The extracellular environment contains a wide assortment of proteoglycans that interact in a complex manner with proteins and peptides resulting in a variety of important biological consequences. Heparan sulfate is one of the most important proteoglycans in the extracellular matrix. Heparin, a related glycosaminoglycan, has been much more extensively studied for the ability to interact with proteins because heparin is a readily available anticoagulant that is a commercial drug. Heparan sulfate and heparin have many similar structural features, and both interact with many extracellular matrix proteins. Experiments over the past 5 years have begun to provide an understanding of the structural requirements for these interactions to occur. Heparin and heparan sulfate binding proteins with specific sites rich in basic residues, often contained in secondary structural domains that bind acidic oligosaccharides with high specificity and affinity. Recent studies suggest that specific sequences also exist within glycosaminoglycans that have high specificity and affinity for binding protein partners. This application is based upon these new observations, and proposes to produce and evaluate "designer" glycosaminoglycans with increased specificity and affinity for binding to proteins. The first grant period allowed this laboratory to develop a much clearer understanding of the structural requirements for glycosaminoglycans and proteins to interact. In this renewal period, the laboratory will evaluate the hypotheses that 1) glycosaminoglycans from various tissues differ in binding affinities and specificities for proteins and peptides and 2) designer glycosaminoglycans can be produced that have enhanced affinity and specificity for bind partners. The primary aims are to: 1) examine tissue specific differences in heparan sulfate; 2) design and construct protein binding sites in glycosaminoglycans; and 3) perform fundamental structure-activity relationship studies to determine in much more detail, requirements for glycosaminoglycans and proteins to interact by studying thermodynamics, kinetics, precise contact points and other important factors that influence these interactions. The principal glycosaminoglycan target will be heparan sulfate, prepared from selected tissues of normal and knockout mice and modified with 3-O-sulfotransferase isoforms to construct structurally unique binding sites. The secondary aims are to develop high-through-put methods for screening glycosaminoglycan-protein interactions; expand the library of structurally defined oligosaccharides; and evaluate new biophysical methods for studying glycosaminoglycan-protein interactions.
NIH ExPorter
NIH ExPorter