Abstract
Phospholipase A2 (PLA2) is one of the representative toxic components of snake venom. PLA2s are categorized into several subgroups according to the amino acid at position 49, which comprises either Asp49, Lys49, Arg49 or Ser49. Previous studies suggested that the Lys49-PLA2 assembles into an extremely stable dimer. Although the behavior on Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing or non-reducing conditions suggested the presence of intermolecular disulfide bonds, these bonds were not observed in the crystal structure of Lys49-PLA2. The reason for this discrepancy between the crystal structure and SDS-PAGE of Lys49-PLA2 remains unknown. In this study, we analyzed a Lys49-PLA2 homologue from Protobothrops flavoviridis (PflLys49-PLA2 BPII), by biophysical analyses including X-ray crystallography, SDS-PAGE, native-mass spectrometry, and analytical ultracentrifugation. The results demonstrated that PflLys49-PLA2 BPII spontaneously oligomerized in the presence of SDS, which is one of the strongest protein denaturants.
Highlights
Previous studies reported that Lys49-Phospholipase A2 (PLA2) exist as either conventional or alternative dimers, based on the results of X-ray crystallography, electrophoresis, dynamic light scattering, and spectroscopy[11,12,14,15,19,23,39,40,41,42]
In contrast to these previous reports, our present crystal structure showed that PflLys49-PLA2 basic protein II (BPII) exists as a monomer in the crystal (Fig. 1)
This was consistent with the results of native-MS (Fig. 3) and analytical ultracentrifugation analyses (Fig. 4), both of which showed that monomeric PflLys49-PLA2 BPII was detected in the absence of sodium dodecyl sulfate (SDS)
Summary
Snakebite induces acute myonecrosis as well as other biological effects including hemolytic, neurotoxic, cardiotoxic, anticoagulant and antiplatelet activities of multiple complex protein assemblies. These protein complexes are composed of various multi-locus gene families that underwent accelerated evolution[1]. Group II PLA2s are further categorized into several subgroups based on the amino acid at position 49. These subgroups contain Asp[49], Lys[49], Arg[49] or Ser[49], among which. Despite the absence of phospholipase activity, Lys49-PLA2 exhibits myonecrotic activity due to the intrinsic function of its C-terminal region that is abundant in positive and hydrophobic residues[7,8,9] which mediate caspase-independent apoptosis[10]. The gyration radius (Rg) calculated from SAXS showed good agreement with the alternative dimer rather than the conventional dimer, suggesting that the alternative dimer has a more stable conformation in solution[21]
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