Purification, sequencing, and phylogenetic analyses of novel Lys-49 phospholipases A(2) from the venoms of rattlesnakes and other pit vipers

Basic phospholipase A(2) homologs with Lys49 substitution at the essential Ca2+-binding site are present in the venom of pit vipers under many genera. However, they have not been found in rattlesnake venoms before. We have no w screened for this protein in the venom of rattlesnakes and other less stu died pit vipers. By gel filtration chromatography and RP-HPLC, Lys49-phosph olipase-like proteins were purified from the venoms of two rattlers, Crotal us atrox and Crotalus m. molossus, and five nonrattlers, Porthidium nummife r, Porthidium godmani, Bothriechis schlegelii, Trimeresurus puniceus, and T rimeresurus albolabris. Their N-terminal amino acid sequences were shown to be characteristic for this phospholipase subfamily. The purified basic pro teins from rattlesnakes caused myonecrosis and edema in experimental animal s. We have also cloned the cDNAs and solved the complete sequences of four novel Lys49-phospholipases from the venom glands of C. atrox, P. godmani, B . schlegelii, and Deinagkistrodon acutus (hundred-pace). Phylogenetic analy ses based on the amino acid sequences of 28 Lys49-phospholipases separate t he pitviper of the New World from those of the Old World, and the arboreal Asiatic species from the terrestrial Asiatic species. The implications of t he phylogeny tree to the systematics of pit vipers, and structure-function relationship of the Lys49-phospholipases are discussed. (C) 2001 Academic P ress.