title: VEGF165-binding sites within heparan sulfate encompass two highly sulfated domains and can be liberated by K5 lyase.
abstract: The vascular endothelial growth factor (VEGF) family of proteins controls the formation and growth of blood vessels. The most potent and widely expressed isoform, VEGF165, 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 VEGF165 for its signaling receptors. We have determined a number of key HS structural features that mediate the specific binding of the VEGF165 dimer. Carboxylate groups and 2-O-, 6-O-, and N-sulfation of HS contributed to the strength of the VEGF165 interaction; however, 6-O-sulfates appeared to be particularly important. Cleavage of HS by heparinase, heparitinase, or heparanase severely reduced VEGF165 binding. In contrast, K5 lyase-cleaved HS retained significant VEGF165 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 VEGF165 monomer. The data presented are consistent with a model whereby the two heparin-binding sites of the VEGF165 dimer interact simultaneously with highly sulfated S-domain regions of the HS chain that can be linked through a stretch of transition sequence.
authors.affiliation: Cancer Research UK and University of Manchester Department of Medical Oncology, Christie Hospital National Health Service Trust, Wilmslow Road, Manchester M20 4BX, United Kindgom. Christopher.Robinson@manchester.ac.uk
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: Robinson
authors.forename: Barbara
authors.initials: B
authors.lastname: Mulloy
authors.forename: John T
authors.initials: JT
authors.lastname: Gallagher
authors.forename: Sally E
authors.initials: SE
authors.lastname: Stringer
mesh_terms: D016475:3T3 Cells; D000818:Animals; D002236:Carbohydrate Conformation; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D019281:Dimerization; D004187:Disaccharides / Q000737:chemistry; D006025:Glycosaminoglycans / Q000378:metabolism; D006497:Heparan Sulfate / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051379:Mice; D008958:Models, Molecular; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012754:Sharks; D042461:Vascular Endothelial Growth Factor A / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004187:Disaccharides; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C467484:VEGFA protein, human