Interisland Evolution of Trimeresurus flavoviridis Venom Phospholipase A 2 Isozymes Takahito Chijiwa, 1 Yoko Yamaguchi, 2 Tomohisa Ogawa, 2 Masanobu Deshimaru, 2 Ikuo Nobuhisa, 2 Kinichi Nakashima, 2 Naoko Oda-Ueda, 1 Yasuyuki Fukumaki, 3 Shosaku Hattori, 4 Motonori Ohno 1 1 Department of Applied Life Science, Faculty of Engineering, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan 2 Laboratory of Biochemistry, Faculty of Science, Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan 3 Institute of Genetic Information, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan 4 Institute of Medical Science, University of Tokyo, Oshima-gun, Kagoshima 894-1531, Japan Received: 12 July 2002 / Accepted: 30 September 2002 Abstract. Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan. A phospholipase A 2 (PLA 2 ), named PL-Y, was isolated from Okinawa T. flavoviridis venom and its amino acid sequence was determined from both protein and cDNA. PL-Y was unable to induce edema. In contrast, PLA-B, a PLA 2 from Tokunoshima T. flavoviridis venom, which is different at only three positions from PL-Y, is known to induce edema. A new PLA 2 , named PLA-B 0 , which is similar to PLA-B, was cloned from Amami-Oshima T. flavoviridis venom gland. Three T. flavoviridis venom basic [Asp 49 ]PLA 2 isozymes, PL-Y (Okin- awa), PLA-B (Tokunoshima), and PLA-B 0 (Amami- Oshima), are identical in the N-terminal half but have one to four amino acid substitutions in the b1-sheet and its vicinity. Such interisland sequence diversities among them are due to isolation in the different en- vironments over 1 to 2 million years and appear to have been brought about by natural selection for point mutation in their genes. Otherwise, a major PLA 2 , named PLA2, ubiquitously exists in the ven- oms of T. flavoviridis snakes from the three islands with one to three synonymous substitutions in their cDNAs. It is assumed that the PLA2 gene is a pro- totype among T. flavoviridis venom PLA 2 isozyme genes and has hardly undergone nonsynonymous mutation as a principal toxic component. Phyloge- netic analysis based on the amino acid sequences re- vealed that T. flavoviridis PLA 2 isozymes are clearly separated into three groups, PLA2 type, basic [Asp 49 ]PLA 2 type, and [Lys 49 ]PLA 2 type. Basic [Asp 49 ]PLA 2 -type isozymes may manifest their own particular toxic functions different from those of the isozymes of the PLA2 type and [Lys 49 ]PLA 2 type. Key words: Interisland evolution — Trimeresurus flavoviridis — Phospholipase A 2 — Amino acid sequence — cDNA — Point mutation — Pro- totype — Phylogeny Introduction Trimeresurus flavoviridis (Crotalinae) snakes inhabit the southwestern islands of Japan: Okinawa, Tok- unoshima, and Amami-Oshima. Their venoms con- tain phospholipase A 2 (PLA 2 ) [EC 3.1.1.4] isoforms as major toxic components. PLA 2 catalyzes the hy- drolysis of the 2-acyl ester bond of 3-sn-phospho- glycerides with the requirement of Ca 2+ to produce 3-sn-lysophosphoglycerides and fatty acids. Snake venom PLA 2 ’s are classified into groups I and II J Mol Evol (2003) 56:286–293 DOI: 10.1007/s00239-002-2400-7 The nucleotide sequences reported in this paper are available from the GenBank/EMBL/DDBJ databases under accession numbers AB072173–AB072175 and AB087496. Correspondence to: Takahito Chijiwa; email: chijiwa@life.sojo-u. ac.jp