Mutagenesis analyses explore residues responsible for the neurotoxic and anticoagulant activities of Trimucrotoxin, a pit-viper venom Asn6-phospholipase A2

Trimucrotoxin (TmCT) is an Asn6-containing phospholipase A2 (PLA2) from Protobothrops mucrosquamatus (pit-viper) venom. In an attempt to characterize the amino acid residues responsible for the neurotoxic and anticoagulant activities of TmCT, the recombinant fusion proteins of TmCT wild type and mutants were expressed in Escherichia coli. Correct refolding and processing of 37 TmCT mutants were confirmed by their HPLC retention times, circular dichroism spectra, and masses obtained from ESI-MS spectrometry. Each mutant was assayed by pH-stat titration using zwitterionic as well as anionic micelle substrates, and the neurotoxicity was evaluated by using the contractile responses of chick biventer cervicis muscles. The results demonstrated that the residues Asn1, Asn6, Lys7, Ile11, Met12, Gly53, Thr79, His108 and Met118 are important to TmCT neurotoxicity. Through various tests, we also confirmed that enzymatic activity, as opposed to binding to Factor Xa, was a necessary part of TmCT’s anticoagulant effect. In addition, pulldown assays of the WT and selected mutants revealed that TmCT’s in vitro binding to crotoxin acidic subunit may involve a broad surface area. We conclude that the hot spot mutations at specific positions 53, 79, 108, and 118 during the pit-viper Asn6-PLA2 evolution regulate their neurotoxicities, and that many of the neurotoxic site residues and the anticoagulant mechanism of TmCT are different from those of ammodytoxin A (a true-viper venom neurotoxic PLA2).

Research highlights► Mutagenesis at residues 1, 6, 7, 11, 12, 53, 79, 108, 118 abolished TmCT neurotoxicity. ► Residues 53, 79, 108, 118 of a N6-PLA2 determine its fate as neurotoxin or myotoxin. ► TmCT's anticoagulant effect depends mainly on its enzymatic activity. ► Neurotoxic sites are different for pitviper TmCT and true viper AtxA.