This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. Surprisingly, 6', 6-disulfolactose was poorly recognized by MECA-79, a carbohydrate- and sulfate-dependent monoclonal antibody that binds competitively to L-selectin ligands. Chen, X., Lee, G. S., Zettl, A., Bertozzi, C. R. Functional glass slides for in vitro evaluation of interactions between osteosarcoma TE85 cells and mineral-binding ligands. Hur, G. H., Meier, J. L., Baskin, J., Codelli, J. Utilizing a biotin-terminated PAH scaffold prepared via RAFT polymerization, we quickly assembled a panel of glycopolymers that we microarrayed on streptavidin-coated glass. Human and Baculovirus-Insect Manufacturing Platforms Generate Chemically and Functionally Distinct AAV Vectors with Sexually Dimorphic Liver Transduction. IsoTaG is therefore an effective platform for identification of intact glycopeptides labeled by alkynyl or azido sugars and will facilitate further studies of the glycoproteome. However, the complexities of glycan biosynthesis prevent the installation of nonnatural moieties at defined, predictable locations within glycoproteins at high levels of incorporation. and Kirk Schanze and Lee, {T. Randall} and Lutkenhaus, {Jodie L.} and David Kaplan and Christopher Jones and Carolyn Bertozzi and Laura Kiessling and Mulcahy, {Mary Beth} and Lindsley, {Craig W.} and Finn, {M. G.} and Blum, {Joel D.} and Prashant Kamat and A chemical transformation that permits the selective formation of covalent adducts among richly functionalized biopolymers within a cellular context is presented. No BPAS is found inside the cells. These challenges have motivated the development of chemoselective ligation, the selective covalent coupling of mutually and uniquely reactive functional groups under mild, aqueous conditions. A significant proportion (>45%) of the identified O-GlcNAc sites lie in close proximity to or coincide with a known phosphorylation site, supporting the potential for PTM crosstalk. Stowell, C. L., Barvian, K. K., Young, P. C., Bigsby, R. M., Verdugo, D. E., Bertozzi, C. R., Widlanski, T. S. Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acids. Kohler, J. J., Czlapinski, J. L., Laughlin, S. T., Schelle, M. W., de Graffenried, C. L., Bertozzi, C. R. Metabolic functionalization of recombinant glycoproteins. The tremendous selectivity of the transformation should permit its execution within a cell's interior, offering new possibilities for probing intracellular interactions. Here, we use methods that combine collision- and electron-based fragmentation to characterize the number of O-glycosites that are present in O-glycopeptides derived from the OpeRATOR digestion of four known O-glycoproteins. A preliminary study of the mechanism of this reaction, and refined conditions for its in vivo execution, are reported. 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. Within mammary epithelia, Gal-1 localized within nuclear Gemini bodies and drove epithelial invasiveness. View details for DOI 10.1002/cbic.200600224, View details for Web of Science ID 000241392400005. GlcNAc6ST-2 is a member of a family of related enzymes that act on similar carbohydrate substrates in vitro but discrete glycoproteins in vivo. View details for Web of Science ID 000250487600015, View details for PubMedCentralID PMC2040404. Enlightened by our proteomic data, we performed further experiments to show that only the LAM from M. tuberculosis inhibits accumulation of autophagic vacuoles in the macrophage, suggesting a new function for this virulence-associated lipid. 364 Lomita Drive Inactivation of the conserved steAB genes (cgp_1603-1604) was also found to confer EMB hypersensitivity and cell division defects. CholA-anchored glycopolymers therefore expand the application of glycocalyx engineering in glycobiology. View details for DOI 10.1021/acsinfecdis.6b00106, View details for Web of Science ID 000388161300007. 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. Presented herein is the synthesis and evaluation of a bisubstrate analogue designed to inhibit estrogen sulfotransferase. Here we present the first synthetic trehalose glycolipids capable of providing desiccation protection to membranes of which they are constituents. Inhibitors of carbohydrate recognition and biosynthesis can reveal the biological functions of the carbohydrate epitope and its cognate receptors. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Two approaches that emphasize developing selective methods to dissect, modify, and control receptor-ligand interactions at the cellular interface are discussed. Our method imparts N-glycopeptide mass envelopes with a pattern that is computationally distinguishable from background ions. Neutralization of acidic organelles directly with ammonium chloride or indirectly with bafilomycin A1 partially abrogated the growth restriction of these drugs. 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. [3] At Stanford University, she holds the Anne T. and Robert M. Bass Professorship in the School of Humanities and Sciences. Surprisingly, the dibenzoselenacycloheptynes also abstracted hydrogen atoms from solvents such as THF or toluene, forming dibenzoselenacycloheptene products. The introduction of chemically unique groups into proteins by means of non-natural amino acids has numerous applications in protein engineering and functional studies. By mutating catalytic residues of two such enzymes, we engineered mucin-selective binding agents with retained glycoform preferences. Importantly, we show that mmpL8 mutants are attenuated for growth in a mouse model of tuberculosis. Metastasis depends upon cancer cell growth and survival within the metastatic niche. Sequestration of peptides derived from an Escherichia coli proteome, pulse labeled with the bio-orthogonal amino acid azidohomoalanine as substitute for methionine, allows identification of numerous newly synthesized proteins. Carolyn Bertozzi, PhD, was one of three scientists to be awarded the 2022 Nobel Prize in Chemistry earlier today for the development of click chemistry and bio-orthogonal chemistry. Bertozzi is a member of the Royal Society and the academies of sciences of Germany and the United States. This concise motif can be installed within heterologous proteins as a genetically encoded "aldehyde tag" for site-specific labeling with aminooxy- or hydrazide-functionalized probes. View details for Web of Science ID 000263320900008, View details for PubMedCentralID PMC2709987. Breidenbach, M. A., Palaniappan, K. K., Pitcher, A. The effort was enabled by a new high-fidelity pattern-searching and glycopeptide validation algorithm termed IsoStamp v2.0, as well as by novel stable isotope probes. This field is now entering a stage of logarithmic growth, during which chemical innovations combined with mass spectrometry advances could make it possible to fully characterize the human glycoproteome. A., Ogorzalek Loo, R. R., Lundberg, E. n., MacCoss, M. J., Mallick, P. n., Mootha, V. K., Mrksich, M. n., Muir, T. W., Patrie, S. M., Pesavento, J. J., Pitteri, S. J., Rodriguez, H. n., Saghatelian, A. n., Sandoval, W. n., Schlter, H. n., Sechi, S. n., Slavoff, S. A., Smith, L. M., Snyder, M. P., Thomas, P. M., Uhln, M. n., Van Eyk, J. E., Vidal, M. n., Walt, D. R., White, F. M., Williams, E. R., Wohlschlager, T. n., Wysocki, V. H., Yates, N. A., Young, N. L., Zhang, B. n. Mapping and quantification of over 2,000 O-linked glycopeptides in activated human T cells with isotope-targeted glycoproteomics (IsoTaG). Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. 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. Modulation of PSA expression by chemical means has a role complementary to genetic and biochemical approaches in the study of complex PSA-mediated events. These findings suggest that high mannose glycans are the major component of cell surface glycosylation with even terminal glucoses. Cell surface reactions of this kind should prove useful in the introduction of new recognition epitopes, such as peptides, oligosaccharides, or small organic molecules, onto cell surfaces and in the subsequent modulation of cell-cell or cell-small molecule binding events. The glycosylphosphatidylinositol (GPI) anchor is a C-terminal posttranslational modification found on many eukaryotic proteins that reside in the outer leaflet of the cell membrane. 1-68A and a 2-dehydroxy analogue, 1-68Aa, inhibit several purified LpxC orthologues. View details for Web of Science ID 000243895200017. 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. In this work, we undertook a mechanistic study of the Staudinger ligation with a focus on factors that affect reaction kinetics and on the identification of intermediates. The most potent examined, 1-68A, is a pH-dependent, two-step, covalent inhibitor of Escherichia coli LpxC that competes with UDP to bind the enzyme in the first step of inhibition. Additionally, we demonstrate that in C. glutamicum, the peripheral peptidoglycan layer at the septal junction remains contiguous throughout septation, forming a diffusion barrier for the fluid mycomembrane. Some members of the family prefer previously gly co sylated peptides (ppGalNAc T7 and T10), whereas others are inhibited by neighboring gly co sy la tion (ppGalNAc T1 and T2). Here, we studied the effects of perturbing O-GlcNAcylation during neural induction of hESCs using the metabolic inhibitor of OGT, peracetylated 5-thio-N-acetylglucosamine (Ac4-5SGlcNAc). View details for Web of Science ID 000280227700012, View details for PubMedCentralID PMC2907714. The lipooligosaccharides (LOS) of Haemophilus ducreyi are highly sialylated, a modification that has been implicated in resistance to host defense and in virulence. Kehoe, J. W., Maly, D. J., Verdugo, D. E., Armstrong, J. I., Cook, B. N., Ouyang, Y. She is a member of the National Academy of Sciences (2005), the Institute of Medicine (2011), and the National Academy of Inventors (2013). In one step, 35S-labeled by-products are then eluted from the membrane, leaving spatially separated 35S-labeled product "dots" for subsequent quantification. 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. 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. 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