WebPrincipal Investigator. Paulick, M. G., Forstner, M. B., Groves, J. T., Bertozzi, C. R. Bioorthogonal click chemistry: Covalent labeling in living systems, Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes. The complex and diverse structures of GPI anchors suggest a rich spectrum of biological functions, but few have been confirmed experimentally because of the lack of appropriate techniques that allow for structural perturbation in a cellular context. View details for Web of Science ID 000304492700020. The study of the metabolome presents numerous challenges, first among them being the cataloging of its constituents. 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. GST-5 has recently been characterized as a novel GalNAc 6-O-sulfotransferase termed chondroitin 6-sulfotransferase-2 (Kitagawa, H., Fujita, M., Itio, N., and Sugahara K. (2000) J. Biol. The leukocyte adhesion molecule L-selection participates in the initial attachment of blood-borne lymphocytes to high endothelial venules (HEVs) during lymphocyte homing to secondary lymphoid organs, and contributes to leukocyte adhesion and extravasation in HEV-like vessels at sites of chronic inflammation. She coined the term "bioorthogonal chemistry" for chemical reactions compatible with living systems. View details for DOI 10.1371/journal.pgen.1008284. Lantos, A. Hatzios, S. K., Baer, C. E., Rustad, T. R., Siegrist, M. S., Pang, J. M., Ortega, C., Alber, T., Grundner, C., Sherman, D. R., Bertozzi, C. R. Imaging the Glycosylation State of Cell Surface Glycoproteins by Two-Photon Fluorescence Lifetime Imaging Microscopy. Here, we develop a "bump-hole" chemical reporter system for studying GalNAc-T activity in vitro. The sulfate esters on these structures are thought to be essential for high-affinity binding to L-selectin. 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. This complex could be stored as a lyophilized powder and then dissociated in organic solvents to produce free DIFBO for in situ kinetic and spectroscopic analysis. 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. Here we review common enrichment strategies used in modern mass spectrometry-based glycoproteomic experiments, including lectins and other affinity chromatographies, hydrophilic interaction chromatography and its derivatives, porous graphitic carbon, reversible and irreversible chemical coupling strategies, and chemical biology tools that often leverage bioorthogonal handles. A major challenge in fundamental studies of galectin-ligand interactions is that their natural ligands comprise a heterogeneous collection of glycoconjugates that share related glycan structures but disparate underlying scaffolds. View details for Web of Science ID 000251457800039, View details for PubMedCentralID PMC2654346. Here we report the design, synthesis, and in vivo applications of Peroxy Caged Luciferin-1 (PCL-1), a chemoselective bioluminescent probe for the real-time detection of H(2)O(2) within living animals. Main sialyl acceptors were identified as mucins by biochemical procedures and protein markers. Furthermore, we determined that the PAT biosynthetic machinery has no cross-talk with that for sulfolipid-1 despite their related structures. bertozzi@stanford.edu. 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. View details for DOI 10.1021/acscentsci.6b00070, View details for PubMedCentralID PMC4827488. Chung, S., Shin, S., Bertozzi, C. R., De Yoreo, J. J. Difluorobenzocyclooctyne: Synthesis, Reactivity, and Stabilization by beta-Cyclodextrin. The azide serves as a bioorthogonal chemical handle for selective modification with biochemical or biophysical probes using the Staudinger ligation. B., Bertozzi, C. R. Membrane proteomics of phagosomes suggests a connection to autophagy. Thus, M. tuberculosis most likely produces PAPS for the sole use of this organism's sulfotransferases. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. This tutorial review will summarize the history of this emerging field, as well as recent progress in the development and application of bioorthogonal copper-free click cycloaddition reactions. Interest in glycoproteomics continues to surge as mass spectrometry instrumentation and software improve, so this review aims to help equip researchers with the necessary information to choose appropriate enrichment strategies that best complement these efforts. Sialic Acid Glycobiology Unveils Trypanosoma cruzi Trypomastigote Membrane Physiology. GRABOWSKI, J. J., Bertozzi, C. R., Jacobsen, J. R., Jain, A., Marzluff, E. M., Suh, A. Y. 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 StcEE447D is a pan-mucin stain derived from enterohemorrhagic Escherichia coli that is tolerant to a wide range of glycoforms. View details for Web of Science ID 000285812000039, View details for PubMedCentralID PMC3018347. We demonstrated that the kinetic parameters of the assembly process depend on DNA sequence complexity, density, and total cell concentration. 1-68A and a 2-dehydroxy analogue, 1-68Aa, inhibit several purified LpxC orthologues. Islet autoantibodies are predominantly measured by radioassay to facilitate risk assessment and diagnosis of type 1 diabetes. We probed the dynamic behavior of cell-bound glycopolymers bearing various hydrophobic anchors and glycan structures using fluorescence correlation spectroscopy (FCS). Upon changing to differentiation media, myotubes formed in the center of the patterned areas with an excellent degree of edge alignment. Although the sulfated xenobiotics bind poorly to the ER, both stimulated the growth of receptor-positive breast tumor cells. Marcaurelle, L. A., Pratt, M. R., Bertozzi, C. R. A new approach to mineralization of biocompatible hydrogel scaffolds: An efficient process toward 3-dimensional bonelike composites, Synthesis of a bisubstrate analogue targeting estrogen sulfotransferase. View details for Web of Science ID 000075388100009. Carolyn Bertozzi Hometown: Palo Alto, California Education: AB, Harvard University, 1988; PhD, University of California, Berkeley, 1993 Current position: Director, Stanford Chemistry, Engineering, and Medicine for Human Health; professor of chemistry, Stanford University; and investigator, Howard Hughes Medical Institute Mesenchymal-like GBMs were highly contractile and expressed elevated levels of glycoproteins that expanded their glycocalyx, and they were surrounded by a stiff extracellular matrix that potentiated integrin mechanosignalling. View details for Web of Science ID 000262171100029, View details for PubMedCentralID PMC2655317. Converse, S. E., Mougous, J. D., Leavell, M. D., Leary, J. Antigen presentation to Tcells in major histocompatibility complex class II (MHC class II) requires the conversion of early endo/phagosomes into lysosomes by a process called maturation. We designed phosphine-luciferin reagent 1 to activate bioluminescence in response to Staudinger ligation with azide-labeled glycans. Proteobacterial ATPS overcomes this energetically unfavorable reaction by associating with a regulatory G protein, coupling the energy of GTP hydrolysis to APS formation. However, the Staudinger ligation has some liabilities, such as the propensity of phosphine reagents to undergo air oxidation and the relatively slow kinetics of the reaction. View details for DOI 10.1021/jacs.0c07700. When the recombinant construct was expressed in stably transfected Drosophila melanogaster S2 cells, the purified protein exhibited transferase activity in vitro. 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 Mycobacterium tuberculosis synthesizes specific polyketide lipids that interact with the host and are required for virulence. Second messengers generated by Fc receptors activated integrins, which formed an actin-tethered diffusion barrier that excluded CD45. Further, we show that cells successfully incorporate synthetic GlcNAc analogs N-azidoacetyglucosamine (GlcNAz) and N-(4-pentynoyl)-glucosamine (GlcNAl) into cell-surface glycans and secreted glycoproteins. Our findings suggest that there is a dynamic and reciprocal link between integrin mechanosignalling and a bulky glycocalyx, implying a causal link towards a mesenchymal, stem-like phenotype in GBMs. Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted disease. Martin, D. D., Vilas, G. L., Prescher, J. The glycan-binding proteins, or lectins, that interact with mucins are often oligomeric receptors with multiple ligand binding domains. While individual CD45 molecules moved randomly, they were displaced from the advancing phagocytic cup by an expanding diffusional barrier. View details for DOI 10.1021/acs.jproteome.6b01053. These data highlight the power of advanced imaging methods to provide molecular and functional insights into glycocalyx biology. Sialome sweet sialome: As sialic acids are involved in many host-pathogen recognition events and are markers of embryonic and malignant tissues, there is great interest in methods for the enrichment and identification of sialylated glycoproteins from complex tissues. Glycoproteins are typically expressed as mixtures of glycoforms, their oligosaccharides being generated by a template-independent biosynthetic process. In these sulfur auxotrophs, heavy sulfate is channeled exclusively into sulfated metabolites. Several important human pathogens are represented in the Corynebacterineae suborder, including Mycobacterium tuberculosis and Corynebacterium diphtheriae. 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. The concept of a folding funnel with kinetic traps describes folding of individual proteins. We report the discovery that boron nitride nanotubes (BNNTs), isosteres of CNTs with unique physical properties, are inherently noncytotoxic. We applied this strategy to the GlcNAc-6-sulfotransferases GlcNAc6ST-1 and GlcNAc6ST-2, which collaborate in the sulfation of L-selectin ligands. Carroll, K. S., Gao, H., Chen, H. Y., Stout, C. D., Leary, J. Here, we report the use of azido-modified unnatural sialic acid to identify those molecules that act as cell surface acceptors of the sialyl residue in the trans-sialidase-catalyzed reaction, which might then be involved in the immune alterations induced. 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