The goal of this proposal is to understand the structure and function of hamster GlcNAc-1-P transferase (GPT). GPT catalyzes the transfer of GlcNAc-1-P from UDP-GlcNAc to dolichol-P to form GlcNAc-P-P-Dolichol. GlcNAc-PP-dolichol is then converted into oligosaccharide-PP-dolichol (with 14 sugars), the immediate precursor for N-linked glycans in eukaryotes. N-linked glycans are important in a broad range of functions, such as cell adhesion, organellar trafficking, and protein folding, and have been implicated in cancer and human diseases such as HEMPAS, CGDS, and I-cell disease. Recent amino acid sequence analyses have linked hamster GPT with a family of similar eukaryotic and prokaryotic sugar-1-P transferases which use UDP-GlcNAc or UDP-MurNAc-pentapeptide as a donor and a polyisoprenyl phosphate as an acceptor. It is essential that GPT activity be regulated within a narrowly defined window. Too little GPT activity prevents sufficient quantities of N-linked glycans from being produced. Too much GPT activity causes excessive competition between GlcNAc-P-P-dolichol synthesis and reactions synthesizing mannose-P-dolichol (MPD) and glucose-P-dolichol (GPD). Insufficient MPD and GPD results in abnormal N-linked glycosylation and defective glycosylphosphatidylinositol (GPI) anchor synthesis. Furthermore, recent data indicate that binding of unfolded proteins to the ER chaperone calnexin, which specifically recognizes Glc1Man9GlcNAc2, would be inhibited.
Five specific aims are proposed to address the following questions about hamster GPT: (1) What are the functions of the conserved sequences found in members of the UDP-GlcNAc/MurNAc transferase family? (2) What determines specificity for different sugar donors and lipid acceptors in the UDP-GlcNAc/MurNAc family of glycosyltransferases? (3) Rather than mediating bind of dolichol, does the """"""""potential dolichol recognition sequence"""""""" (PDRS) promote a complex of enzymes which act early in the dolichol pathway? (4) How is GPT stabilized by its sequence-specific, bipartite C-terminus? (5) What is the topological orientation of GPT in the ER membrane? The answers to these questions will not only provide new fundamental information about GPT and its role in N-linked glycosylation, but will also have direct implications for a number of structurally related proteins.
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