The long range objective of this proposal is to gain insight into the mechanism of proteoglycan assembly and its regulation. Altered proteoglycan synthesis correlates with the onset of several deceases, including neoplastic transformation, atherosclerosis, and various growth disorders associated with connective tissues. Thus, any insight into the mechanisms that cells use to therapeutic agents. To achieve these long- range goals, we propose a series of genetic, biochemical and molecular genetic experiments to probe the biosynthetic pathways of heparan sulfate synthesis in Chinese hamster ovary (CHO) cells. In particular, we plan to execute the following studies: 1. Regulation of GAG composition. We plan to purify alpha-G1cNAc transferase 1, which catalyzes the first committed step in heparan sulfate biosynthesis. We will also examine the way its activity depends on tetrasaccharide and glycopeptide substrates to establish how this enzyme and beta-GaINAc transferase 1 determine if heparan sulfate or chondroitin sulfate assembles at attachment sites in natural proteoglycans. The enzymes will be localized in Golgi sub compartments by glycosylating trapped intermediates. 2. Polymerization of heparan sulfate. Studies of pgsD mutants have established that a single protein contains both alpha.G1cNAc transferase II and G1cUA transferase, which together catalyze heparan sulfate polymerization. Mutant alleles have been found that alter both enzyme activities or only G1cUA transferase. This finding suggests that the mutations in these strains alter different functional domains of the transferase complex. We plan to clone cDNAs corresponding to pgsd alleles to test this hypothesis and to define how the mixed transferase gives rise to heparin sulfate. 3. Modification of heparan sulfate. A new mutant (clone 17) was found to lack 2-O-sulfated uronic acids in heparan sulfate. Enzymatic characterization of this strain and new mutants altered in N-sulfation and 6-O-sulfation will provide insight into how cells regulate the fine structure of heparan sulfate. The mutant also provide important new tools for studying heparan sulfate-protein interactions.
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