Island specific expression of a novel [Lys49]phospholipase A2 (BPIII) in Protobothrops flavoviridis venom in Amami–Oshima, Japan

In search of the transcripts expressed in Protobothrops flavoviridis venom gland, 466 expressed sequence tags (ESTs) were generated from the venom gland cDNA library of P. flavoviridis in Amami–Oshima, Japan. The sequencing of randomly selected cDNA clones followed by identification in similarity search against existing databases led to the finding of a novel lysine-49-phospholipase A2 ([Lys49]PLA2) clone. It coded for one amino acid-substituted BPII homologue or two amino acids-substituted BPI homologue in which BPII and BPI are [Lys49]PLA2s contained in Amami–Oshima and Tokunoshima P. flavoviridis venoms. This isozyme, named BPIII, was isolated from Amami–Oshima P. flavoviridis venom. BPIII gave a specific [M + 2H]2+ peak of m/z 736.3 on mass spectrometry (MS) analysis after S-carboxamidomethylation and trypsin digestion when compared with BPII. It became evident from MS analysis after S-carboxamidomethylation and trypsin digestion of the mixed protein peaks ranging from BPI to BPII obtained by fractionation on a carboxymethyl cellulose column of Amami–Oshima and Tokunoshima P. flavoviridis venoms that BPIII protein is contained in Amami–Oshima P. flavoviridis venom but not in Tokunoshima P. flavoviridis venom. It is for the first time that a protein present in Amami–Oshima P. flavoviridis venom is not found in Tokunoshima P. flavoviridis venom.