Bothropstoxin-I (BthTx-I) is a myotoxic phospholipase A2 variant present in the venom of Bothrops jararacussu, in which the Asp49 residue is replaced with a lysine, which damages artificial membranes by a Ca2+-independent mechanism. Wild-type BthTx-I and the mutants Lys49 → Asp, His48 → Gln and Lys122 → Ala were expressed in Escherichia coli BL21(DE3) cells, and the hydrolytic, myotoxic and membrane-damaging activities of the recombinant proteins were compared with native BthTx-I purified from whole venom. The Ca2+-independent membrane-damaging and myotoxic activities of the native and wild-type recombinant BthTx-I, His48Gln and Lys49Asp mutants were similar; however, the Lys122Ala mutant demonstrated reduced levels of both activities. Although a low hydrolytic activity against a mixed phospholipid substrate was observed with native BthTx-I, no substrate hydrolysis was detected with the wild-type recombinant enzyme or any of the mutants. In the case of the Lys49Asp mutant, this demonstrates that the absence of catalytic activity in Lys49-PLA2 is not a consequence of the single Asp49 → Lys replacement. Furthermore, these results provide unambiguous evidence that the Ca2+-independent membrane-damaging and myotoxic activities are maintained in the absence of hydrolysis. The evidence favours a model for a hydrolysis-independent, membrane-damaging mechanism involving an interaction of the C-terminal region of BthTx-I with the target membrane.
Abbreviations used: ASPC, 1-stearoyl-2-arachidonyl-sn-glycero-3-phosphocholine; BthTx-I, bothropstoxin-I; ITFE, intrinsic tryptophan fluorescence emission; rBthTx-I, recombinant bothropstoxin-I; CK, creatine kinase; DMPA, dimyristroylphosphatidic acid; EYPC, egg-yolk phosphatidylcholine; (Lys49-/Asp49-)PLA2s, phospholipase A2s with either lysine or aspartate at residue position 49; Rh, hydrodynamic radius.