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.
