VEGF165-binding Sites within Heparan Sulfate Encompass Two Highly Sulfated Domains and Can Be Liberated by K5 Lyase

Christopher J Robinson,Barbara Mulloy,John T Gallagher,Sally E Stringer

doi:10.1074/jbc.m510760200

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.

Highlights

Logical conditions that are driven by inappropriate or excessive angiogenesis, such as diabetic retinopathy, rheumatoid arthritis, and the growth of solid tumors [3]
Specificity of the VEGF165 Interaction with heparan sulfate (HS)—A number of structurally related GAG species are present at the cell surface and in the extracellular matrix, all of which could potentially interact with VEGF165
To investigate whether VEGF165 binds preferentially to HS, a variety of unlabeled GAGs were tested in filter binding assays (FBAs; see “Experimental Procedures”) for their ability to compete with 3H-labeled NIH 3T3 fibroblast HS (3T3-HS) for VEGF165 (Fig. 1A)

Summary

EXPERIMENTAL PROCEDURES

All GAG samples were desalted on a Sepharose PD-10 column prior to use in filter binding assays. Intact HS chains and the HS polysaccharide products of heparinase I, K5 lyase, and platelet extract digests were resolved on a Sepharose CL-6B column (1.5 ϫ 100 cm) or a Superdex 75 HR 10/30 fast protein liquid chromatography column. The Sepharose CL-6B column was run at a flow rate of 6 ml/h in 0.25 M (NH4)HCO3, and 1-ml (15 min) fractions were collected. HS saccharides produced by low pH nitrous acid or heparinase III treatment were resolved on a Bio-Gel P-10 column (1 ϫ 120 cm) at a flow rate of 4 ml/h in 0.25 M (NH4)HCO3, and 1-ml (15 min) fractions were collected. Samples pooled from the ProPac PA1 column (dp – subspecies) were desalted again (Sepharose PD-10) prior to use in VEGF165 binding assays.

RESULTS

NS disaccharides

DISCUSSION