Dermatan sulfate (DS) proteoglycans are characteristic components of fibrous connective tissues and are found in skin, heart valves, blood vessels, lung, sclerra, and many other tissues. The physiological functions of these compounds are essentially unknown, but certain specific interactions with other biomolecules are well established. For example, DS binds to low density lipoproteins, and in view of its presence in artery walls it has been suggested that DS may contribute to the development of atherosclerotic plaques. Our long-term objective is to elucidate the processes whereby DS proteoglycans are synthesized and to determine the mechanisms by which these processes are regulated. Today, there is no specific information about the biosynthesis of the core proteins, and knowledge of the assembly of the polysaccharide chains is scanty. At least 10 enzymes particpapte in this latter process (6 glycosyltransferases, 3 sulfotransferases, and one uronosyl 5-epimerase), but none of these enzymes has ever been purified from a DS-synthesizing tissue. We propose a) to isolate the DS-synthesizing enzymes, b) to study their fundamental molecular and catalytic properties, c) to determine whether the various enzymes are capable of interacting with each other to form efficient multi-enzyme complexes; and d) to initiate studies of the metabolism of the biosynthetic enzymes themselves. In the process, it will be necessary to develop a substantial body of new methodology, mainly in the areas of substrate preparation, enzyme assays, and enzyme purification by specific affinity methods. In the proposed investigations, particular emphaisis will be placed on the newly discovered uronosyl 5-epimerase which catalyzes formation of the iduronic acid residues in DS from glucuronic acid units already incorporated into the polymer. This unique enzyme occupies a key position in the biosynthetic process, since it conveys the structural individuality and the functional properties to DS which distinguish this polysaccharide from the chondroitin sulfates. It is therefore deemed especially important to examine the factors which regulate epimerase activity, and this problem will be approached by determining the effects of various agents (ionophores such as monensin; glucocorticoids; and prostaglandin F2) on glucuronic acid/iduronic acid ratios in DS synthesized by cultured skin fibroblasts.
