MAPPING THE HEPARIN-BINDING SITES ON TYPE-I COLLAGEN MONOMERS AND FIBRILS

The glycosaminoglycan chains of cell surface heparan sulfate proteogly cans are believed to regulate cell adhesion, proliferation, and extrac ellular matrix assembly, through their interactions with heparin-bindi ng 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. S pring, 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, rem ains undescribed. Here we used heparin, a structural and functional an alogue of heparan sulfate, as a probe to study the nature of the hepar an sulfate proteoglycan-binding site on type I collagen. We used affin ity 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. U sing affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (K-d's similar to 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Comple xes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of h eparin binding was observed near the NH2 terminus of procollagen. Nati ve or reconstituted type I collagen fibrils showed one region of signi ficant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the ''a'' bands re gion. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-b inding site near the NH,, terminus of the collagen monomer. Correlatin g 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 hepari n-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed asse mbly or restructuring of the collagenous extracellular matrix.