An important class of macromolecules in mammalian cells is glycoproteins, including: membrane-bound enzymes such as HMG-CoA reductase (endoplasmic reticulum, ER), sialyl and galactosyl transferases (Golgi), and Na/K ATPase (plasma membrane); cell surface receptors for growth factors (transferrin, insulin, EGF, FGF) and for low density lipoproteins; cation-independent mannose 6-phosphate receptor, involved in targeting of lysosome enzymes in fibroblasts and soluble proteins such as secretory products (hormones, fibronectin, collagen, laminin) and lumenal lysosomal enzymes. The role of the glycose moiety differs from protein to protein; it can be important for stability, solubility, final conformation, targeting, and/or intermolecular interactions. Oligosaccharides are attached to glycoproteins via asparagine or serine/threonine residues. The biosynthesis of asparagine-linked oligosaccharides is a complex series of at least fifty membrane- associated reactions with four major steps. First is the synthesis of the polyisoprene, dolichyl phosphate. Second, an oligosaccharide is synthesized attached to dolichyl phosphate by stepwise additions of N-acetylglucosamine, mannose, and glucose from sugar nucleotides and monoglycosylated, phosphorylated dolichols. Third, the oligosaccharide is transferred as a unit to nascent proteins that have the sequence Asn-X-Ser(Thr). Fourth, glucose and some mannose residues are removed and in some cases N- acetylglucosamine, galactose, fucose, and sialic acid residues are added, giving the final glycan structure found on mature glycoproteins. We are dissecting this multistep, multiorganellar biosynthetic pathway using biochemical genetics. During this project period, we will determine the precise enzymatic defect in three Chinese hamster ovary cell mutants. One of these mutants, F2A8, is defective in the synthesis of dolichol, and accumulates neutral polyisoprenol. We plan to determine if this accumulation, which results in a high ratio of neutral prenols to phosphorylated prenols, correlates with the presence of smooth ER in the cell. A second mutant, CHB11-1-3, appears defective in the translocation of oligosaccharide-lipid intermediates from the cytoplasmic to the lumenal face of the ER. A third mutant Lec9 synthesizes forty-fold less labeled (G1c)3(Man)9(G1cNAc)2-P-P-lipid compared to parental cells. Finally, we will isolate new glycosylation mutants.
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