AM Session
11:00 am – 11:30 am Hung-Wen Liu (UT Austin)
11:30 am – 12:00 pm Poster Talks
12:00 pm – 12:30 pm Lina Cui (University of Florida)
12:30 pm – 13:45 pm Poster Session/Lunch Break
PM Session
13:45 pm – 14:15 pm Young Ho Rhee (POSTECH, Korea)
14:15 pm – 14:45 pm Nicole Snyder (Davidson)
14:45 pm – 15:15 pm Poster Session/Coffee Break
15:15 pm – 15:45 pm Stephen Withers (UBC)
15:45 pm – 16:15 pm Xing Chen (Peking University)

Topics & Abstract

Hung-Wen Liu (UT Austin)
Topic: Mechanism of the radical SAM dehydratase BlsE involved in the biosynthesis of blasticidin S.
Abstract: The biosynthesis of blasticidin S has drawn attention due to the participation of the radical SAM enzyme BlsE. The original assignment of BlsE as a radical mediated, redox neutral decarboxylase is unusual, because this reaction appears to serve no biosynthetic purpose and would need to be reversed by a subsequent carboxylation step. Furthermore, with the exception of BlsE, all other radical SAM decarboxylases reported to date are oxidative in nature. Careful analysis of the BlsE reaction, however, demonstrates that BlsE is not a decarboxylase but instead a lyase that catalyzes the dehydration of cytosylglucuronic acid (CGA) to form cytosyl-4′-keto-3′-deoxy-Dglucuronic acid, which can rapidly decarboxylate nonenzymatically in vitro. Analysis of substrate isotopologs, fluorinated analogs, as well as computational models based on X-ray crystal structures of the BlsE•SAM (2.09 Å) and BlsE•SAM•CGA (2.62 Å) complexes suggests that BlsE catalysis likely proceeds via direct elimination of water from the CGA C4′ a-hydroxyalkyl radical as opposed to 1,2-migration of the C3′-hydroxyl prior to dehydration. Biosynthetic and mechanistic implications of the revised assignment of BlsE will be presented in the lecture.

Lina Cui (University of Florida)
Topic: Targeting an emerging therapeutic biomarker, heparanase.
Abstract: Heparan sulfate proteoglycans (HSPGs), major components in the extracellular matrix (ECM) of all tissue types, maintain ECM structural integrity and regulate cellular signaling via binding with ECM components and protein ligands such as growth factors and chemokines. Heparanase, the only known enzyme that can cleave the heparan sulfate (HS) side chains of HSPGs, regulates many cellular processes including ECM remodeling and homeostasis of cell-associated HS, and controls the bioavailability and activity of molecules attached to HS. Increased heparanase activity is mostly linked with cancer progression, angiogenesis, metastasis, and various types of inflammation. Our research focuses on the development of molecular tools that can target and visualize the spatiotemporal activities of heparanase in both cells and living organisms. We apply our molecular probes of heparanase to both diagnostics and drug discovery.

Young Ho Rhee (POSTECH, Korea)
Topic: Developing New Glycoside Synthesis Based Upon Asymmetric Metal Catalysis.
Abstract: Glycosides represent one of the most fundamental and highly useful structural motif not only in chemistry but also in other related fields. In this presentation, a conceptually new approach towards O- and N-glycoside synthesis will be introduced. A key feature of this de novo method is highlighted by the Pd-catalyzed asymmetric coupling reaction of various alcohol and N-heterocycle nucleophiles with alkoxyallenes under very mild conditions. Upon combination with the subsequent Ru-catalyzed ring-closing-metathesis, this reaction can generate glycosidic bonds in a highly efficient manner with no need of Lewis acid activators. Notably, stereochemical information of the anomeric center is controlled by the chiral ligand. Thus, this method is particularly well-suited for the synthesis of challenging 2-deoxyoligosaccharides and bioactive nucleoside derivatives. Our most recent results in this area will be disclosed.

Nicole Snyder (Davidson)
Topic: Glycopolymers for Pathogen Targeting.
Abstract:

Stephen Withers (UBC)
Topic: New and improved glyco-enzymes for glycoprotein analysis via metagenomics and mutagenesis.
Abstract: Analysis of the glycans present on glycoproteins typically requires, at some stage, the release of the glycans from the glycoproteins or derived glycopeptides prior to structural analysis. Enzymatic release processes are typically employed when possible as they minimize any damage to the glycan or the peptide. Several different classes of enzyme are available for effecting release of N-glycans. These either cleave the amide bond between the asparagine and the GlcNAc (PNGase F type) or cleave between the two core GlcNAc residues leaving a GlcNAc “stub” (Endo-H etc.). The only such enzymes available for release of O-glycans are the GH101 endo-GalNAcases, but these restricted to cleavage of the T-antigen disaccharide. Longer glycans must be removed using harsh chemical approaches. We have been attempting to address this deficiency, as well as the dearth of enzymes for analysis of mucins, through high-throughput screening of metagenomic libraries, based on the assumption that these enzymes are there to find. Our first (minor) success was in finding new enzymes for cleavage of the Tn antigen; in this case surprisingly from the GH31 family, which normally is home to alpha-glucosidases and alpha-xylosidases. Using T-antigen and sialyl-T-antigen based substrates we were able to identify some members of GH101 that will cleave off the intact sialyl-T-antigen, but only very slowly. Through several rounds of directed evolution we improved those activities substantially. However not all mutants were improved in their performance on glycoproteins, since “you get what you screen for”. To address this problem we have developed a flexible new FACS-based high-throughput screen for discovery and engineering of enzymes that function on glycoproteins. This can not only screen for the glycosidases of interest but also for the GalNAcTs that add the initial GalNAc of O-glycans or for mucin glycopeptidases. If you want to know how it works you will have to come to the talk or wait till the paper appears!!!

Xing Chen (Peking University)
Topic: Deciphering the brain glycocode by chemical biology approaches.
Abstract: The human brain accounts for ~2% of the body weight, but consumes as high as 20% of glucose. As a result, the brain possesses the highest level of glycosylation, such as sialylation and O-GlcNAcylation, among all the organs. The glycans in the brain have been implicated in neural connectivity and neurodegenerative diseases. Aiming to elucidate the functional roles of brain glycosylation, we have developed chemical tools for glycan labeling, imaging, and glycoproteomics. To enable in vivo visualization of the sialoglycans in the mouse brain, we used stealth liposomes to shuttle azidosugars into the brain for metabolic labeling of brain sialoglycans, followed by click-labeling with imaging probes. Termed liposome-assisted bioorthogonal reporter (LABOR), this strategy, for the first time, enables click-labeling and imaging of brain sialoglycans in living mice. Furthermore, to implement expansion microscopy (ExM) for brain glycan imaging, we developed click-ExM, which integrates click labeling into ExM to enable super-resolution fluorescence imaging of glycans. We demonstrated click-ExM imaging of sialoglycans in cultured neurons and in brain tissues with super-resolution. Finally, we performed glycoproteomic analysis of protein O-GlcNAcylation in primary neurons, which revealed that O-GlcNAc is enriched at the synapses and regulates synapse activation.

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