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.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
3R01CA020421-16S1
Application #
3165281
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1976-12-01
Project End
1994-01-31
Budget Start
1993-09-01
Budget End
1994-01-31
Support Year
16
Fiscal Year
1993
Total Cost
Indirect Cost
Name
Johns Hopkins University
Department
Type
Schools of Public Health
DUNS #
045911138
City
Baltimore
State
MD
Country
United States
Zip Code
21218
Acosta-Serrano, Alvaro; O'Rear, Jessica; Quellhorst, George et al. (2004) Defects in the N-linked oligosaccharide biosynthetic pathway in a Trypanosoma brucei glycosylation mutant. Eukaryot Cell 3:255-63
Pu, Lixia; Scocca, Jane R; Walker, Brian K et al. (2003) A single point mutation resulting in an adversely reduced expression of DPM2 in the Lec15.1 cells. Biochem Biophys Res Commun 312:555-61
Pu, Lixia; Scocca, Jane R; Walker, Brian K et al. (2003) The divergent 5' ends of DPM2 mRNAs originate from the alternative splicing of two adjacent introns: characterization of the hamster DPM2 gene. Biochem Biophys Res Commun 312:817-24
Cacan, R; Duvet, S; Labiau, O et al. (2001) Monoglucosylated oligomannosides are released during the degradation process of newly synthesized glycoproteins. J Biol Chem 276:22307-12
O'Rear, J L; Scocca, J R; Walker, B K et al. (1999) Chinese hamster ovary cells with reduced hexokinase activity maintain normal GDP-mannose levels. J Cell Biochem 72:56-66
Quellhorst Jr, G J; O'Rear, J L; Cacan, R et al. (1999) Nonglucosylated oligosaccharides are transferred to protein in MI8-5 Chinese hamster ovary cells. Glycobiology 9:65-72
Krag, S S (1998) The importance of being dolichol. Biochem Biophys Res Commun 243:1-5
Quellhorst Jr, G J; Piotrowski, J S; Steffen, S E et al. (1998) Identification of Schizosaccharomyces pombe prenol as dolichol-16,17. Biochem Biophys Res Commun 244:546-50
Walker, B K; Lei, H; Krag, S S (1998) A functional link between N-linked glycosylation and apoptosis in Chinese hamster ovary cells. Biochem Biophys Res Commun 250:264-70
Kaiden, A; Rosenwald, A G; Cacan, R et al. (1998) Transfer of two oligosaccharides to protein in a Chinese hamster ovary cell B211 which utilizes polyprenol for its N-linked glycosylation intermediates. Arch Biochem Biophys 358:303-12

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