VEGF-A165 displays multiple effects through binding to KDR (VEGFR-2). Heparin/heparan sulfate-like molecules are known to greatly modulate their interaction. In fact, VEGF-A lacking a C-terminal heparin-binding region exhibits significantly reduced mitogenic activity. We recently found novel heparin-binding VEGFs in snake venom, designated VEGF-Fs, which specifically recognize KDR, rather than other VEGF receptors. VEGF-Fs virtually lack the C-terminal heparin-binding region when compared with other heparin-binding VEGF subtypes, despite their heparin-binding potential. The C-terminal region does not exhibit any significant homology with other known proteins or domains. In this study, we attempted to identify the heparin-binding region of VEGF-F using synthetic peptides. The C-terminal peptide of vammin (one of the VEGF-Fs, 19 residues) bound to heparin with similar affinity as native vammin. We then evaluated the effects of this peptide on the biological activity of VEGF-A165. The C-terminal peptide of VEGF-F exhibited specific blockage of VEGF-A165 activity both in vitro and in vivo. These observations demonstrate that the short C-terminal region of VEGF-F functions fully as the active heparin-binding domain and the corresponding peptide specifically blocks VEGF-A165, thus suggesting that the C-terminal heparin-binding region of VEGF-F recognizes similar heparin/heparan sulfate molecules as VEGF-A165. The present results will provide novel insight into VEGF-heparin interaction and may facilitate the design of new anti-VEGF drugs based on novel strategies.
Vascular endothelial growth factor-A165 (VEGF-A165) exhibits diverse biological effects through binding to its receptor KDR (VEGFR-2). Heparin-like molecules are known to modulate their interaction. There have been reports that VEGF-A lacking the C-terminal heparin binding region significantly reduced mitogenic activity. Recently, we found novel heparin-binding VEGFs from snake venoms, designated VEGF-Fs, which specifically recognize kinase domain containing receptor (KDR). The C-terminal heparin-binding region is almost completely absent in VEGF-Fs when compared with other heparin-binding VEGFs, despite their heparin-binding potential. In this congress, we report that the C-terminal heparin-binding region of VEGF-F specifically/preferentially interacts with the VEGF-bondable heparin/heparan sulfate, but not with those associated with bFGF or TFPI. We also present the identification of a VEGF receptor-binding protein from the venom of eastern cottonmouth (Agkistrodon piscivorus piscivorus). Sequence analysis revealed the isolated KDR-binding protein (designated KDR-bp) is identical to Lys49PLA2, an inactive PLA2 homologue with strong myoxicity. KDR-bp binds to the extracellular domain of KDR with subnanomolar affinity. The interaction between KDR-bp and KDR was blocked by VEGF-A165, and KDR-bp specifically inhibited VEGF-A165-stimulated endothelial cell proliferation, indicating KDR-bp is an antagonistic ligand for KDR. This is the first observation demonstrating that an exogenous factor antagonizes the VEGF receptor, furthermore, it is the first identification of the target molecule of the myotoxic PLA2 from viper venom.
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