Heparin and heparan sulfate proteoglycans are biologically active macromolecules composed of a core protein and one or more polysaccharide chains attached via an O-glycosidic linkage between xylose and serine. Among several distinct biological activities, the anticoagulant activity displayed by some heparins and heparan sulfates is the most conspicuous, and this property is now known to reside in an antithrombin-binding pentasaccharide segment of unique structure. The biosynthesis of heparins and heparan sulfates encompasses 15 enzymatic steps, which take place in three phases: a) formation of a specific carbohydrate-protein linkage region; b) assembly of repeating disaccharides (GlcUA-GlcNAc); and c) polymer modifications leading to the generation of the biological activities of the polysaccharides. Basic knowledge about the properties of the enzymes catalyzing these reactions is meager, and, to date, only two of the enzymes have been purified to homogeneity. Our long-term objectives are to establish the structures and mechanisms of action of some of these enzymes, to determine how the synthetic apparatus is organized in the intracellular membranes, and to elucidate how the synthesis of the polysaccharides is regulated by intrinsic mechanisms at the molecular and cellular levels as well as by extrinsic influences on the intact cells. In the immediate future, our investigations will be focused on two reactions, glucuronysyl transfer to galactose residues during linkage region formation, which may be a rate-limiting step in the overall assembly process, and uronosyl 5-epimerization, which is a polymer modification essential to the formation of the antithrombin- binding sites. The following specific aims will be pursued: 1) purification of heparosan N-sulfate D-glucuronosyl 5-epimerase; 2) characterization of the purified enzyme with respect to molecular and catalytic properties; 3) development of simplified methodology for the assay of glucuronosyltransferase I and the closely related transferase involved in the biosynthesis of the HNK-1 antigen; 4) purification of the two glucuronosyltransferases; and 5) investigations of the metabolism of the 5-epimerase, with initial emphasis on its turnover in cultured skin fibroblasts and hepatocytes. Research in this general area has an intrinsic value, inasmuch as it advances our basic knowledge of important processes in the human body, but it also has a direct and proven clinical value in that it has led to improved forms of antithrombotic therapy and has aided in the diagnosis of genetic metabolic deficiencies.
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