The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed assembly or restructuring of the collagenous extracellular matrix.

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