The long-range objective of this proposal is to gain insight into the mechanism of heparan sulfate proteoglycan assembly and its regulation. Heparan sulfate proteoglycans participate in a variety of physiological processes by binding, activation or immobilization of various protein ligands. These interactionsdepend to a large extent on the composition and fine structure of the heparan sulfate chains, which in turn depend on the substrate specificity of the various biosynthetic enzymes and regulatory factors. Thus, insight into the mechanismsthat cells use to synthesize and to regulate heparan sulfate composition may provide new avenues for developing agents to enhance or modulate protein- carbohydrate interactions. This information may lead to novel approaches for treating human diseases associated with altered heparan sulfate synthesis, such as neoplastic transformation, atherosclerosis, and various growth disorders associated with connective tissues. To achieve these long-range goals, we propose a series of genetic and biochemical experiments to probe the biosyntheticpathways of heparan sulfate synthesis in Chinese hamster ovary (CHO) cells and mice. Specifically,we plan to execute the following studies: 1. Identify genes affecting heparan sulfate biosynthesis. We will continueto isolate and characterize CHO cell mutants altered in glycosaminoglycansynthesis in order to characterize genes involved in chain initiation, polymerization and sulfation, with particularemphasis on heparan sulfate. In addition, we will search for regulatory genes in the system using multi-copy plasmidsto inactivatethe pathway through gene dosage effects. 2. Analyze the function of GIcA transferase-I in mice. W will target GlcAT-I in mice using the Cre-loxP system in order to ablate the gene in a tissue specific manner. Targeting the gene in this way will allow us to address whether a related isozyme (GlcAT-P) participatesin glycosaminoglycanformation in the brain. The mouse mutant would open up the possibility of studying the function of glycosaminoglycans in other tissues as well. 3. Determine the relationship between heparan sulfate copolymerase and pgsD. A controversy exists regarding the identity of pgsD and EXT, the putative heparan sulfate copolymerase. To resolve this problem, we will obtain the cDNA that encodes the pgsD locus by cloningthe gene from a stable correctant or by a sib-screening procedure. Characterizing various mutant alleles of pgsD that alter both GlcNAc and GIcA transferase activities or only the GlcA transferase may provide insight into structure and function of this interestinglocus. 4. Explore the specificity of the new NDST isozymcs in heparan sulfate processing. We will finish cloningthe fourth member of the N-deacetylase/N-sulfotransferase family and examine the substrate specificity of each isozyme by trapping and characterizing acceptor oligosaccharides. In additionwe will explore how the individual isozymes affects heparan sulfate biosynthesis in transfected cells.
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