Abstract

The interaction of fibronectin with cell surface heparan sulfate proteoglycans is important biologically in inducing reorganization of the cytoskeleton and the assembly of focal adhesions. The major heparan sulfate-binding site in fibronectin, which is also implicated in these morphological events, is the COOH-terminal Hep-2 domain. We describe the first extensive study of the structural determinants required for the interaction between heparan sulfate/heparin and Hep-2. It is clear that, in heparan sulfate, there is a very prominent role for N-sulfate groups, as opposed to a relatively small apparent contribution from carboxyl groups. Furthermore, a minimal octasaccharide binding sequence appeared to contain at least two 2-O-sulfated iduronate residues, but no 6-O-sulfate groups. However, affinity was enhanced by the presence of 6-O-sulfates, and the interaction with Hep-2 also increased progressively with oligosaccharide size up to a maximum length of a tetradecasaccharide. This overall specificity is compatible with recent information on the structure of Hep-2 (Sharma, A., Askari, J. A., Humphries, M. J., Jones, E. Y., and Stuart, D. I. (1999) EMBO J. 18, 1468-1479) in which two separate, positively charged clusters, involving up to 11 basic amino acid residues (mostly arginines with their preferential ability to co-ordinate sulfate groups), could form a single extended binding site.