This proposal seeks support for a comprehensive research program In the area of peptide glycosylation. The problems selected for investigation comprise a long range plan to uncover detailed knowledge about (l) peptide- carbohydrate interactions, and (2) the enzymic mechanism of peptide glycosylation. Asparagine glycosylation is a cotranslational protein modification catalyzed by oligosaccharyl-transferase (OST). which results in oligosaccharide attachment to the Asn gamma-carboxamide in Asn-Xaa- Ser/Thr consensus sequences. These N-linked glycoproteins mediate a variety of normal and pathological processes, including protein folding and cell- cell adhesion. Glycoproteins have been proposed to be critical for such cancer processes as tumor metastasis and viral infectivity. We propose to use organic synthesis. multi-dimensional NMR, and mechanistic enzymology to study peptide N-glycosylation. A major goal is to define the structure of Asn glycopeptides and obtain information about intramolecular peptide-sugar interactions. Our strategy relies on synthesis of structurally defined glycopeptide analogs, systematically modified in both the sugar and peptide domains. These molecules will be well suited for multi-dimensional and multinuclear NMR structural and dynamic studies. We also propose to determine, at a basic level, the peptide N-glycosylation mechanism as catalyzed by OST. Besides basic knowledge about glycopeptide structure and function, the proposed research may lead to glycosylation inhibitors with therapeutic value.
