Interisland variegation of venom [Lys49]phospholipase A2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan

• Three [Asp49]PLA2s and one [Lys49]PLA2, named BPI, were identified from Protobothrops tokarensis (Kodakarajima island) venom.
• The cDNAs encoding three [Asp49]PLA2s and BPI and the gene encoding BPI were cloned and sequenced.
• P. flavoviridis (Amami-Oshima island) venom have three [Lys49]PLA2s, thus showing interisland variegation in their numbers.
• PtBPI gene has LINE-1 fragment inserted into its second intron, thus duplication of PtBPI gene appears to be restricted.

Protobothrops tokarensis (Pt), a Crotalinae snake, inhabits only Takarajima and Kodakarajima islands of the Tokara Islands located in the immediate north of Amami-Oshima island of Japan. Kodakarajima P. tokarensis venom gland cDNA library gave four types of phospholipase A2 (PLA2) cDNAs encoding neutral [Asp49]PLA2, basic [Asp49]PLA2, highly basic [Asp49]PLA2, and [Lys49]PLA2. As the amino acid sequences encoded by their open reading frames (ORFs) were identical to those of PLA2, PLA-B, PLA-N, and BPI (a [Lys49]PLA2), respectively, from Amami-Oshima P. flavoviridis (Pf) venom, they were named PtPLA2, PtPLA-B, PtPLA-N, and PtBPI. Chromatography of P. tokarensis venom gave three PLA2 isozymes, PtPLA2, PtPLA-B, and PtBPI. However, BPII and BPIII ([Lys49]PLA2s) expressed in Amami-Oshima P. flavoviridis venom were not found in P. tokarensis venom. Genomic polymerase chain reaction (PCR) for P. tokarensis liver DNAs with the unique primers gave PtBPI gene. Notably it was found that LINE (long interspersed nuclear element)-1 fragment is inserted into second intron of PtBPI gene. The LINE-1 fragment may prevent duplication of PtBPI gene and thus formation of plural [Lys49]PLA2 genes in P. tokarensis genome. The interisland variegation of venom [Lys49]PLA2 isozyme genes in Protobothrops genus snakes in the southwestern islands of Japan is discussed.