Two vascular apoptosis-inducing proteins from snake venom are members of the metalloprotease/disintegrin family

Hemorrhagic snake venom induces apoptosis in vascular endothelial cells. In a previous report, we described the purification of a vascular apoptosis-inducing protein (VAP) from Crotalus atrox [Masuda, S., Araki, S., Kaji, K. & Hayashi, H. (1997) Biochem. Biophys. Res, Commun. 235, 59-63]. We report here the identification of a second vascular apoptosis-inducing protein, VAP2, in venom from C. atrox. When we fractionated crude venom from C. atrox by isoelectric focusing, we found two proteins with apoptosis-inducing activity, one was basic and the other acidic. The basic protein corresponded to VAP, and we named the acidic protein VAP2. VAP2 was a monomeric protein with molecular mass of 55 kDa and an isoelectric point of 4.5. VAP2 killed vascular endothelial cells in culture, and the death of cells exhibited the characteristic features of apoptotic activity of VAP2, seemed to be specific to endothelial cells, as reported for VAP. The half-lethal doses of VAP and VAP2 were 0.3 mu g/ml and 0.1 mu g/ml, respectively. Analysis of the partial amino acid sequences of VAP and VAP2 revealed similarities to members of the metalloprotease/disintegrin family. The sensitivity of VAP2 to changes in pH and temperature was distinct from that of VAP. Our results suggest that VAP and VAP2 are members of the metalloprotease/disintegrin family.