One class of glycoproteins has oligosaccharides attached to asparagine residues in an Asn-X-Ser(Thr) consensus sequence via an N-glycosidic bond. N-linked glycoproteins are ubiquitous components of a eukaryotic cell's secretory pathway. Examples of intracellular N-linked glycoproteins include membrane components of the endoplasmic reticulum, Golgi apparatus, and lysosome, lumenal lysosomal hydrolases, and cell surface receptors. N- Linked glycoproteins are also secreted from cells and are components of the extracellular matrix. N-linked glycoproteins have diverse functions and the carbohydrate moiety on those proteins have different functions. The mature oligosaccharide found attached to asparagine residues in proteins can exist as a variety of structures. Importantly, regardless of their mature structure, different oligosaccharide moieties are synthesized utilizing common steps involving a polyisoprenoid lipid. Initially, an oligosaccharide is preassembled on a lipid carrier and transferred en bloc to an asparagine residue of a nascent protein. Subsequently, protein-bound oligosaccharides are processed to a myriad of known structures. Delineating and understanding the regulation of the common biosynthetic steps are crucial for understanding N-linked glycosylation. The goals of the work in my laboratory are first to define the steps in the biosynthesis of the lipid carrier, dolichyl phosphate, and the steps in the assembly of oligosaccharide-lipid intermediates. The second goal is to understand how the levels of dolichol-intermediates and hence N-linked glycosylation are regulated. We use several complementary approaches, including in vitro and in vivo biochemical experiments, somatic cell genetics using Chinese hamster ovary (CHO) cells, and molecular biology. During the proposed grant period, we will do the following: (1) Isolate, sequence, and express the cDNA for polyprenol reductase, a key enzyme in dolichyl phosphate synthesis. (2) Determine normal cellular mechanisms responsible for short-term regulation of N-glycosylation in response to an increased or decreased requirement for N-glycosylation. (3) Determine the enzymatic defect in various CHO glycosylation mutants which we recently isolated using procedures designed to detect mutants in the early, common biosynthetic pathway for N-linked glycoproteins.
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