Cloning, expression, and apoptotic activity of a PIII-SVMP isolated from Agkistrodon contortrix contortrix venom gland cDNA

Disintegrins and disintegrin-like peptides interact with various cell surface integrins and block cell-cell and cell-matrix interactions and induce apoptosis in various cell types. A novel disintegrin-like snake venom gene, Acocostatin, was isolated from the venom gland mRNA of Agkistrodon contortrix contortrix. The cDNA was cloned and expressed, and the role of the recombinant protein was tested on human umbilical vein cells (HUVECs). Translation of the cloned cDNA sequence revealed that acocostatin belongs to the PIII-SVMP subfamily of disintegrin proteins. It is composed of 139 amino acids with a Glu-Cys-Asp (ECD) disintegrin-like domain and conserved cysteine residues. Acocostatin showed 93% amino acid identity with other PIII-SVMPs such as VAP-1 (vascular apoptosis-inducing protein-1) and crotastatin-1. The recombinant protein had a molecular weight of 16 kDa when analyzed by SDS-PAGE. Incubation of actively proliferating HUVECs with 5 muM GST-acocostatin for 24 h induced 19.68% of early apoptotic cells. Incubation of HUVECs with 5 muM captothecin revealed 35% early apoptotic cells. Untreated and 5 muM GST-treated cells showed 1.2% and 2.4% early apoptotic cells, respectively, as detected by annexin V-FITC staining and flow cytometric analysis. Typical apoptotic changes such as nuclear condensation and DNA fragmentation were also observed when GST-cocostatin treated cells were stained with Hoechst 33342 and observed under fluorescence microscopy.