Structural requirements of echistatin for the recognition of alpha(v)beta(3) and alpha(5)beta(1) integrins

There are key differences between the amino acid residues of the RGD loops and the C termini of echistatin, a potent antagonist of alpha(IIb)beta(3), alpha(v)beta(3) and alpha(5)beta(1), and eristostatin, a similar disintegri n selectively inhibiting alpha(IIb)beta(3),. In order to identify echistati n motifs required for selective recognition of alpha(v)beta(3) and alpha(5) beta(1) integrins, we expressed recombinant echistatin, eristostatin, and 1 5 hybrid molecules. We tested them for their ability to inhibit adhesion of different cell lines to fibronectin and von Willebrand factor and to expre ss ligand-induced binding site epitope. The results showed that Asp(27) and Met(28) support recognition of both alpha(v)beta(3) and alpha(5)beta(1). R eplacement of Met(28) with Asn completely abolished echistatin's ability to recognize each of the integrins, while replacement of Met(28) with Leu sel ectively decreased echistatin's ability to recognize alpha(5)beta(1) only. Eristostatin in which C-terminal WNG sequence was substituted with HKGPAT e xhibited new activity with alpha(5)beta(1), which was 10-20-fold higher tha n that of wild type eristostatin, A hypothesis is proposed that the C termi nus of echistatin interacts with separate sites on beta(1) and beta(3) inte grin molecules.