Cell-surface associated 0-linked glycoproteins are key transmitters of information between cells and the surrounding environment. For example, 0-linked glycoproteins present carbohydrate ligands for leukocyte adhesion molecules and contribute to T-cell activation during an immune response. Changes in the presentation of 0-linked glycoproteins are associated with inflammatory disease and tumor cell metastasis. Whereas studies of N-linked glycosylation have benefited tremendously from the availability of chemical tools for use in cell-based assays, such tools have been lacking for studies of 0-linked glycosylation. As a result, the spectrum of cellular events governed by 0-linked glycans, and the molecular basis of those that have been identified, remain poorly understood. The goal of this proposal is to develop chemical approaches for studying 0-linked glycosylation in cellular systems.We are focusing our efforts on two key enzyme families involved in the biosynthesis of these glycans. The UDP-GlcNAc-C4-epimerase generates UDP-GalNAc from UDP-G1cNAc, and the family of polypeptidyl-N-acetylgalactosaminyltransferases (ppGalNAcTs) initiate 0-linked glycosylation using UDP-GalNAc as a substrate.
The specific aims of this proposal are fourfold.
The first aim i s to discover/develop small molecule inhibitors of both families of enzymes. This will be accomplished by high-throughput screening of libraries based on the structures of preferred substrates.
The second aim i s to identify unnatural GalNAc analogs tolerated by the ppGalNAcTs as substrates for metabolic engineering of unnatural 0-linked glycans. The metabolic modifications will allow us to label cellular 0-linked glycoproteins and characterize the spectrum of proteins that bear this modification in the presence and absence of specific ppGalNAcT inhibitors.
The third aim focuses on determining the preferred substrates of the ppGalNAcTs both in vitro using glycopeptide substrate libraries, and in vivo by convergent enzyme/substrate engineering. The fourth and final aim is to synthesize irreversible inhibitors of ppGalNAcTs that will validate the proposed mechanism of action and facilitate structural studies.This series of experiments will advance our understanding of 0-linked glycoprotein biosynthesis and provide tools for modulating the process in cells. As 0-linked glycans participate in numerous disease states, these studies may also offer new avenues for therapeutic intervention.
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