Proteoglycans major components of the extracellular matrix, bind a vast array of proteins that regulate diverse biological processes. Heparan sulfate is one of the important proteoglycans found in the extracellular matrix. Past studies have focused on heparin, a related glycosaminoglycan widely used as a drug and available in large quantities. Heparin and heparan sulfate glycosaminoglycans have many of the same structural features and bind a number of proteins that are found in the extracellular matrix including vitronectin and fibroblast growth factors. Clearly, binding of acidic polysaccharides to proteins is a common biological event that may have very important consequences. Unfortunately, the primary structural requirements, for either protein or oligosaccharide, that give rise to this binding are largely unknown. Molecular modeling and some experimental data predict that heparin-binding proteins contain specific patterns of basic amino acids (consensus sequences) with high affinity for acidic oligosaccharides. Nevertheless, these requirements are only now being studied and the oligosaccharide requirements for binding to protein or peptide have not been reported. This study will examine the hypothesis that certain oligosaccharide sequences in heparin and heparan sulfate have high affinity for consensus peptide sequences in proteins present in the extracellular matrix. A variety of peptides based on sequences in acidic fibroblast growth factor and vitronectin, predicted to have high affinity for oligosaccharide, will be synthesized. These peptides will be used to isolate high affinity oligosaccharides, which will be examined for composition and sequence. Oligosaccharide-peptide interactions will be examined for: l. binding affinity (Kd): 2. binding specificity; 3. binding thermodynamics; 4. binding orientation; and 5. binding site structural requirements. Peptides will also be synthesized to contain variations of the consensus sequence and modified oligosaccharides will be synthesized to help establish the structure activity relationship for oligosaccharide-peptide interaction. Native protein will be compared with synthetic peptide to confirm specificity of binding. Vitronectin and recombinant acidic fibroblast growth factor are the proteins to be studied because of the important biological consequences of their binding to heparin/heparan sulfate. Ultimately, these studies should lead to custom designed specific oligosaccharide molecules that have increased protein binding affinity and custom designed peptides that have increased oligosaccharide binding affinity to enhance or attenuate the activities associated with oligosaccharide-protein interaction. These oligosaccharides and peptides represent the starting point for new drug design focused on peptido- mimetics and sulfated oligosaccharide-mimetics. Such new therapeutic agents might be used to regulate a variety of biological processes including angiogenesis, metastasis, atherogenesis, immune responses, HIV infection, and thrombosis.
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