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 interactions depend 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 mechanisms that 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 level 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 biosynthetic pathways 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 continue to isolate and characterize CHO cell mutants altered in glycosaminoglycan synthesis in order to characterize genes involved in chain initiation, polymerization and sulfation, with particular emphasis on heparan sulfate. In addition, we will search for regulatory genes in the system using multi-copy plasmids to inactive the pathway through gene dosage effects. 2. Analyze the function of GlcA transferase-I in mice. We 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) participates in glycosaminoglycan formation in the brain. The mouse mutant would open up the possibility of studying the function of glycosaminoglycan in other tissues as well. 3. Determine the relationship between heparan sulfate co-polymerase and pgsD. A controversy exists regarding the identity of pgsD and EXT, the putative heparan sulfate co-polymerase. To resolve this problem, we will obtain the cDNA that encodes the pgsD locus by cloning the gene from a stable correctant or by a sib-screening procedure. Characterizing various mutant alleles of pgsD that alter both GlcNAc and GlcA transferase activities or only the GlcA transferase may provide insight into structure and function of this interesting locus. 4. Explore the specificity of the new NDST isozymes in heparan sulfate processing. We will finish cloning the further member of the N- deacetylase/N-sulfotransferase family and examine the substrate specificity of each isozyme by trapping and characterizing acceptor oligosaccharides. In addition we will explore how the individual isozymes affects heparan sulfate biosynthesis in transfected cells.
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