Binding affinities of vascular endothelial growth factor (VEGF) for heparin-derived oligosaccharides

Abstract

Heparin and heparan sulphate (HS) exert their wide range of biological activities by interacting with extracellular protein ligands. Among these important protein ligands are various angiogenic growth factors and cytokines. HS-binding to vascular endothelial growth factor (VEGF) regulates multiple aspects of vascular development and function through its specific interaction with HS. Many studies have focused on HS-derived or HS-mimicking structures for the characterization of VEGF165 interaction with HS. Using a heparinase 1-prepared small library of heparin-derived oligosaccharides ranging from hexasaccharide to octadecasaccharide, we systematically investigated the heparin-specific structural features required for VEGF binding. We report the apparent affinities for the association between the heparin-derived oligosaccharides with both VEGF165 and VEGF55, a peptide construct encompassing exclusively the heparin-binding domain of VEGF165. An octasaccharide was the minimum size of oligosaccharide within the library to efficiently bind to both forms of VEGF and that a tetradecasaccharide displayed an effective binding affinity to VEGF165 comparable to unfractionated heparin. The range of relative apparent binding affinities among VEGF and the panel of heparin-derived oligosaccharides demonstrate that VEGF binding affinity likely depends on the specific structural features of these oligosaccharides including their degree of sulphation and sugar ring stereochemistry and conformation. Notably, the unique 3-O-sulpho group found within the specific antithrombin binding site of heparin is not required for VEGF165 binding. These findings afford new insight into the inherent kinetics and affinities for VEGF association with heparin and heparin-derived oligosaccharides with key residue specific modifications and may potentially benefit the future design of oligosaccharide-based anti-angiogenesis drugs.

Synergistic Binding of Vascular Endothelial Growth Factor-A and Its Receptors to Heparin Selectively Modulates Complex Affinity

“…Free protein concentration ([protein] F ) at each inhibitor (i) concentration was calculated using Equation 4; briefly, the ratio of maximal binding response of protein plus inhibitor (R max i) to protein alone (R max ) times the total protein concentration ([protein] T ). The values were plotted against inhibitor concentration and data points were fit to Equation 5 to obtain apparent binding constants (K D ) for each oligosaccharide and modified heparins as previously described (39).…”

“…The structural requirements for heparin to bind to NRP-1 are distinct from those for binding VEGF 165 determined in previous studies (37,39). Thus, we were interested in determining the size and structural features required for heparin to enhance VEGF 165 binding to VEGFR-2 and NRP-1.…”

Pharmacology of Heparin and Related Drugs

“…Crystal structure of FGF-2/FGFR1/heparin (1FQ9.pdb) FGF-2/FGFR1/heparin decamer 2:2:2 (Schlessinger et al, 2000) FGF-4, FGF-7, FGF-8b Affinity chromatography of HS fragments on immobilized FGF All three FGFs bind to the same HS fragments with similar affinity; no evidence for differential binding to specific sequences (Kreuger et al, 2005) FGFR NMR spectroscopy and molecular modeling Heparin induces dimerization of FGFR heparin binding domain without FGF ) VEGF SPR on immobilized heparin Heparin octasaccharide minimum; the AT-binding motif not necessary (Zhao et al, 2012) NMR titrations and molecular modeling…”

Triethyl orthoformate covalently cross-linked chitosan-(poly vinyl) alcohol based biodegradable scaffolds with heparin-binding ability for promoting neovascularisation

“…In particular they have used heparin loaded with VEGF to bind and stabilize VEGF which has a short half -life when added to wound beds on its own [14][15][16] .…”