Endocrine Abstracts

VEGF is an endothelial-cell specific mitogen and is a critical regulator of developmental and reproductive angiogenesis. VEGF165 is the most abundant isoform, with the N-terminal 110 aa domain containing the receptor binding site and the C-terminal 55 aa domain containing a heparin-binding site. The activity of the molecule is mediated through its interaction with two main receptors, VEGFR-1 and VEGFR-2, the binding to which is modulated by both cell surface-associated glycosaminoglycans and exogenous heparin or heparan sulphate (HS). Structural and functional characterisation of the interaction between VEGF and heparin/HS may lead to the development of specific inhibitors of pathological angiogenesis. Applying a docking algorithm with model heparin oligosaccharide probes, a heparin-binding site was predicted to lie in a semi-circular protein cleft in the C-terminal domain. This hypothesis was tested by site-directed mutagenesis of selected arginine and lysine residues of VEGF165, both within the predicted heparin-binding region and adjacent to it. A heparin-coated microplate assay was developed to assess the heparin binding properties of the mutants and wild-type protein. Single-site mutations of the four residues predicted to be most likely involved in the interaction (R124, K140, R145, R159) had no significant effect on heparin-binding. However increasing effects on the attenuation of binding were seen with double mutants e.g. R124/R159 and almost complete abolition of heparin binding was attained with alteration of all four residues. Reduction in heparin binding was not due to complete disruption of molecular structure: the mutants retained biological activity as measured by VEGF-dependent in vitro survival of HUVECs (human umbilical vein endothelial cells), indicating integrity of the receptor binding sites.

Modulation of heparin binding and bioactivity were compatible with the theoretical predictions, identifying a heparin binding site occupying the central part of the elongated C-terminal domain.