Many proteins in eukaryotic cells have oligosaccharide moieties covalently attached to asparagine residues. Examples of glycosylated proteins include components of organellar and cell surface membranes, secreted proteins such as hormones, and enzymes such as the soluble lysosomal hydrolases. The biosynthesis of these oligosaccharide sidechains involves dolichol- linked mono- and oligosaccharide intermediates and a minimum of forty steps occurring in three different subcellular compartments. An understanding of this complex pathway and its regulation by endogenous and exogenous agents will require purification of the enzymes involved and reconstitution of the system in vitro. As a first step, we propose to purify two enzymes ad the genes which encode them from Chinese hamster ovary cells; both proteins catalyze early reactions in the biosynthetic scheme and utilize both sugar nucleotide and dolichyl phosphate as substrates. Both enzymes, UDP-N-acetylglucosamine:dolichyl phosphate N- acetylglucosamine-1-phosphate transferase and mannosylphosphoryldolichol synthase, are integral membrane proteins, are present in small amounts in the endoplasmic reticulum of cells, and catalyze potential regulatory steps in glycoprotein synthesis. Purification of the genes which encode for these enzymes will provide the amounts of protein needed for characterization and reconstitution studies. We will isolate the gene for the mannosylphosphoryldolichol synthase by utilizing B4- 2-1, a Chinese hamester ovary mutant cell line which we have characterized as lacking synthase activity. Compared to parental cells, this mutant has ten-fold less mannose 6-phosphate- dependent uptake of exogenous lysosomal enzymes and ten-fold less endogenous alpha-iduronidase activity than wild-type cells. Transfected cells expressing the synthase gene will be detected by an autoradiographic screen for uptake of labelled lysosomal enzymes or a fluorescent screen for alpha-iduronidase activity. The second enzyme, glucosamine phosphate transferase, will be purified from 3E11 cells, a clone of tunicamycin-resistant Chinese hamster ovary cells which were isolated and characterized in this laboratory. Membranes for a tunicamycin-resistant population and clones (such as 3E11) from that population had fifteen times the specific activity of the transferse as did membranes from wild-type cells. Antiserum against the purified enzyme will be produced to facilitate the initial isolation of fragments and finally the entire gene for the transferase. Finally, 3E11 and B4-2-1 will be used to study specific aspects of the regulation of asparagine- linked glycoprotein biosynthesis.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
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Physiological Chemistry Study Section (PC)
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Johns Hopkins University
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Scocca, J R; Krag, S S (1997) Aspartic acid 252 and asparagine 185 are essential for activity of lipid N-acetylglucosaminylphosphate transferase. Glycobiology 7:1181-91
Zou, J; Krag, S S (1995) Method identifying hybridizing regions of DNA within an insert. Biotechniques 18:402-4
Zou, J; Scocca, J R; Krag, S S (1995) Asparagine-linked glycosylation in Schizosaccharomyces pombe: functional conservation of the first step in oligosaccharide-lipid assembly. Arch Biochem Biophys 317:487-96
Scocca, J R; Zou, J; Krag, S S (1995) Genomic organization and expression of hamster UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosaminyl phosphoryl transferase. Glycobiology 5:129-36
Crick, D C; Scocca, J R; Rush, J S et al. (1994) Induction of dolichyl-saccharide intermediate biosynthesis corresponds to increased long chain cis-isoprenyltransferase activity during the mitogenic response in mouse B cells. J Biol Chem 269:10559-65
Kaiden, A; Krag, S S (1992) Dolichol metabolism in Chinese hamster ovary cells. Biochem Cell Biol 70:385-9
Scocca, J R; Krag, S S (1990) Sequence of a cDNA that specifies the uridine diphosphate N-acetyl-D-glucosamine:dolichol phosphate N-acetylglucosamine-1-phosphate transferase from Chinese hamster ovary cells. J Biol Chem 265:20621-6
Rosenwald, A G; Stoll, J; Krag, S S (1990) Regulation of glycosylation. Three enzymes compete for a common pool of dolichyl phosphate in vivo. J Biol Chem 265:14544-53
Scocca, J R; Hartog, K O; Krag, S S (1988) Evidence of gene amplification in tunicamycin-resistant Chinese hamster ovary cells. Biochem Biophys Res Commun 156:1063-9