Heparin-like glycosaminoglycans (GAGs) are highly complex acidic polysaccharides. The complexity arises due to heterogeneity in chemical composition of the repeat unit and conformational flexibility of the sugar unit. As a consequence of the structural and compositional polymorphism, they interact with several proteins in the extra-cellular matrix and influence a variety of cellular processes including cell adhesion, migration, growth, proliferation, and differentiation. Heparinases are bacterial enzymes that depolymerize heparin-like molecules; different heparinases recognize and cleave at specific sites or regions of the substrate hence, they have unique substrate specificities. Heparinases are important reagents in the tool kit of molecular and cell biology, and are also as emerging therapeutic agents. These enzymes are used in clinical applications in the monitoring (approved by the FDA) and neutralization of heparin in blood (in phase II clinical trials). As biochemical reagents, heparinases have become indispensable in studying the role of heparin-like GAGs in several physiological as well as pathological processes, including the extracellular matrix, regulation of growth factor and cytokine function, inflammation, cancer metastasis and infection. Despite the well established role and significance of heparinases, these enzymes belong to a class of polysaccharide degrading lyases for which little mechanistic information has been obtained. The preliminary studies carried out by our group on the cloning, recombinant expression and biochemical characterization of heparinase I and the cloning of heparinase II and II, provide a framework for an in-depth analysis of heparin degrading enzymes as proposed in this application. This analysis would yield valuable information on how this important class of molecules act. Therefore, this grant aims at investigating the structure-function relationship of heparinases with the goal of elucidating the catalytic mechanism and substrate specificities governing degradation of heparin-like GAGs. This study will not only provide the necessary background to our understanding of heparin degradation and of protein-complex polysaccharide interactions, but will facilitate the development of novel heparinase mutzymes with altered specificities, which could be potentially useful for different applications including sequencing heparin-like GAGs. In conclusion, this study represents the first detailed investigation of the catalytic mechanism of complex polysaccharide degrading enzymes.
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