Heparan sulfates (HS) are abundantly expressed on the cell surface and in the extracellular matrix as proteoglycans with differential sulfation pattern, chain length and number of chains leading to vast structural features. Each chain interacts simultaneously with numerous proteins including a variety of signaling molecules, morphogens, receptors, etc and orchestrates various biological functions. Modulation of their biosynthetic pathways has begun to shed light on their role in such model organisms as zebra fish, c. elegans, drosophila, etc. However, the lack of efficient synthetic approaches coupled with a small amount of HS chains isolated from animal models has limited our knowledge of how these molecules regulate various physiological and pathological processes at the molecular level. The central hypothesis of this proposal is that multiple oligosaccharide sequences with conserved critical groups may elucidate the same biological response or that a given oligosaccharide sequence with a set of critical groups may elucidate more than one biological response. Determining those critical groups and their spatial distance would be sufficient to determine the rules that dictate HS-protein interactions. Development of a general enzymatic approach for the rapid assembly of HS of various sizes should allow us to test our hypothesis and to delineate the rules that dictate HS-protein interactions. We propose to construct the oligosaccharide libraries and screen their ability to form signaling complex with various growth factors and receptors that play a central role in heart, lung and brain development. With the aid of stable isotopes, we will then develop novel structural biology approaches to elucidate the critical groups that attribute a biological function. The knowledge gained in synthesizing ATIII binding sites will be used in designing diverse size-based HS libraries that should be the key to defining all interacting proteins and elucidating the chemical rules by which such interactions take place. This proposed study should establish a firm foundation for the glycobiology of proteoglycans. ? ? ?
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