The long range objective of this proposal is to gain insight into the mechanism of proteoglycan assembly. Altered proteoglycan synthesis correlates with the onset of several diseases, including neoplastic transformation, atherosclerosis, and various growth disorders associated with connective tissues. Thus, any insight into the mechanisms that cells use to synthesize and to regulate proteoglycan composition may provide new avenues for the development of therapeutic agents. To achieve these long-range goals, we propose a series of genetic, biochemical and molecular biologic experiments to probe the biosynthetic pathways of heparan sulfate and chondroitin sulfate synthesis in Chinese hamster ovary (CHO) cells. In particular we plan to execute the following studies: 1. Intermediates in Proteoglycan Biosynthesis. A comparison of proteo- glycans and biosynthetic intermediates in CHO mutants defective in chain initiation and polymerization may provide clues about the factors that control glycosaminoglycan composition. 2. Assembly of Heparan Sulfate. Studies of the reactions that lie at the branchpoint between the pathways of heparan sulfate and chondroitin sulfate synthesis may reveal how intermediates distribute into the individual pathways. Biochemical studies and genetic correction experiments of GlcNAc and GlcA transferase-deficient CHO cells should better define the relationship of these transferases. 3. Modification of Heparan Sulfate. Biochemical and genetic characterization of new mutants altered in bFGF binding should lead to a better understanding of polymer modification reactions involved in heparan sulfate synthesis. 4. Heparan Sulfate Structure and Biological Activity. Cell adhesion assays and binding experiments with heparin-binding proteins will help decide if unique saccharide sequences mediate the interaction of heparan sulfate with these ligands.
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