Oligosaccharides are attached to asparagine residues in a wide variety of eukaryotic soluble and membrane-associated proteins such as enzymes, hormones, receptors, extracellular matrix proteins, and the proteins of the plasma membrane and subcellular organelles. N-linked glycoproteins vary as to their biological function and the role of the glycan in that function differs from protein to protein. In some cases, the glycan plays a direct role in the glycoprotein's biological activity and in other cases the glycan affects the protein's physicochemical properties. Many of the initial steps in the complex biosynthetic pathway of the glycan moiety are common regardless of the final processed structure. Recent work has focused on understanding the regulation of this pathway. The approach to be used is a biochemical one: to characterize and reconstitute the activity of purified enzymes in vitro and to determine how alterations in an individual enzyme level in vivo effects the activity of the entire glycosylation pathway. Dr. Krag is concentrating on two enzymes, UDP-N-acetylglucosamine:dolichyl phosphate N-acetylglucosamine-1-phosphate transferase and mannosylphosphoryldolichol synthase. The transferase is an enzyme early in the reaction pathway and the synthase is positioned at a branch point in the pathway. The hypothesis is that both these enzymes are important regulatory enzymes in the biosynthetic pathway of the glucose moiety of N-linked glycoproteins. The studies outlined in this proposal will directly test this hypothesis.

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