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, 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.
Robinson
Christopher J
CJ
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
Mulloy
Barbara
B
Gallagher
John T
JT
Stringer
Sally E
SE
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*
D004187:Disaccharides; D006025:Glycosaminoglycans; D006497:Heparan Sulfate; D011994:Recombinant Proteins; D042461:Vascular Endothelial Growth Factor A; C467484:VEGFA protein, human