N-linked glycosylation is an essential protein modification reaction associated with all eukaryotic systems. Central to this modification is the co-translational transfer of a carbohydrate from a lipid-linked pyrophosphate donor to the side chain of an asparagine within the -Asn-Xaa-Ser/Thr- tripeptide recognition motif. The multimeric, membrane-associated enzyme oligosaccharyl transferase (OT) catalyzes this transformation. The biological roles of the oligosaccharides in N-linked glycoconjugates are highly diverse. Carbohydrate markers are critical in intracellular targeting and intercellular recognition phenomena. Glycoproteins play a pivotal role in all aspects of cell development and function. Carbohydrates are also known to be involved in a decoy function and protect the protein surface from the immune system, proteolysis, and microbial attack. Accordingly, N-linked glycosylation also has implications in the biotechnology industry since it influences the structure, secretion, antigenicity, and clearance of many pharmaceutically important glycoproteins. N-linked glycosylation is also implicated in facilitating protein folding and/or stabilizing a folded motif.
The specific aims of this proposal are presented in four categories namely: 1.Protein Biochemistry and Reconstitution of Oligosaccharyl Transferase: These studies focus on defining the catalytic machinery and molecular mechanism of N-linked glycosylation through the development of efficient heterologous expression systems and reconstitution protocols. Related studies include expression and investigation of soluble OT subunits and the isolation and characterization of an archaebaterial OT. 2.Development of a Bioavailable OT Inhibitor and Mechanistic Studies with Substrate Analogs: Significant progress has been made in the development of potent (nMKi) inhibitors of OT. A key objective in this proposal is the redesign of these inhibitors to make them cellularly available. Additional peptide analogs which probe the active site machinery of OT are also proposed. 3.Biophysical and Biochemical Studies on Physiologically Significant Glycoproteins: Future studies are aimed at developing an understanding of the biophysical consequences of glycosylation in polypeptide motifs and small protein domains. The two systems that we shall examine include beta-turn peptides from hemagglutinin and subdomains of the prion protein (PrP).
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