Elucidation of the structural features of heparan sulfate important for interaction with the Hep-2 domain of fibronectin

The interaction of fibronectin with cell surface heparan sulfate proteoglyc ans is important biologically in inducing reorganization of the cytoskeleto n and the assembly of focal adhesions. The major heparan sulfate-binding si te 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 s ulfate/heparin and Hep-a. 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 octasacc haride binding sequence appeared to contain at least two 2-O-sulfated iduro nate residues, but no 6-O-sulfate groups. However, affinity was enhanced by the presence of 6-O-sulfates, and the interaction with Hep-a also increase d progressively with oligosaccharide size up to a maximum length of a tetra decasaccharide, This overall specificity is compatible with recent informat ion on the structure of Hep-a (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 fl basic amino acid residues (mostly arginines with their preferential ability to co-ordinate s ulfate groups), could form a single extended binding site.