The development of bioorthogonal reactions has classically focused on bond-forming ligation reactions. Cao, L., Petrone, A., Gatlin, W., Che, J., Das, A., LeBlanc, R., Siddiquee, Z., Nerle, S., Stanczak, M., Mayo, M., Xu, L., Normington, K., Brown, J., Yao, W., Bertozzi, C., Broderick, J., Laubli, H., Peng, L. Physical Principles of Membrane Shape Regulation by the Glycocalyx. View details for Web of Science ID 000167162100006, View details for Web of Science ID 000166873000031. 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. WebBio. With the aid of density functional theory calculations reported previously by Nagano and co-workers, we identified azidofluorescein derivatives that were predicted to undergo an increase in fluorescence quantum yield upon Cu-catalyzed or Cu-free cycloaddition with linear or cyclic alkynes, respectively. Using high-performance liquid chromatography, we quantified the degree of accumulation and reversibility upon acidic compartment neutralization in macrophages and observed that accumulation was greater in infected than in uninfected macrophages. View details for Web of Science ID 000082564500003. These results shed light on the physical barriers that modulate phagocytosis, which have been heretofore underappreciated. These results suggest that glycopolymer microarrays can reveal discrete higher-order binding preferences beyond the recognition of individual glycan epitopes. However, because they are internalized within hours, these glycopolymers could not be employed to probe processes that occur on longer time scales. View details for Web of Science ID 000296036700058, View details for PubMedCentralID PMC3187659. In this report, we seek to expand the functional repertoire of such transformations by introducing a new bond-cleaving reaction between N-oxide and boron reagents. 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. Rabuka, D., Parthasarathy, R., Lee, G. S., Chen, X., Groves, J. T., Bertozzi, C. R. Vaccine efficacy of an attenuated but persistent Mycobacterium tuberculosis cysH mutant. View details for Web of Science ID 000259139900020, View details for PubMedCentralID PMC2721638. Increased levels of circulating saturated free fatty acids, such as palmitate, have been implicated in the etiology of type II diabetes and cancer. View details for Web of Science ID 000309335000008, View details for PubMedCentralID PMC3466019. In this study, we performed a proteomic analysis of the membrane fraction from latex bead-containing (LBC) phagosomes isolated from macrophages. View details for Web of Science ID 000088039800042. Cohen, A. S., Dubikovskaya, E. A., Rush, J. S., Bertozzi, C. R. Visualizing enveloping layer glycans during zebrafish early embryogenesis. The primary sequence of APS reductase is distinguished by a conserved iron-sulfur cluster motif, -CC-X( approximately )(80)-CXXC-. Collectively, these results provide evidence that polySia is involved in hematopoietic development. Using this procedure, we found that the rate constant for the cycloaddition reaction of DIFBO with an azide exceeds those for difluorinated cyclooctyne (DIFO) and dibenzocyclooctyne (DIBO). Mougous, J. D., Leavell, M. D., Senaratne, R. H., Leigh, C. D., Williams, S. J., Riley, L. W., Leary, J. We observed crosslinking between FG-repeat nucleoporins and nuclear transport factors, suggesting that O-GlcNAc residues are intimately associated with essential recognition events in nuclear transport. Here we review common enrichment strategies used in modern mass spectrometry (MS)-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography (HILIC) and its derivatives, porous graphitic carbon (PGC), reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. Perez-Vilar, J., Mabolo, R., McVaugh, C. T., Bertozzi, C. R., Boucher, R. C. Molecular basis for G protein control of the prokaryotic ATP sulfurylase. Lastly, we show that sialic acid depletion enhances ADC lysosomal delivery and killing in diverse cancer cell types, including with FDA (US Food and Drug Administration)-approved trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Furthermore, Rv0516c disruption modified peptidoglycan thickness, enhanced antibiotic resistance, and activated genes in the regulon of the alternative -factor SigF. We apply the strategy to a particularly redundant yet disease-relevant human glycosyltransferase family, the polypeptide N-acetylgalactosaminyl transferases. View details for Web of Science ID 000296075300003. Accurate mass measurements indicated an oxidation state of +2 for the 4Fe-4S cluster, with no disulfide bond in the holoenzyme. In June 2015, she joined the faculty at Stanford University as an Institute Scholar at Sarafan ChEM-H. Prof. Bertozzi's research interests span the disciplines of chemistry and biology with an emphasis on studies of cell surface glycosylation pertinent to disease states. These modified proteins integrated into the plasma membranes of a variety of mammalian cells and were internalized and directed to recycling endosomes similarly to GFP bearing a native GPI anchor. 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. 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. Cyclopropane Modification of Trehalose Dimycolate Drives Granuloma Angiogenesis and Mycobacterial Growth through Vegf Signaling. A., Qi, Y. n., Yasumoto, Y. n., Schmitz, C. O., Mumbach, M. R., Limaye, A. n., Wei, J. n., Alfajaro, M. M., Parker, K. R., Chang, H. Y., Horvath, T. L., Carette, J. E., Bertozzi, C. n., Wilen, C. B., Satpathy, A. T. Enhanced Bactericidal Effects of Pyrazinamide Toward Mycobacterium smegmatis and Mycobacterium tuberculosis upon Conjugation to a {Au(I)-triphenylphosphine}+ Moiety. A major obstacle to tuberculosis (TB) control is the problem of chronic TB infection (CTBI). 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. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. The C-type lectins dectin-1 and dectin-2 contribute to innate immunity against microbial pathogens by recognizing their foreign glycan structures. We introduced the peptide sequence recognized by the endoplasmic reticulum (ER)-resident formylglycine generating enzyme (FGE), which can be as short as 6 residues, into heterologous proteins expressed in mammalian cells. Moreover, these studies show that to properly exploit the sialic acid biosynthetic pathway for metabolic oligosaccharide engineering in H. ducreyi and possibly other prokaryotes that share similar pathways, precursors based on sialic acid and not mannosamine must be used. We developed a new fluorophore-trehalose conjugate to visualize trehalose monomycolates of the mycomembrane using super-resolution microscopy. N-Propanoylmannosamine (ManProp), which differs from ManBut by a single methylene group, did not inhibit PSA biosynthesis. A critical branch point in mucin-type oligosaccharides is the beta 1-->3 glycosidic linkage to the core alpha-N-acetylgalactosamine (GalNAc) residue. A convergent synthesis of trehalose. Choi, J., Wagner, L. S., Timmermans, S. E., Malaker, S. A., Schumann, B., Gray, M. A., Debets, M. F., Takashima, M., Gehring, J., Bertozzi, C. R. CRISPR-Cas9 screens identify regulators of antibody-drug conjugate toxicity. We seeded collagen-glycosaminoglycan meshes, a widely used tissue engineering scaffold material, with human IMR-90 fibroblasts and compared transcript levels with control cells grown on tissue culture polystyrene. In this protocol, we describe a novel mass-independent chemical glycoproteomics platform for characterizing intact, metabolically labeled glycopeptides from complex proteomes, termed Isotope Targeted Glycoproteomics (IsoTaG). Overall, our findings provide a quantitative characterization of O-GlcNAc glycoproteins and their corresponding modification sites in primary human T cells, which will facilitate mechanistic studies into the function of O-GlcNAc in T cell activation. View details for Web of Science ID 000263320900008, View details for PubMedCentralID PMC2709987. 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. 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. Hsiao, S. C., Shum, B. J., Onoe, H., Douglas, E. S., Gartner, Z. J., Mathies, R. A., Bertozzi, C. R., Francis, M. B. Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications. This heterogeneity precludes enrichment strategies that can be universally beneficial for all glycan classes. The nematode Caenorhabditis elegans is an excellent model organism for studies of glycan dynamics, a goal that requires tools for imaging glycans in vivo. 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. The rapid assembly of "core 1"and "core 3" O-linked glycopeptide mimetics was accomplished in this fashion. Previously we showed that the epithelium of healthy mouse corneas becomes vulnerable to Pseudomonas aeruginosa adhesion if it lacks the innate defense protein MyD88 (myeloid differentiation primary response gene 88), or after superficial injury by blotting with tissue paper. This Cu-free click reaction possesses comparable kinetics to the Cu-catalyzed reaction and proceeds within minutes on live cells with no apparent toxicity. The unsymmetrical (2'-, 3'-, 4'- and 6'-) monodeoxy analogs were synthesized by desymmetrization of alpha,alpha-trehalose and subsequent deoxygenation under radical conditions. Notably, we observed that the transcription factors c-JUN and JUNB show higher levels of O-GlcNAc glycosylation and higher levels of expression in activated T cells. The development of chemical strategies for decorating cells with defined carbohydrate epitopes would greatly facilitate studies of carbohydrate-mediated cell surface interactions. 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