Isolation of two basic phospholipases A2 from Bothrops diporus snake venom: Comparative characterization and synergism between Asp49 and Lys49 variants

Bothrops diporus, previously considered a subspecies of the B. neuwiedi complex, is a medically relevant viperid in Northeastern Argentina. The venom of this species causes local tissue damage characterized by myonecrosis, hemorrhage, blistering, and edema. In the present study, two basic phospholipases A(2) (PLA(2)-I and PLA(2)-II) were isolated from this venom, and their pathological effects upon murine skeletal muscle and myogenic cells in culture were analyzed. Partial amino acid sequencing showed that PLA(2)-I and PLA(2)-II are Asp49 and Lys49 PLA(2)s, respectively. In agreement with this, PLA(2)-I showed PLA(2) activity, whereas PLA(2)-II did not. Functional assays revealed differences in their myotoxicity, cytotoxicity, and anti-adhesion activity, and in the ability to inhibit cell migration, all of which were greater for the Lys49 variant. Native electrophoresis showed that PLA(2)-I was less basic than PLA(2)-II. The two proteins act synergistically to affect the integrity of C2C12 myogenic cells, providing a further example of the concerted action of coexisting snake venom components. PLA(2)-I and PLA(2)-II, together with additional basic PLA(2)s revealed by RP-HPLC, probably play an important role in myonecrosis after envenomation by B. diporus.