Amino acid sequence of a basic aspartate-49-phospholipase A 2 from Trimeresurus flavoviridis venom and phylogenetic analysis of Crotalinae venom phospholipases A 2 Takahito Chijiwa a , Kazuki Abe a , Tomohisa Ogawa b , Nikolai N. Nikandrov c , Shosaku Hattori d , Naoko Oda-Ueda a , Motonori Ohno a, * a Department of Applied Life Science, Faculty of Engineering, Sojo University, Kumamoto 860-0082, Japan b Department of Biomolecular Science, Graduate School of Life Science, Tohoku University, Sendai 981-8555, Japan c Division of Biological Resources and Environmental Science, Graduate school of Kyushu University, Higashi-ku, Fukuoka 812-8581, Japan d Institute of Medical Science, University of Tokyo, Oshima-gun, Kagoshima 894-1531, Japan Received 4 March 2005; accepted 4 April 2005 Available online 20 June 2005 Abstract Trimeresurus flavoviridis snakes inhabit the southwestern islands of Japan: Amami-Oshima, Tokunoshima and Okinawa. A phospholipase A 2 (PLA 2 ) of basic nature (pI 8.5) was isolated from the venom of Amami-Oshima T. flavoviridis. Its amino acid sequence determined by the ordinary procedures was completely in accord with that predicted from the nucleotide sequence of the cDNA previously cloned from Amami-Oshima T. flavoviridis venom gland, which was named PLA-B 0 . It consists of 122 amino acid residues and has aspartate at position 49. It induced edema in a mouse footpad assay and caused necrosis in mouse skeletal muscles. PLA-B 0 is similar in sequence to PLA-B (Tokunoshima) and PL-Y (Okinawa), both basic [Asp 49 ]PLA 2 s, with a few amino acid substitutions, indicating occurrence of interisland mutation. Although PLA 2 s of Crotalinae subfamily were phylogenetically classified into four types, PLA2 (acidic or neutral [Asp 49 ]PLA 2 ) type, basic [Asp 49 ]PLA 2 type, neurotoxic [Asp 49 ]PLA 2 type and [Lys 49 ]PLA 2 type, it was ascertained that PLA 2 s of PLA2 type and [Lys 49 ]PLA 2 type are most essential as toxic components for Crotalinae snake venoms and that basic [Asp 49 ]PLA 2 -type PLA 2 s are uniquely contained only in the venoms of T. flavoviridis species. Prediction of physiological activities of some PLA 2 s was made based on their location in the phylogenetic tree. Relationship of divergence of PLA 2 s via accelerated evolution followed by less rapid mutation and physiological activities was discussed. q 2005 Elsevier Ltd. All rights reserved. Keywords: Phospholipase A 2 ; Amino acid sequence; Trimeresurus flavoviridis; Crotalinae subfamily; Evolution; Phylogenetic tree 1. Introduction Phospholipase A 2 (PLA 2 , EC 3.1.1.4) catalyses the hydrolysis of the 2-acyl ester bond of 3-sn-phosphogly- cerides with the requirement of Ca 2C to produce 3-sn-lysophosphoglycerides and fatty acids (Dijkstra et al., 1981, 1983). Snake venoms contain PLA 2 isoforms as major toxic components. Snake venom PLA 2 s are classified into groups I and II based on the mode of disulfide pairings (Dufton and Hider, 1983). Group I PLA 2 s are found in Elapidae (Elapinae and Hydrophiinae) venoms, while group II PLA 2 s are in Viperidae (Viperinae and Crotalinae) venoms. Group II PLA 2 s are divided into two subgroups, [Asp 49 ]PLA 2 forms and [Lys 49 ]PLA 2 Toxicon 46 (2005) 185–195 www.elsevier.com/locate/toxicon 0041-0101/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.toxicon.2005.04.004 * Corresponding author. Tel.: C81 96 326 3111; fax: C81 96 323 1331. E-mail address: chijiwa@life.sojo-u.ac.jp (M. Ohno).