The long-term objectives of the research proposed are to elucidate the function and biological roles of mammalian hyaluronan synthases. Hyaluronan (HA) is a linear, unbranched glycosaminoglycan of repeating disaccharide units of [D-glucuronic acid(beta-1,3)N-acetylglucosamine(beta-1,4)]x. HA is considered a critical component of the extracellular matrix in vertebrates. HA-containing matrices define spaces in the developing embryo, providing environments favorable for cell migration such as endocardial cushions. Specific cellular receptors for HA, including CD44, RHAMM (Receptor for Hyaluronate Mediated Motility) and hepatic endothelial cell receptors mediate cell migration, growth and HA turnover. Biochemical and cell biological studies have revealed that HA, in contrast to other glycosaminoglycans, is synthesized on the inner surface of the plasma membrane of eukaryotic cells and exported as a polymer into the extracellular matrix. However, until recently, the enzymes responsible for synthesizing HA in eukaryotes have resisted molecular identification. The applicant group has utilized degenerate RT-PCR to identify cDNA clones encoding a novel protein, termed hyaluronan synthase 2 (Has2), with several putative membrane spanning sequences and highly conserved regions conserved in enzymes catalyzing beta(1-4) bond formation. Transfection of the cDNA encoding this protein results in the de novo appearance of HA synthase activity in cell membranes. They have targeted the gene in the mouse, developed antibodies, and begun to explore the expression of mHas2 in vivo. The currently proposed studies seek to establish the catalytic function and properties of purified mHas2, map its expression in the mouse in relationship to the known distribution of HA, and study the phenotype of the targeted null mutation that they have created. It is suggested by the applicant that the planned studies may elucidate the function, expression and developmental role of a new mammalian enzyme, whose putative extracellular matrix product is considered vital to normal mammalian growth and development, and implicated widely in the pathophysiology of inflammation and tissue injury.
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