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. Finally the lack of an endogenous human orthologue of cysH and its possible role in defence against adaptive immunity renders CysH an attractive enzyme for further studies as a target for therapeutics active against CTBI. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). The vast majority of H. ducreyi strains contain high levels of sialic acid (N-acetylneuraminic acid, NeuAc) in their LOS. These data show that the normal distribution of surface glycosylation requires IL-1R, but not MyD88, and is not sufficient to prevent bacterial binding. We evaluated a panel of four lectins (Soybean agglutinin (SBA), Wisteria floribunda lectin (WFL), Vicia villosa-B-4 agglutinin (VVA), and Helix pomatia agglutin (HPA)) with specificity for -N-acetylgalactosamine (-GalNAc), an epitope displayed on mucins overexpressed in many adenocarcinomas. Shin, S., Chung, S., Sanii, B., Comolli, L. R., Bertozzi, C. R., De Yoreo, J. J. Here we show that MmpL8, a member of a large family of predicted lipid transporters in M. tuberculosis, is required for SL-1 production. Our editors will review what youve submitted and determine whether to revise the article. Further, we demonstrate an ideal counter-screening strategy, which relies on a target peptide from an unrelated protein, the Src-family kinase Fyn. An intriguing example is Sulfolipid-1 (SL-1), a sulfated glycolipid that has been implicated in Mtb pathogenesis, although no direct role for SL-1 in virulence has been established. Her research group profiles changes in cell surface glycosylation associated with cancer, inflammation and bacterial infection, and uses this information to develop new In mice, ScTyrY43G and MmPheT413G label the melanoma tumor proteome and plasma secretome. She is an elected member of the Institute of Medicine, National Academy of Sciences, and American Academy of Arts and Sciences. We introduce a non-natural substrate biosynthetic pathway and use engineered glycosyltransferases to incorporate chemically tagged sugars into the cell surface glycome of the living cell. 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. View details for Web of Science ID 000225233600024. 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. B., Bertozzi, C. R. Membrane proteomics of phagosomes suggests a connection to autophagy. A., Rajaiah, G., Falck, J. R., Bertozzi, C. R., Berthiaume, L. G. Identification of palmitoylated mitochondrial proteins using a bio-orthogonal azido-palmitate analogue. Selective chemical reactions enacted within a cellular environment can be powerful tools for elucidating biological processes or engineering novel interactions. Riley, N. M., Bertozzi, C. R., Pitteri, S. J. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. These studies are currently in progress in our laboratory. 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. The assay was validated by measuring K(M) values for PAPS and K(I) values for PAP, the product of sulfuryl transfer. A goal of tissue engineering is to produce a scaffold material that will guide cells to differentiate and regenerate functional replacement tissue at the site of injury. Riley, N. M., Malaker, S. A., Driessen, M. n., Bertozzi, C. R. An enzymatic toolkit for selective proteolysis, detection, and visualization of mucin-domain glycoproteins. This report describes a general strategy for engineering the display of chemically defined oligosaccharides on cell surfaces that combines the concepts of metabolic engineering and selective chemical reactivity. Cholesterol Catabolism by Mycobacterium tuberculosis Requires Transcriptional and Metabolic Adaptations. 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. In all assays, activity appeared glycosylation independent. View details for DOI 10.1016/j.dib.2016.05.060, View details for PubMedCentralID PMC4908283. Upon exposure to mycobacterial cell wall lipids, 166 macrophage proteins showed differential expression. A., Engleman, E. G., Bertozzi, C. R. A bulky glycocalyx fosters metastasis formation by promoting G1 cell cycle progression. Recent studies suggest that the mucin granule lumen consists of a matrix meshwork embedded in a fluid phase. The repertoire of sialic acids presented by cells can be expanded to include unnatural variants by intercepting the sialic acid biosynthetic pathway with unnatural precursors. Indeed, O-glycopeptides modified exclusively at the N-terminus would enable O-glycoproteomic methods to rely solely on collision-based fragmentation rather than electron-driven dissociation because glycan-retaining peptide fragments would not be required for localization. Thus, choice of enrichment strategy has profoundimplications on experimental outcomes. 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. Protein-carbohydrate interactions are known to mediate cell-cell recognition and adhesion events. 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. 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. There is growing interest in extending this progress to O-glycoproteomics, which necessitates comparisons of method performance for the two classes of glycopeptides. Therapeutic strategies that target tumor-associated sialosides may therefore potentiate antitumor immunity. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. Using this knowledge, glycosyltransferase assembly lines have been redesigned for the production of specific glycan structures using protein engineering and chemical approaches. Molecular level analysis of cell-surface phenomena could benefit from model systems comprising structurally defined components. The surface-bound glycopolymers bind lectins in a ligand-specific manner. Chang, P. V., Dube, D. H., Sletten, E. M., Bertozzi, C. R. Progress and challenges for the bottom-up synthesis of carbon nanotubes with discrete chirality, Identification of glycoproteins targeted by Trypanosoma cruzi trans-sialidase, a virulence factor that disturbs lymphocyte glycosylation. 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. Pratt, M. R., Hang, H. C., Ten Hagen, K. G., Rarick, J., Gerken, T. A., Tabak, L. A., Bertozzi, C. R. Expanding the diversity of unnatural cell-surface sialic acids. Patterson, B. n., Dinkele, R. n., Gessner, S. n., Morrow, C. n., Kamariza, M. n., Bertozzi, C. R., Kamholz, A. n., Bryden, W. n., Call, C. n., Warner, D. F., Wood, R. n. Marschallinger, J. n., Iram, T. n., Zardeneta, M. n., Lee, S. E., Lehallier, B. n., Haney, M. S., Pluvinage, J. V., Mathur, V. n., Hahn, O. n., Morgens, D. W., Kim, J. n., Tevini, J. n., Felder, T. K., Wolinski, H. n., Bertozzi, C. R., Bassik, M. C., Aigner, L. n., Wyss-Coray, T. n. Updates to the Symbol Nomenclature for Glycans guidelines, Neelamegham, S., Aoki-Kinoshita, K., Bolton, E., Frank, M., Lisacek, F., Luetteke, T., O'Boyle, N., Packer, N. H., Stanley, P., Toukach, P., Varki, A., Woods, R. J., Darvill, A., Dell, A., Henrissat, B., Bertozzi, C., Hart, G., Narimatsu, H., Freeze, H., Yamada, I., Paulson, J., Prestegard, J., Marth, J., Vliegenthart, J. G., Etzler, M., Aebi, M., Kanehisa, M., Taniguchi, N., Edwards, N., Rudd, P., Seeberger, P., Mazumder, R., Ranzinger, R., Cummings, R., Schnaar, R., Perez, S., Kornfeld, S., Kinoshita, T., York, W., Knirel, Y., SNFG Discussion Grp, Engineering Orthogonal Polypeptide GalNAc-Transferase and UDP-Sugar Pairs. View details for DOI 10.1146/annurev.biochem.71.110601.135334, View details for Web of Science ID 000177352600021. We show that a recently discovered sialidase family, whose first member EnvSia156 was isolated from hot spring metagenomes, defines an unusual structural fold and active centre constellation, not previously described in sialidases. DIFBO was so reactive that it spontaneously trimerized to form two asymmetric products that we characterized by X-ray crystallography. Here we describe a biarylazacyclooctynone (BARAC) that has exceptional reaction kinetics and whose synthesis is designed to be both modular and scalable. Bertozzi subsequently optimized the bioorthogonal reaction using an azide as a binding partner for the fluorescent tag. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures. The immobilization procedure itself was found not to activate primary T-cells, in contrast to previously reported antibody- and lectin-based methods. View details for DOI 10.1016/j.jmb.2006.08.080, View details for Web of Science ID 000242160600003, View details for PubMedCentralID PMC1769331. Schilling, B., Goon, S., Samuels, N. M., Gaucher, S. P., Leary, J. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance. CalFluors: A Universal Motif for Fluorogenic Azide Probes across the Visible Spectrum. A readily available bis-Weinreb amide of D-tartrate served as a key intermediate. 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. In previous work, we demonstrated that H. ducreyi scavenges sialic acid from the extracellular milieu and incorporates those residues into LOS. 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