The attachment of carbohydrate is a profound modification which a broad array of proteins with diverse functions undergo during the course of their biosynthesis. Indeed eukaryotes have evolved an elaborate enzymatic machinery for assembling saccharide units of striking diversity and this provides numerous sites for physiological regulation as well as for malfunction in disease states such as neoplasis and diabetes. Furthermore it has recently become evident that a number of glycoproteins are attached to the cell membrane by a functionally intriguing glycan-lipid anchor. It is our objective to continue as well as to expand our multifaceted research program on glycoprotein biosynthesis with emphasis on the following topics, assembly, processing and sulfation of Asn-linked carbohydrate units; enzymatic mechanism and subcellular localization of glycosyl-phosphatidylinositol (GPI) membrane anchor formation; and glycosylation of type IV (basement membrane) collagen and regulation of its production. We will examine a number of aspects of N-linked oligosaccharide biosynthesis, including: exploration in HepG2 and thyroid cells of the physiological role of the recently discovered endo-alpha-D- mannosidase and of the alternate glycoprotein processing route it initiates, purification of this unique processing enzyme and its use in characterization of biosynthetic intermediates; isolation by affinity chromatography of the RER oligosaccharide-lipid Glc-transferase and evaluation of our hypothesis that RER transmembrane movement of G1c is effected by a shuttle involving multiple Bol-P molecules; isolation of the thyroid PAPS; galactose 3-O-sulfotransferase and assessment in cultured cells of its action (cooperative or competitive) with other transferases in attaching capping groups. Using as a model a major thyroid cell surface glycoprotein we will follow GPI-anchor biosynthesis in cultured cells, look for glycan-PI intermediates; and characterize the enzymes responsible for glycan assembly. In glomerular and lens epithelial cells we will explore the regulation of type IV collagen production at transcriptional, translational and postranslational levels with emphasis on factors which could be responsible for the basement membrane changes of diabetes, the subcellular locale and sequence of collagen Hy1- and Asn-glycosylation will also be investigated.
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