Complete Structure of an Increasing Capillary Permeability Protein (ICPP) Purified from Vipera lebetina Venom

Abstract

The partial sequence of the increasing capillary permeability protein (ICPP) purified from Vipera lebetina venom revealed a strong homology to vascular endothelial growth factor (VEGF)-A. We now report its complete amino acid sequence determined by Edman degradation and its biological effects on mouse and human vascular endothelial cells. ICPP is a homodimeric protein linked by cysteine disulfide bonds of 25115 Da revealed by mass spectrometry. Each monomer is composed of 110 amino acids including eight cysteine residues and a pyroglutamic acid at the N-terminal extremity. ICPP shares 52% sequence identity with human VEGF but lacks the heparin binding domain and Asn glycosylation site. Besides its strong capillary permeability activity, ICPP was found to be a potent in vitro angiogenic factor when added to mouse embryonic stem cells or human umbilical vein endothelial cells. ICPP was found to be as potent as human VEGF165 in activating p42/p44 MAPK, in reinitiation of DNA synthesis in human umbilical vein endothelial cells, and in promoting in vitro angiogenesis of mouse embryonic stem cells. All these biological actions, including capillary permeability in mice, were fully inhibited by 1 M of a new specific VEGF receptor tyrosine kinase inhibitor (ZM317450) from AstraZeneca that belongs to the anilinocinnoline family of compounds. Indeed, up to a 30 times higher concentration of inhibitor did not affect platelet-derived growth factor, epidermal growth factor, FGF-2, insulin, ␣-thrombin, or fetal calf serum-induced p42/p44 MAPK and reinitiation of DNA synthesis. Therefore, we conclude that this venom-derived ICPP exerts its biological action (permeability and angiogenesis) through activation of VEGF receptor signaling (VEGF-R2 and possibly VEGF-R1).

A Novel Snake Venom Vascular Endothelial Growth Factor (VEGF) Predominantly Induces Vascular Permeability through Preferential Signaling via VEGF Receptor-1

“…During the course of our attempt to clarify the molecular actions of TfsvVEGF, two novel VEGF-like proteins derived from snake venom were reported by two groups, viz. ICPP (increasing capillary permeability protein) from Vipera lebetina venom (24) and vammin from Vipera ammodytes ammodytes venom (32). Both ICPP and vammin stimulate the proliferation of vascular endothelial cells ϳ1.5 times more potently than VEGF 165 .…”

“…Recently, several VEGF-like proteins have been isolated from snake venoms (22,23) and reported to have biological activity similar to VEGF 165 (24). However, several fundamental questions remain unanswered.…”

Response of endothelial cells to a dual tyrosine kinase receptor inhibition combined with irradiation

“…VTKI is a specific inhibitor of VEGFR-2 belonging to the anilinocinnoline family of compounds with an IC 50 of 0.05 F 0.01 Amol/L for VEGFR-2 and 0.5 F 0.04 Amol/L for VEGFR-1 (18).…”

“…After 24 hours, drugs gefitinib (1 Amol/L) and/or VTKI (10 Amol/L) were added 2 hours before growth factors (either 20 ng/mL EGF or 30 ng/mL VEGF). These drug concentrations were selected according to previous data (15,18). Cells were stimulated by growth factors for 10 or 20 minutes, lysed in Laemmli buffer, and analyzed by Western blotting for levels of phospho-EGFR, phosphoextracellular signal-regulated kinase-1/2 (phospho-p42/ p44), and phospho-Akt.…”

Identification of the Heparin-Binding Region of Snake Venom Vascular Endothelial Growth Factor (VEGF-F) and Its Blocking of VEGF-A165

“…Proline residues typically have considerable effect on peptide and protein structure, for example, in turn or helix formation. These residues are also conserved in VR-1 and other snake venom VEGFs (31,32), except for svVEGFs from the venom of Bothrops species (33), thus suggesting some role in conformation and heparin interaction.…”