and determine their suitability for both N- and O-glycoproteomic applications. In addition, GalNAz efficiently labeled mucin-type O-linked glycoproteins expressed at endogenous levels. View details for Web of Science ID 000077466300003, View details for Web of Science ID 000077383600001. Atoms out of Blobs: CryoEM Takes the Nobel Prize in Chemistry. Mycobacterium tuberculosis ( Mtb) produces a number of sulfur-containing metabolites that contribute to its pathogenesis and ability to survive in the host. This work provides a method to study the biosynthesis of fucosylated glycans in vivo. Scherman, M. S., Winans, K. A., Stern, R. J., Jones, V., Bertozzi, C. R., McNeil, M. R. Preparation of pHEMA-CP composites with high interfacial adhesion via template-driven mineralization, Characterization and mutagenesis of Gal/GlcNAc-6-O-sulfotransferases. She coined the term "bioorthogonal chemistry"[2] for chemical reactions compatible with living systems. 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. Antibodies are widely used biomarkers for the diagnosis of many diseases. A cell metabolic labeling experiment can be completed in approximately 4 d. View details for DOI 10.1038/nprot.2007.422, View details for Web of Science ID 000253140000033. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. 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. View details for Web of Science ID 000304837800041. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. Cell surface sialosides constitute a central axis of immune modulation that is exploited by tumors to evade both innate and adaptive immune destruction. BT4244E575A derived from Bacteroides thetaiotaomicron is selective for truncated, asialylated core 1 structures commonly associated with malignant and premalignant tissues. ppGalNAc T1 and T2 revealed no significant enhancements suggesting Ser/Thr-O-GalNAc was inhibitory at most positions for these isoforms. A. Shieh, P., Dien, V. T., Beahm, B. J., Castellano, J. M., Wyss-Coray, T., Bertozzi, C. R. Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. 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. The biophysical properties of the system are characterized, and the technique is used to form complex cellular patterns with single-cell line widths and self-assembled cellular microarrays. Physical and Materials Sciences, Energy and the Environment, Shadow Program (for High School students). Rv3406 was identified based on its homology to the alkyl sulfatase AtsK from Pseudomonas putida. Jewett, J. C., Sletten, E. M., Bertozzi, C. R. Targeted metabolic labeling of yeast N-glycans with unnatural sugars. Symbol Nomenclature for Graphical Representations of Glycans. This gene was mapped to mouse chromosome X at band XA3.1-3.2. A key tool we developed for this study is a cell-permeable, small molecule inhibitor of GlcNAc 2-epimerase designed based on mechanistic principles. Furthermore, the study of SL-1 has led to questions regarding the significance of sulfation in mycobacteria. This protocol details the syntheses of the azido sugars N-azidoacetylmannosamine (ManNAz), N-azidoacetylgalactosamine (GalNAz), N-azidoacetylglucosamine (GlcNAz) and 6-azidofucose (6AzFuc), and the detection reagents phosphine-FLAG and phosphine-FLAG-His6. Thus, we hypothesize that the species-specific phenotype of the stf0 mutant is reflective of differences in antimycobacterial effector mechanisms of macrophages. Here we develop a combined genetic and chemical approach that enables introduction of the diazirine photocrosslinker onto the O-GlcNAc modification in cells. Andres, L. M., Blong, I. W., Evans, A. C., Rumachik, N. G., Yamaguchi, T., Pham, N. D., Thompson, P., Kohler, J. J., Bertozzi, C. R. Bioorthogonal Labeling of Human Prostate Cancer Tissue Slice Cultures for Glycoproteomics. Malaker, S. A., Quanico, J., Raffo-Romero, A., Kobeissy, F., Aboulouard, S., Tierny, D., Bertozzi, C. R., Fournier, I., Salzet, M. The CD22-IGF2R interaction is a therapeutic target for microglial lysosome dysfunction in Niemann-Pick type C. Pluvinage, J. V., Sun, J., Claes, C., Flynn, R. A., Haney, M. S., Iram, T., Meng, X., Lindemann, R., Riley, N. M., Danhash, E., Chadarevian, J. P., Tapp, E., Gate, D., Kondapavulur, S., Cobos, I., Chetty, S., Paca, A. M., Paca, S. P., Berry-Kravis, E., Bertozzi, C. R., Blurton-Jones, M., Wyss-Coray, T. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. Here we present an ultrasensitive solution-phase method for detecting antibodies, termed antibody detection by agglutination-PCR (ADAP). We identify and characterize C18ORF8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Selective chemical reactions that are orthogonal to the diverse functionality of biological systems have become important tools in the field of chemical biology. This approach bestows a gain-of-chemical-functionality modification on cells, where the products of individual glycosyltransferases can be selectively characterized or manipulated to understand glycan contribution to major physiological processes. A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of blomolecules in living systems. Proof of principle was performed by using various heparin/HS samples isolated from bovine and porcine tissues. Studying posttranslational modifications classically relies on experimental strategies that oversimplify the complex biosynthetic machineries of living cells. View details for Web of Science ID 000257257100003, View details for PubMedCentralID PMC2663890. View details for DOI 10.1096/fj.201601198R, View details for Web of Science ID 000401553400015, View details for PubMedCentralID PMC5434651, View details for DOI 10.1021/acscentsci.7b00204, View details for PubMedCentralID PMC5445543. The identification of certain cell surface oligosaccharides as potent antigens has prompted their use in tumor vaccines, and inspired new approaches to the management of tissue rejection subsequent to xenotransplantation. Imbert, P. R., Saric, A. n., Pedram, K. n., Bertozzi, C. R., Grinstein, S. n., Freeman, S. A. Electron-Based Dissociation Is Needed for O-Glycopeptides Derived from OpeRATOR Proteolysis. These findings provide a platform for defining the molecular basis of SMK's role in M. tuberculosis pathogenesis. Fusion of these domains to the proteins FRB and FKBP enabled their induced assembly by the natural product rapamycin. View details for Web of Science ID 000256043200016, View details for PubMedCentralID PMC2711008. In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Williams, S. J., Senaratne, R. H., Mougous, J. D., Riley, L. W., Bertozzi, C. R. A 96-well dot-blot assay for carbohydrate sulfotransferases, Sulfotransferases and sulfatases in mycobacteria. The concepts outlined herein lay a foundation for future development of peptide tags in the context of site-selective modification of lysine residues within engineered microenvironments. This work furnishes new tools for mammalian residue-specific bioorthogonal chemistry, and enables more robust and comprehensive cell-type-specific proteomics in live mammals. The challenge of engineering glycosylation has been confronted by synthetic chemists, biochemists and cell biologists, each with the primary goal of optimizing glycoconjugates for therapeutic applications. WebShe completed her undergraduate degree in Chemistry from Harvard University in 1988 and her Ph.D. in Chemistry from UC Berkeley in 1993. View details for DOI 10.1074/jbc.M111619200, View details for Web of Science ID 000174400600071. Kohler, J. J., Czlapinski, J. L., Laughlin, S. T., Schelle, M. W., de Graffenried, C. L., Bertozzi, C. R. Metabolic functionalization of recombinant glycoproteins. Consistent with this view, increased levels of MMCoA led to increased abundance and mass of both PDIM and SL-1. This position statement originated from a working group meeting convened on April 15, 2015, by the NHLBI and incorporates follow-up contributions by the participants as well as other thought leaders subsequently consulted, who together represent research fields relevant to all branches of the NIH. Saxon, E., Luchansky, S. J., Hang, H. C., Yu, C., Lee, S. C., Bertozzi, C. R. Carbohydrate sulfotransferases of the GalNAc/Gal/GlcNAc6ST family, Kinetic analysis of NodST sulfotransferase using an electrospray ionization mass spectrometry assay. 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. 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). 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