We demonstrate that gna1Delta strains require a GlcNAc supplement and that expression plasmids containing both exogenous components of the salvage pathway, GlcNAc transporter NGT1 from Candida albicans and GlcNAc kinase NAGK from Homo sapiens, are required for rescue in this context. GST-5 was also expressed in soluble epitope-tagged form and found to catalyze 6-O-sulfation of GlcNAc residues in synthetic acceptor structures. The lack of a defined consensus sequence for the ppGalNAcTs makes the prediction of mucin-type O-linked glycosylation difficult based on primary sequence alone. a nanoscience research center at Lawrence Berkeley National Laboratory. Schilling, B., Goon, S., Samuels, N. M., Gaucher, S. P., Leary, J. WebBertozzi is a professor of Humanities and Sciences, and of Chemical and Systems Biology and of Radiology. Tian, E., Ten Hagen, K. G., SHUM, L., Hang, H. C., Imbert, Y., Young, W. W., Bertozzi, C. R., Tabak, L. A. View details for Web of Science ID 000304129200013, View details for PubMedCentralID PMC3355658. In vivo, BPA appears to have greater activity than is suggested by its estrogen receptor (ER) binding affinity. Proteins bearing this "aldehyde tag" were chemically modified by selective reaction with hydrazide- or aminooxy-functionalized reagents. Surprisingly, for three of the enzymes, significant activity was observed with sialylated LacNAc, and two of the enzymes were capable of detectable sulfation of GlcNAc in the context of sialyl Lewis x. However, SL-1 per se is not required for establishing infection as pks2 mutants, which are defective in an earlier step in SL-1 biosynthesis, have no obvious growth defect. Selective protein-protein interactions between nonribosomal peptide synthetase (NRPS) proteins, governed by communication-mediating (COM) domains, are responsible for proper translocation of biosynthetic intermediates to produce the natural product. Metastasis depends upon cancer cell growth and survival within the metastatic niche. BPA is a substrate for estrogen sulfotransferase, and bisphenol A sulfate (BPAS) and disulfate are substrates for estrone sulfatase. 2023 Chuan He Hiroaki Suga Jeffery W. Kelly This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biological chemistry. This technique has attracted significant attention recently for the synthesis of biological macromolecules of defined homogeneous composition, the design of self-assembling drugs and the chemical remodeling of cell surfaces. Studies of 4Fe-4S cluster stability and cysteine reactivity in the presence and absence of substrates, and in the free enzyme versus the covalent enzyme-intermediate (E-Cys-S-SO(3)(-)), suggest a structural rearrangement that occurs during the catalytic cycle. To increase the utility of bioaerosol sampling, we present advances in bioaerosol collection and Mtb identification that improve detection yields.A previously described Respiratory Aerosol Sampling Chamber (RASC) protocol, or "RASC-1", was modified to incorporate liquid collection of bioaerosol using a high-flow wet-walled cyclone (RASC-2). [8] Since 2021 she has been a member of the Accademia dei Lincei. The compound potentiates the cytotoxicity of carfilzomib, a clinically used proteasome inhibitor, against MM and T cell-derived acute lymphoblastic leukemia (T-ALL) cell lines. Methods capable of directing orientation, as well as an understanding of the underlying physical mechanisms are, however, lacking. 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. The concept of a folding funnel with kinetic traps describes folding of individual proteins. Wild world of bioorthogonal chemistry, Polymerization of glycosylated NCAs for preparation of biomedical materials and synthetic glycoproteins. Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging of nonprotein biomolecules. Goon, S., Schilling, B., Tullius, M. V., Gibson, B. W., Bertozzi, C. R. GlcNAc 2-epimerase can serve a catabolic role in sialic acid metabolism. The identification of this enzyme in T. gondii demonstrates that this human parasite has its own enzymatic machinery for the O-glycosylation of toxoplasmal proteins. Robust surface mineral layers a few microns thick were obtained. Molecular level analysis of cell-surface phenomena could benefit from model systems comprising structurally defined components. In addition, the mode of inhibition for PAP was rapidly determined. Parthasarathy, R., Rabuka, D., Bertozzi, C. R., Groves, J. T. Copper-free click chemistry for dynamic in vivo imaging. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. Protein glycosylation can have an enormous variety of biological consequences, reflecting the molecular diversity encoded in glycan structures. This novel assay may improve pediatric testing compliance and facilitate easier community-wide screening for islet autoantibodies. View details for DOI 10.1016/j.bmc.2005.04.085, View details for Web of Science ID 000231341900006. 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. The optimal configuration of sulfate esters on the N-acetyllactosamine (Galbeta1-->4GlcNAc) core of sulfosialyl-Le(X), however, remains unsettled. We further generated rapamycin-inducible chimeric enzymes comprising the localization domain of a sulfotransferase and the catalytic domain of a glycosyltransferase, demonstrating the generality of the system among other Golgi enzymes. In this paper, we describe experiments in which the conformations of structurally well-defined polymers anchored to fluid lipid membranes were probed using Fluorescence Interference Contrast Microscopy (FLIC), an optical technique that provides topographic information with few-nanometer precision. Furthermore, ADAP correctly identified 95% high-risk individuals with two or more autoantibodies by radioassay amongst 39 relatives of T1D patients tested. A., Bertozzi, C. R. Synthetic Trehalose Glycolipids Confer Desiccation Resistance to Supported Lipid Monolayers. To enable noninvasive, real-time, spatiotemporal quantitative imaging of fatty acid fluxes in animals, we created a bioactivatable molecular imaging probe based on long-chain fatty acids conjugated to a reporter molecule (luciferin). Furthermore, we demonstrate that the metabolic diversity of nature enables the use of naturally occurring functional groups that display inherent biocompatibility alongside abiotic components for organism-specific applications. [92] She has a wife and three sons.[93]. Yarema, K. J., Mahal, L. K., Bruehl, R. E., Rodriguez, E. C., Bertozzi, C. R. Synthesis of an oxime-linked neoglycopeptide with glycosylation-dependent activity similar to its native counterpart. Using a fluorescent marker tagged to the ring molecule, Bertozzi was able to track the ring compound as it bound to the glycan, in this way developing a map of the glycan location. The controlled addition of structurally defined components to live cell membranes can facilitate the molecular level analysis of cell surface phenomena. 5 '-adenosinephosphosulfate lies at a metabolic branch point in mycobacteria. Most progress has occurred in the area N-glycoproteomics. To elucidate the important sites of sulfation on Le(x) with respect to L-selectin recognition, we have synthesized six sulfated Le(x) analogs and determined their abilities to block binding of a recombinant L-selectin-Ig chimera to immobilized GlyCAM-1. Additionally, we observe enhanced glycocalyx height in response to epithelial-to-mesenchymal transition and to oncogenic KRAS activation. Several protein lysine methyltransferases (PKMTs) modify histones to regulate chromatin-dependent cellular processes, such as transcription, DNA replication and DNA damage repair. Disruption of cysH rendered Mtb auxotrophic for cysteine and methionine, and attenuated virulence in BALB/c and C57BL/6 immunocompetent mice. Examples from the past decade suggest that a promising strategy for bioorthogonal reaction development begins with an analysis of functional group and reactivity space outside those defined by Nature. Here we report two engineered aminoacyl-tRNA synthetases for mammalian bioorthogonal labeling: a tyrosyl ( ScTyrY43G) and a phenylalanyl ( MmPheT413G) tRNA synthetase that incorporate azide-bearing noncanonical amino acids specifically into the nascent proteomes of host cells. Chp1 is fully active in vitro, but in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8. This was confirmed by enzymatic assay of the partially purified enzyme with unnatural substrates. View details for DOI 10.1074/jbc.M204613200, View details for Web of Science ID 000177859000029. We devised an experimental model that mimics the structure of mycobacterial envelopes in which an immobile hydrophobic layer supports a TDM-rich, two-dimensionally fluid leaflet. The kinetic constants of Stf0 were measured, and the catalytic mechanism of the sulfuryl group transfer reaction was investigated in initial rate kinetics and product inhibition experiments. Strikingly, we found that cholesterylamine (CholA) anchored glycopolymers are internalized into vesicles that serve as depots for delivery back to the cell surface, allowing for the display of cell-surface glycopolymers for at least ten days, even while the cells are dividing. Malaker, S. A., Pedram, K., Ferracane, M. J., Bensing, B. Here we describe a method for the site-specific introduction of aldehyde groups into recombinant proteins using the 6-amino-acid consensus sequence recognized by the formylglycine-generating enzyme. View details for DOI 10.1073/pnas.0811481106, View details for Web of Science ID 000262263900006, View details for PubMedCentralID PMC2629201. Chen, Q., Zhang, D., Somorjai, G., Bertozzi, C. R. Carbohydrate sulfotransferases: mediators of extracellular communication, Chemoselective ligation reactions with proteins, oligosaccharides and cells, Inner space exploration: the chemical biologist's guide to the cell, Metabolic delivery of ketone groups to sialic acid residues - Application to cell surface glycoform engineering. View details for DOI 10.1002/anie.201508861, View details for Web of Science ID 000368061800024, View details for PubMedCentralID PMC4715665. The objective of these methods is to make glycoconjugate synthesis accessible to a broader community, thereby accelerating progress in glycobiology. B., Stein, G. S., Ayers, D. C., Bertozzi, C. R. Chemical Approaches To Perturb, Profile, and Perceive Glycans, PapA3 Is an Acyltransferase Required for Polyacyltrehalose Biosynthesis in Mycobacterium tuberculosis. Antibodies bind to and agglutinate synthetic antigen-DNA conjugates, enabling ligation of the DNA strands and subsequent quantification by qPCR. Bule, P. n., Chuzel, L. n., Blagova, E. n., Wu, L. n., Gray, M. A., Henrissat, B. n., Rapp, E. n., Bertozzi, C. R., Taron, C. H., Davies, G. J. Sulfolipid-I (SL-I) is an abundant metabolite found in the cell wall of Mycobacterium tuberculosis that is comprised of a trehalose 2-sulfate core modified with four fatty acyl substituents. IsoTaG is therefore positioned to enhance structural understanding of the glycoproteome. We postulate that covalent modification and perhaps inhibition of various mitochondrial enzymes by palmitoyl-CoA could lead to the metabolic impairments found in obesity-related diseases. View details for Web of Science ID 000171601400045. Here we present a unified design, based on the principle of photoinduced electron transfer, to access a panel of highly fluorogenic azide probes that are activated by conversion to the corresponding triazoles via click chemistry. Each enzyme preferred a terminal GlcNAc residue, and was impeded by the addition of a beta1,4-linked Gal residue (i.e., terminal LacNAc). But in M. tuberculosis, its function is potentiated by the previously identified sulfolipid transporter MmpL8 and disulfate substrates. Folding of individual proteins the partially purified enzyme with unnatural substrates for was! Web of Science ID 000177859000029 ( BPAS ) and disulfate are substrates for estrone.! Structural understanding of the DNA strands and subsequent quantification by qPCR can have an enormous variety of consequences! Here we introduce click-EM, a labeling technique for correlative light microscopy and EM imaging nonprotein! 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