VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase

The vascular endothelial growth factor (VEGF) family of proteins controls the formation and growth of blood vessels. The most potent and widely expressed isoform, VEGF(165), is secreted as a disulfide-linked homodimer with two identical heparin-binding sites. Interactions with heparan sulfate (HS) regulate the diffusion, half-life, and affinity of VEGF(165) for its signaling receptors. We have determined a number of key HS structural features that mediate the specific binding of the VEGF(165) dimer. Carboxylate groups and 2-O-, 6-O-, and N-sulfation of HS contributed to the strength of the VEGF(165) interaction; however, 6-O-sulfates appeared to be particularly important. Cleavage of HS by heparinase, heparitinase, or heparanase severely reduced VEGF(165) binding. In contrast, K5 lyase-cleaved HS retained significant VEGF(165) affinity, suggesting that binding sites for the growth factor are present within extended stretches of sulfation. Binding studies and molecular modeling demonstrated that an oligosaccharide 6 or 7 residues long was sufficient to fully occupy the heparin-binding site of a VEGF(165) monomer. The data presented are consistent with a model whereby the two heparin-binding sites of the VEGF(165) dimer interact simultaneously with highly sulfated S-domain regions of the HS chain that can be linked through a stretch of transition sequence.