Diversity of Phospholipases A2 from Bothrops atrox Snake Venom: Adaptive Advantages for Snakes Compromising Treatments for Snakebite Patients

Abstract

The evolution of snake venoms resulted in multigene toxin families that code for structurally similar isoforms eventually harboring distinct functions. PLA₂s are dominant toxins in viper venoms, and little is known about the impact of their diversity on human envenomings and neutralization by antivenoms. Here, we show the isolation of three distinct PLA₂s from B. atrox venom. FA1 is a Lys-49 homologue, and FA3 and FA4 are catalytic Asp-49 PLA₂s. FA1 and FA3 are basic myotoxic proteins, while FA4 is an acid non-myotoxic PLA₂. FA3 was the most potent toxin, inducing higher levels of edema, inflammatory nociception, indirect hemolysis, and anticoagulant activity on human, rat, and chicken plasmas. FA4 presented lower anticoagulant activity, and FA1 had only a slight effect on human and rat plasmas. PLA₂s presented differential reactivities with antivenoms, with an emphasis on FA3, which was not recognized or neutralized by the antivenoms used in this study. Our findings reveal the functional and antigenic diversity among PLA₂s from B. atrox venom, highlighting the importance of assessing venom variability for understanding human envenomations and treatment with antivenoms, particularly evident here as the antivenom fails to recognize FA3, the most active multifunctional toxin described.

Keywords: PLA2; antivenom; diversity; snake venom; snakebites.

Figure 1

Chromatographic and electrophoretic profiles of the PLA₂s isolated from B. atrox venom. B. atrox venom was submitted to RP-HPLC in a C18 column using a gradient elution of solution A (0.1% TFA) and solution B (100% acetonitrile) at a flow rate of 2.0 mL/min (a). Three fractions (peaks 3, 8, and 9) were collected, dried in a SpeedVac, resuspended in 1 mL of solution A, and then rechromatographed individually using the same protocol: peak 3 (FA1) eluted at 55.6 min (b); peak 8 (FA3) eluted at 65.8 min (c); and peak 9 (FA4) eluted at 67.49 min (d). Isolated toxins were subjected to 1D-SDS-PAGE in 15% polyacrylamide gels under reducing (R) and non-reducing (NR) conditions (FA1-e; FA3-f; FA4-g) and 2D-PAGE (FA1-h; FA3-i; FA4-j), with a pH gradient of 3–10 in the first dimension, and 15% polyacrylamide-SDS gels for the second dimension. Gels were stained with Coomassie Blue G250. The mobility of molecular weight (MW) standards is shown on the left, with values indicated in kDa.

Figure 2

Sequence alignment of identified peptides with complete B. atrox PLA₂ sequences. Peptides of FA1, FA2, and FA3 identified using mass spectrometry were aligned against PLA₂ sequences previously characterized in B. atrox venom using the CLUSTAL W multiple alignment package. Dots represent the uncovered regions, positions not elucidated by MS/MS, of BATXPLA002 (Accession number JAV01882.1), BATXPLA006 (Accession number JAV01878.1), and BATXPLA001 (Accession number JAV01883.1) by peptides identified in FA1, FA3, and FA4, respectively. Asterisk indicates the amino acid residue at 49 position (*).

Figure 3

Characterization of catalytic and myotoxic activities of isolated PLA₂s. The PLA₂ catalytic activity (a) was determined using NOBA as substrate and 0.3 µg/20 μL of PLA₂ samples or crotoxin B (CB), as a positive control or 20 μL of standard buffer (C−, negative control). The absorbance was measured at 425 nm, and the results are expressed as Abs/min/µg of protein. Symbols indicate differences that are statistically significant for p ≤ 0.005: (*) CB compared to FA1, FA4, and negative control; (#) FA3 compared to FA4 fraction and negative control; (Ф) FA3 compared to FA1 fraction; (∆) FA3 compared to FA4 fraction. (α) FA4 fraction compared to negative control. Myotoxic activity (b) was measured via CK levels in the serum of mice (n = 5), after intramuscular injection in the gastrocnemius muscle of 25 µg of the PLA₂s, dissolved in 50 µL of PBS. Control groups received injections containing 50 µL of PBS only (negative control) or 50 µg/50 µL of B. jararacussu venom (Jssu) as a positive control. After 3 h, blood samples were collected and the CK levels were assayed in serum samples using a commercial kit CK-UV (Bioclin) and with CK levels expressed in U/L. Symbols indicate differences that are statistically significant for p ≤ 0.005: (*) PBS group compared to Jssu; (#) Jssu group compared to FA4; (&) FA1 group compared to PBS; (Ф) FA4 compared to FA1; (Δ) FA3 group compared to FA4. Results are expressed as mean ± SD or mean ± SEM of three independent experiments for PLA₂ activity and myotoxic activity, respectively.

Figure 4

Evaluation of edema formation after intraplantar injection of the PLA₂s. Groups of five mice were injected subcutaneously into the footpad of the right hind paw with samples of the fractions (FA1, FA3, and FA4) containing 2 µg (a) or 10 µg (b) dissolved in 30 µL sterile PBS. Control groups were injected with the same volume of PBS (negative control) or B. atrox venom (BaV) at the same doses as fractions. The edematogenic activity was evaluated at different times: 0 min (before the treatment), 30 min, 1, 2, 4, 24, and 48 h (after treatment). Edema was estimated using the increase in paw thickness after the injection of the fractions, using a plethysmometer to measure the difference in volume displaced (µL). The data represent the mean ± SEM of three independent experiments. Symbol (*) indicates differences statistically significant for p ≤ 0.005 compared to the PBS control group.

Figure 5

Nociceptive response in mice after intraplantar injection of the PLA₂s. Fractions (2, 5, or 10 µg/30 µL) were dissolved in sterile PBS injected (i.pl.) into the right hind paw of mice (n = 5). Control groups were injected with 30 μL of sterile PBS only. The nociceptive response, evaluated by the reactivity of animals to lick the injected foot, was scored in seconds: (a) response in the early or neurogenic phase (from 0 to 5 min after the injection); (b) late or inflammatory phase (from 15 to 30 min after the injection). The data represent the mean ± SEM of three independent experiments. Symbols indicate differences statistically significant for p ≤ 0.005: (&) PBS group compared to FA1, FA3, and FA4; (#) FA1 group compared to FA3 and FA4; (∆) FA3 group compared to FA4; (*) FA3 group compared to FA1.

Figure 6

Evaluation of the hemolytic activity induced by the PLA₂s. The hemolytic activity of the PLA₂s was tested using washed human red blood cells. Serially diluted concentrations (96, 48, 24, 12, 6, 3, and 1.5 µg/mL) of the fractions FA1, FA3, and FA4 were added to 100 µL of erythrocyte suspensions (1 × 10⁸) and incubated for 1 h with gentle mixing at 37 °C. Next, plates were added with 50 µL of human serum of the same donor for the indirect test (a,b), or with 50 µL of Tris-sucrose buffer for the evaluation of indirect hemolytic activity (c,d), and incubated under the same experimental conditions. For the controls, 50 µL of 3% Triton X-100 (positive) or 50 µL of Tris-sucrose (negative) were used. The samples were centrifuged (200× g for 5 min, 25 °C), and the absorbance of the supernatant was measured in a spectrophotometer at 550 nm. The experiments were performed in triplicate, and the results represent the mean ± SD of three independent experiments. Symbols indicate differences that are statistically significant for p ≤ 0.005: (*) positive control compared to Tris-sucrose, and to FA1, FA3, and FA4 fractions; (&) Negative control (Tris-sucrose) compared to FA3 and FA4; (∆) and (#) FA1 compared to FA3 and FA4, respectively.

Figure 7

Anticoagulant action of the PLA₂s on the plasma of birds, rodents and humans. The anticoagulant activity was assessed on recalcified and activated plasmas of chicken, rat and humans. Samples containing 0.5 µg/10 µL of each toxin were pipetted onto specific cups containing 20 µL of 20 mM CaCl₂ and 60 µL of the coagulation activator ellagic acid. Next, 240 µL of plasma were added to the reaction mixture, and the clotting time (CT), the clot formation time (CFT) and maximum clot firmness (MCF) were recorded by thromboelastometry for 1 hour. The same volume (10 µL) of PBS with or without ellagic acid-based activator of coagulation, were used as positive and negative controls, respectively. The green line (CT) represents the beginning clot formation; pink and blue backgrounds represent clot formation time (CFT) and maximum clot firmness (MCF), respectively. The data shown are representative of three experiments.

Figure 8

Antigenic reactivity of the PLA₂s with different antivenoms, evaluated via immunoblotting. Samples containing 10 µg of each PLA₂ were separated in a 15% SDS-PAGE gel under reducing conditions using DTT (+) and non-reducing (−) conditions. The proteins were transferred onto nitrocellulose membranes, which were blocked and incubated with (a) SAB (1:1000); (b) anti-ATX (1:100); or (c) anti-PLA₂ (1:40). Immunoreactivity was detected using IgG conjugated to peroxidase of anti-horse (SAB), anti-rabbit (anti-ATX), or anti-mice (anti-PLA₂) antivenoms, diluted 1:1000. The reactive bands were developed with 0.05% 4-chloro-1-naphthol in 15% (v/v) methanol, in the presence of 0.03% (v/v) H₂O₂.

Figure 9

Neutralization by antivenoms of the myotoxic activity induced by FA1 and FA3 PLA₂ fractions. The neutralizing efficacies of the different antivenoms (SAB, Anti-ATX, and Anti-PLA₂) were evaluated in groups of 5 mice, intramuscularly (i.m.) injected into the gastrocnemius muscle, with samples containing 25 µg/50 µL of the B. jararacussu venom (a), B. atrox venom (b) or the myotoxins FA1 (c) or FA3 (d), 50 µL of PBS (negative control), previously incubated with SAB, anti-ATX or anti-PLA₂, as appropriate (0.2 µL of AV/µg of PLA₂). Three hours after inoculation, blood samples were collected from the mice orbital plexus and centrifuged (2000× g for 5 min, 4 °C) to obtain the sera. CK levels of the sera were measured using a commercial kit, CK-UV (Bioclin), and expressed as U/L. The data shown represent mean ± SD of three experiments. Symbols indicate differences statistically significant for p ≤ 0.005: (&) PBS group compared to Jssu, and Jssu + SAB; (*) Jssu group compared to Jssu + SAB, and Jssu + anti-PLA₂; (∆) Jssu + SAB group compared to Jssu + anti-PLA₂. (&) PBS group compared to Jssu and Jssu + SAB; (*) Jssu group compared to Jssu + SAB, and Jssu + anti-PLA₂; (∆) Jssu + SAB group compared to Jssu + anti-PLA₂. (&) PBS group compared to FA1, FA1 + SAB, FA1 + anti-PLA₂, and FA1 + anti-ATX; (#) FA1 group compared to FA1 + SAB, FA1+ anti-PLA₂, and FA1 + FA1 + anti-ATX; (ϕ) FA1 + SAB group compared to FA1 + anti-PLA₂, and FA1 + anti-ATX; (∆) FA1 + SAB group compared to FA1 + anti-PLA₂. (&) PBS group compared to FA3, FA3 + SAB, FA3 + anti-PLA₂, and FA3 + anti-ATX.