The azide was detected and quantified by Staudinger ligation with a phosphine probe in a microtiter plate format. A strain-promoted [3+2] azide-alkyne cycloaddition for covalent modification of blomolecules in living systems. Expression of large tumour-associated glycoproteins in non-transformed mammary cells promoted focal adhesion assembly and facilitated integrin-dependent growth factor signalling to support cell growth and survival. WebCarolyn R. Bertozzi Benjamin F. Cravatt United States United States United States: for their seminal contributions to understanding the chemistry of cellular communication and inventing chemical methodologies to study the role of carbohydrates, lipids, and proteins in such biological processes. Finally, the reaction must be tested in a variety of environments, escalating from aqueous media to biomolecule solutions to cultured cells and, for the most optimized transformations, to live organisms. Tsui, C. K., Barfield, R. M., Fischer, C. R., Morgens, D. W., Li, A., Smith, B. 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. We applied this "azido-ELISA" to the family of polypeptide alpha-N-acetylgalactosaminyltransferases (ppGalNAcTs), all of which were able to transfer N-azidoacetylgalactosamine (GalNAz) from the unnatural nucleotide sugar donor UDP-GalNAz. This work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance. 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 Agard, N. J., Prescher, J. Azido sugars are fed to cells and integrated by the glycan biosynthetic machinery into various glycoconjugates. Additionally, the isotopic signature imparted by the dibromide tag was detectable on a 12-kDa protein, suggesting applications in identifying large peptide fragments, such as those containing multiple or large posttranslational modifications (e.g., glycosylation). Detection of metabolites and post-translational modifications can be achieved using the azide as a bioorthogonal chemical reporter. View details for DOI 10.1002/anie.200806319, View details for Web of Science ID 000266415400022, View details for PubMedCentralID PMC2868584. View details for Web of Science ID 000229578100018. Individual GalNAc-Ts were rationally engineered to contain an enlarged active site (hole) and probed with a newly synthesized collection of 20 (bumped) uridine diphosphate N-acetylgalactosamine (UDP-GalNAc) analogs to identify enzyme-substrate pairs that retain peptide specificities but are otherwise completely orthogonal to native enzyme-substrate pairs. With this technique, we studied the dynamics of glycan trafficking and identified a population of sialoglycoconjugates with unexpectedly rapid internalization kinetics. Myoblast cells were patterned with high efficiency and remained undifferentiated after surface attachment. Here we investigate the biosynthetic origin of H. ducreyi sialosides by metabolic incorporation studies using a panel of N-acylmannosamine and sialic acid analogues. Under these conditions, Nrf1 is inactive in regulating proteasome subunit gene expression in response to proteasome inhibition. Bhat, R., Belardi, B., Mori, H., Kuo, P., Tam, A., Hines, W. C., Quynh-Thu Le, Q. T., Bertozzi, C. R., Bissell, M. J. In the mouse genome we have found a homologous ORF that predicts a novel murine GlcNAc 6-O-sulfotransferase with 88% identity to the human enzyme. We engineered mammalian cells to produce diazirine-modified O-GlcNAc by expressing a mutant form of UDP-GlcNAc pyrophosphorylase and subsequently culturing these cells with a cell-permeable, diazirine-modified form of GlcNAc-1-phosphate. She was a postdoctoral fellow at the University of California, San Francisco, from 1993 to 1995. In particular, GST-5 was found to catalyze 6-O-sulfation of beta-benzyl GlcNAc but not alpha- or beta-benzyl GalNAc. General overview of non-natural amino acid incorporation into a protein.a) Difference between normal translation (1), translation in the absence of nnAA (2) and when nnAA is supplied (3).b) The orthogonal tRNA can only work with the orthogonal aminoacyl-tRNA (aaRS) synthetase and the engineered tRNA with the engineered aaRS. A. Sequential assembly of the septal cell envelope prior to V snapping in Corynebacterium glutamicum. Shon, D., Malaker, S. A., Pedram, K., Yang, E., Krishnan, V., Dorigo, O., Bertozzi, C. R. On-tissue microscale glycoproteomics and N-glycan imaging reveal global dysregulation of canine glioma glycoproteomic landscape. 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. 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. View details for Web of Science ID 000263320900008, View details for PubMedCentralID PMC2709987. Key advantages of DNA-directed cell binding include the ability to immobilize both adherent and non-adherent cells, to capture cells selectively from a mixed population, to tune the binding properties of the cells, and to reuse substrates prepared with widely available DNA printing technologies. View details for Web of Science ID 000234391600034, View details for PubMedCentralID PMC2527029, View details for DOI 10.1002/ANIE.200502421, View details for Web of Science ID 000235246600005. View details for Web of Science ID 000259675500001, View details for PubMedCentralID PMC2709988. Upon exposure to mycobacterial cell wall lipids, 166 macrophage proteins showed differential expression. O-GlcNAc transferase (OGT) catalyzes the addition of N-acetylglucosamine (O-GlcNAc) onto a diverse array of intracellular proteins. This phenotype probably reflects a decreased capacity of the ST8Sia IV(-/-) progenitors to escape from the bone marrow niche. CDG-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder. The FGE recognition sequence, or aldehyde tag, can be inserted into heterologous recombinant proteins produced in either prokaryotic or eukaryotic expression systems. The mammalian glycocalyx is a heavily glycosylated extramembrane compartment found on nearly every cell. Monomeric sialyl Lewis(X) (sLe(x)) and sLe(x)-like oligosaccharides are minimal structures capable of supporting selectin binding in vitro. Control of enzyme activity is achieved using a small molecule to regulate association of the two domains. In this paper, we report on a general synthetic strategy for the assembly of glycopolymers that capitalizes on the intrinsic reactivity of reducing glycans toward hydrazides to form stable cyclic N-glycosides. Comparison of the cDNA and genomic DNA sequences reveals that this transferase is encoded by 10 exons in a 10 kb region. New therapies are therefore needed to treat diseases caused by these organisms, and a better understanding of the mechanisms of envelope assembly should aid in their discovery. Given the sensitivity and negligible background provided by bioluminescence imaging (BLI), we reasoned that 1 might be able to overcome some of the limitations encountered with fluorescent phosphine probes. VIDEO ABSTRACT. However, current bioaerosol sampling approaches have reported low detection yields in sputum-positive TB cases. Cell-surface glycans are attractive targets for molecule imaging due to their reflection of cellular processes associated with development and disease progression. In conclusion, the new ADAP assay can reliably detect the three cardinal islet autoantibodies/antibodies in 1muL serum with high sensitivity. The 35S-labeled products are then captured on an appropriate membrane, taking advantage of their polymeric architecture. Her recent efforts include synthesis of chemical tools to study cell surface sugars called glycans and how they affect diseases such as cancer, inflammation, and viral infections like COVID-19. Changes in O-linked protein glycosylation are known to correlate with disease states but are difficult to monitor in a physiological setting because of a lack of experimental tools. View details for DOI 10.1073/pnas.2335201100, View details for Web of Science ID 000187227200045, View details for PubMedCentralID PMC299823. We demonstrate that cyp128 and stf3 are sufficient for the biosynthesis of SMK from menaquinone and rv2269c exhibits promoter activity in M. tuberculosis. The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. A recently developed technology, termed metabolic oligosaccharide engineering, enables glycan labeling with probes for visualization in cells and living animals, and enrichment of specific glycoconjugate types for proteomic analysis. Our glycan-specific assay can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein levels. Electron-based dissociation methods are necessary to capture the O-glycopeptide diversity present in OpeRATOR digestions. Glioblastoma multiforme (GBMs) are recurrent lethal brain tumours. [8] Since 2021 she has been a member of the Accademia dei Lincei. View details for DOI 10.1016/j.chembiol.2004.02.023, View details for Web of Science ID 000220502900010. de Almeida, G., Townsend, L. C., Bertozzi, C. R. Mycobacterium tuberculosis Rv3406 Is a Type II Alkyl Sulfatase Capable of Sulfate Scavenging. Among the biomolecules that might be targeted for imaging analysis, glycans occupy a privileged position by virtue of their primary location on the cell surface. Living organisms produce hydrogen peroxide (H(2)O(2)) to kill invading pathogens and for cellular signaling, but aberrant generation of this reactive oxygen species is a hallmark of oxidative stress and inflammation in aging, injury, and disease. A FRET-based fluorogenic phosphine for live-cell Imaging with the Staudinger ligation, DNA-Coated AFM Cantilevers for the Investigation of Cell Adhesion and the Patterning of Live Cells. View details for DOI 10.1016/j.biomaterials.2004.01.025, View details for Web of Science ID 000222040300008, View details for DOI 10.1002/cbic.200400156, View details for Web of Science ID 000224453500014. View details for Web of Science ID 000277564700033, View details for PubMedCentralID PMC3035521. The reaction of azides with strained alkynes, such as cyclooctynes, readily forms a triazole product without the need for a toxic catalyst. The machinery responsible for sulfatase activation is poorly understood in prokaryotes. Our strategy makes use of an alkyne-bearing S-adenosylmethionine (SAM) analogue, which is accepted by the PKMT, SETDB1, as a cofactor, resulting in the enzymatic attachment of a terminal alkyne to its substrate. Born: 10 October 1966, Boston, MA, USA. View details for DOI 10.1073/pnas.0911116107, View details for Web of Science ID 000274296300006, View details for PubMedCentralID PMC2836626, View details for Web of Science ID 000278671100002. Circumventing the steps of purification from native cell membranes, this methodology facilitates the reconstitution of membrane-associated proteins. Here we explored their effect on corneal surface glycosylation using a metabolic label, tetra-acetylated N-azidoacetylgalactosamine (Ac4GalNAz). Work therefore extends the use of bioorthogonal labeling strategies to problems of clinical relevance a bioorthogonal reporter! Can be paired with traditional proximity ligation assays to simultaneously determine the change in total protein.. Cdg-Tre showed high selectivity for mycobacteria over other clinically prevalent species in the Corynebacterineae suborder glycosylated extramembrane found. In conclusion, the new ADAP assay can be inserted into heterologous proteins! 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