PhTX-II a basic myotoxic phospholipase A₂ from Porthidium hyoprora snake venom, pharmacological characterization and amino acid sequence by mass spectrometry

Abstract

A monomeric basic PLA₂ (PhTX-II) of 14149.08 Da molecular weight was purified to homogeneity from Porthidium hyoprora venom. Amino acid sequence by in tandem mass spectrometry revealed that PhTX-II belongs to Asp49 PLA₂ enzyme class and displays conserved domains as the catalytic network, Ca²⁺-binding loop and the hydrophobic channel of access to the catalytic site, reflected in the high catalytic activity displayed by the enzyme. Moreover, PhTX-II PLA₂ showed an allosteric behavior and its enzymatic activity was dependent on Ca²⁺. Examination of PhTX-II PLA₂ by CD spectroscopy indicated a high content of alpha-helical structures, similar to the known structure of secreted phospholipase IIA group suggesting a similar folding. PhTX-II PLA₂ causes neuromuscular blockade in avian neuromuscular preparations with a significant direct action on skeletal muscle function, as well as, induced local edema and myotoxicity, in mice. The treatment of PhTX-II by BPB resulted in complete loss of their catalytic activity that was accompanied by loss of their edematogenic effect. On the other hand, enzymatic activity of PhTX-II contributes to this neuromuscular blockade and local myotoxicity is dependent not only on enzymatic activity. These results show that PhTX-II is a myotoxic Asp49 PLA₂ that contributes with toxic actions caused by P. hyoprora venom.

Figure 1

Chromatographic and electrophoretic profile of Porthidium hyoprora venom fractioning on a µ-Bondapack C18 column, monitoring elution profile at 280 nm. Emphasized in black is fraction 11 (*) characterized as PhTX-II PLA₂; Insert: Electrophoretic profile in Tricine SDS-PAGE (1) Molecular mass markers; (2) PhTX-II not reduced; (3) PhTX-II reduced with DTT (1 M).

Figure 2

Mass determination of PhTX-II PLA₂ by mass spectrometry using a Q-Tof Ultima API ESI/MS (TOF MS mode). The MH⁺ and MH₂⁺⁺ species are shown.

Figure 3

MS/MS spectrum of the tryptic peptides of m/z 2157.9189 (A) and 1504.54 (B); Series of y fragment ions of 11-residue-long tryptic peptide (CCFVHDCCYGK) containing the aspartic acid residue at position 49 (*) in the amino acid sequence (B) and Ca²⁺-binding loop NAL/IPFYAFYGCYCGWGGR, highly conserved region in the amino acid sequences of PLA₂ (A).

Figure 4

(A) Alignment of the amino acid sequence of the PhTX-II PLA₂ with D49-PLA₂ by Edit Seq version 5.01© Program (DNASTAR Inc., Madison, WI, USA, 2001). LmTX-I from Lachesis muta muta (P0C942.1) [12]; Cdr12 from Crotalus durissus ruruima (P0CAS3.1) [13]; Piratoxin III from Bothrops pirajai (1GMZ_A) [14]; PhTX-I from Porthidium hyoprora [6]; BmTX-I from Bothrops moojeni (P0C8M1) [15]; BthTX-II from Bothrops jararacussu (2OQD_B) [16]; BbTX-III from Bothrops brazili [17]. The asterisk corresponds to Asp49 position; the program introduces gaps to maximize alignments; (B) Phylogeny relationship of PhTX-II to other PLA₂ isoforms statistically evaluated by Bootstrap method.

Figure 5

Far-UV circular dichroism spectra of PhTX-II PLA₂.

Figure 6

(A) PLA₂ activity of P. hyoprora venom and PhTX-II PLA₂ (B) Effect of substrate concentration on the PLA₂ activity of PhTX-II; (C) Effect of pH on the PLA₂ activity of PhTX-II; (D) Effect of temperature on the PLA₂ activity of PhTX-II; (E) Influence of ions (10 mM each) on PLA₂ activity of PhTX-II in the absence or presence of 1 mM Ca²⁺; (F) Effect inhibitory of heparin, EDTA crotapotins (F2 and F3) and chemical modification with BPB on PLA₂ activity of PhTX-II. In each case, the concentration of PhTX-II was 5 μM. The results of all experiments are the mean ± SEM of three determinations (p < 0.05).

Figure 7

Neuromuscular blockade of chick biventer cervicis preparations incubated with PhTX-II PLA₂ (0.35, 0.7, 1.4 μM and modified with BPB) at 37 °C (A). Panel B shows the effect of PhTX-II PLA₂ on muscle contractures evoked by KCl and acetylcholine (ACh). Panels C shows a representative recording from a preparation treated with 0.7 μM of PhTX-II PLA₂. Tissue responses to KCl (■ 20 mM) and acetylcholine (ACh, ● 110 μM) were obtained before and after toxin addition. The points are the mean ± SEM of six experiments. * (p < 0.05) compared to the twitch-tension before toxin addition.

Figure 8

Time-course of the increments in plasma CK activity after intramuscular injection of PhTX-II PLA₂ (5.20, 50 μg and modified with BPB/100 μL) in mice. Controls were injected with 100 μL of PBS. At different times, blood was collected, and serum levels were measured. Values are means ± SEM of five mice at each time point (A); Edema-forming activity of PhTX-II PLA₂ (2.5, 10 μg and modified with BPB/50 µL) in mice. Edema by toxin was injected s.c. in the footpad of mice. At various time intervals the increase in footpad volume, as compared to controls, was expressed as percent edema (B). Each point represents the mean ± SEM of five animals.