Bame 9418559 Cell associated heparan sulfate proteoglycans have been implicated in a number of processes, including cell adhesion, growth and transformation. The regulation of these activities will be determined. Initially, cell surface heparan sulfate proteoglycans are internalized, the glycosaminoglycans is removed from the protein core and cleaved into small chains by heparanases. These small chains are secreted or transported to other sites in the cell where they have been implicated in regulating proteoglycan functions or other cellular processes. To determine how the metabolism of proteoglycans regulates, it is important to understand how many heparanases are involved in heparan sulfate proteoglycan catabolism, how the enzymes recognize and cleave the heparan sulfate substrate, and the functions of the short chains produced by the enzyme reaction. To determine the number of enzymes involved, heparanase activities will be purified from Chinese hamster ovary (CHO) cells, using enzyme assays that differentiate between a heparanse activity that degrades free heparan sulfate chains and heparanase activity that cleaves glycosaminoglycans off core proteins. The recognition sequence cleaved by the purified heparanase(s) will be analyzed to establish the modifications important for the enzymes(s) to bind to and cleave the glycosaminoglycan chain, while the size of the heparan sulfate formed in reactions will be obtained to determine if the glycosaminoglycan is cleaved randomly or progressively. The short heparan sulfate chains in CHO cells will be purified and characterized to determine whether heparanases generate populations of glycosaminoglycans enriched in specific modifications or locations inside cells, and to determine their ability to bind molecules that interact with heparan sulfate to determine if heparanases produce short chains that in turn regulate proteoglycan functions. %%% Heparan sulfate proteoglycans, found inside cells or at cell surfaces, are composed of one or more g lycosaminoglycan, or sugar, chains attached to a protein. They have been implicated in cell processes, including adhesion, growth and differentiation. Little is known about how they work. It appears that heparan sulfate proteoglycans are degraded inside cells by enzymes called heparanases. Heparanase-dependent release the heparan sulfate glycosaminoglycans produced from larger proteins are then degraded to short oligosaccharides. Some of these short heparan sulfate oligosaccharide chains are secreted from cells and affect the binding of proteins to the cell surface. The aims of this project are to (1) purify heparanases from Chinese hamster ovary cells, (2) to characterize how the enzymes work to cleave the heparan sulfate glycosaminoglycans, and (3) determine if the short heparan sulfate chains produced by the heparanases regulate binding of growth factors at the surface of cells. ***
