Active-site mutagenesis of a Lys49-phospholipase A2: biological and membrane-disrupting activities in the absence of catalysis

Abstract

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.

Inhibition of Myotoxic Activity of Bothrops asper Myotoxin II by the Anti-trypanosomal Drug Suramin

“…30,46 With the biochemical and structural information currently available we are now able to probe the structure-function relationship, and the mode of action and inhibition of Lys49 PLA 2 analogues by addressing the roles of the C-terminal region and the nominal active site in myotoxicity.…”

“…The controversy as to whether these enzymes possess catalytic activity [27][28][29] has been clarified, since no substrate hydrolysis was detected with the wildtype recombinant protein. 30 …”

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action

“…At the present time, it cannot be excluded that FAs could play a role in myotoxin intoxication of muscle cells, through uncoupling of mitochondria, since they are highly dependent on the activity of these organelles. The Lys49 PLA 2 myotoxins are devoid of enzymatic activity [17,46,81], and therefore their membrane action has to be explained on grounds different from direct hydrolysis of phospholipids. These PLA 2 homologues have a highly cationic face [82 -84].…”

Protein Engineering in Structure-Function Studies of Viper's Venom Secreted Phospholipases A2

“…The main structural feature of snake venom sPLA 2 -like myotoxins is the substitution of the highly-conserved aspartic acid residue at position 49, effectively preventing Ca 2+ binding and thus abolishing catalytic activity (Petan et al, 2007;Ward et al, 2002). The most common substitution is Lys, but AtnL is one of the two known Ser49 sPLA 2 homologues.…”