Characterization, amino acid sequence and evolution of edema-inducing, basic phospholipase A₂ from Trimeresurus flavoviridis venom

Two phospholipases A₂ (PLA₂s) were purified from the venom of Trimeresurus flavoviridis (Crotalinae) inhabiting Tokunoshima island, Japan, and named PLA-A and PLA-B in the order of elution on a cation-exchange column. Lipolytic activities of PLA-A and PLA-B toward mixed micelles and liposomes were substantially lower than that of PLA2 (an [Asp⁴⁹]PLA₂) which had been isolated from the same venom. Both PLA-A and PLA-B consisted of 122 amino acids and contained aspartate at position 49 (the numbering according to the aligned sequences of PLA₂s in Fig. 8), thus belonging to an [Asp⁴⁹]PLA₂ subgroup. PLA-A and PLA-B were identical in sequence with an exception at position 79. PLA-B contained Asn-Gly at positions 79 and 80 which are located in the β-sheet region. On the other hand, PLA-A had β-Asp-Gly and α-Asp-Gly in high and low proportion, respectively, at the corresponding positions which were produced from Asn-Gly through the base-catalyzed formation and hydrolysis of the succinimide type intermediate. Thus, PLA-A is derived from PLA-B. PLA-B is similar in sequence to PL-X, which had been purified from the venom of T. flavoviridis inhabiting Amami-Oshima island, Japan, and to PL-X′, whose cDNA had been cloned from Tokunoshima T. flavoviridis venom gland, rather than PLA2. PLA-B showed strong edema-inducing activity, while PLA-A exhibited rather lower activity. The sequence around position 79 which constitutes a β-turn segment seems to be crucial for edema-inducing activity. Phylogenetic tree of Tokunoshima T. flavoviridis venom PLA₂ isozymes indicated that PLA-B and PL-X′ diverged from PLA2 after branching of [Asp⁴⁹]PLA₂ forms and [Lys⁴⁹]PLA₂ forms.