The Venom of Vipera ammodytes ammodytes: Proteomics, Neurotoxic Effect and Neutralization by Antivenom

Abstract

Deep proteomic analyses identified, in total, 159 master proteins (with 1% FDR and 2 unique peptides) from 26 protein families in the venom of Vipera ammodytes ammodytes (Vaa). Data are available via ProteomeXchange with the identifier PXD056495. The relative abundance of PLA2s is 11.60% of the crude venom, of which 4.35% are neurotoxic Ammodytoxins (Atxs). The neurotoxicity of the venom of Vaa and the neutralizing effect of the antivenom were tested on the neuromuscular preparation of the diaphragm (NPD) of rats. The activity of PLA2 in the venom of Vaa and its neutralization by the antivenom were determined under in vitro conditions. The Vaa venom leads to a progressive decrease in NPD contractions. We administered pre-incubated venom/antivenom mixtures at various ratios of 1:2, 1:10 and 1:20 (w/w) and observed the effects of these mixtures on NPD contractions. The results show that the mean effective time (ET₅₀) for NPD contractions with the 1:20 mixture is highly significantly different (p < 0.001) from the ET₅₀ for the venom and the ET₅₀ for the 1:2 and 1:10 mixture ratios. We also found a highly significant (p < 0.001) reduction in Na⁺/K⁺-ATPase activity in the NPD under the influence of the venom. The reduction in the activity of this enzyme was reversible by the antivenom. Under in vitro conditions, we have achieved the complete neutralization of PLA2 by the antivenom. In conclusion, the antivenom abolished the venom-induced progressive decrease in NPD contractions in a concentration-dependent manner. Antivenom with approximately the same mass proportion almost completely restores Na⁺/K⁺-ATPase activity in the NPD and completely neutralizes the PLA2 activity of the venom in vitro.

Keywords: Na+/K+-ATPase; Vipera ammodytes ammodytes; antivenom; diaphragm; neurotoxicity; proteomics; venom.

Figure 1

Vipera ammodytes ammodytes. Original photo: Institute of Virology, Vaccines and Sera “Torlak”, Belgrade, Serbia.

Figure 2

(A) Distribution of identified proteins for fraction 0 when different protein FASTA databases were used in the analysis; (B) Distribution of identified proteins for all fractions (0, 3A, 5A, 8A, 9A and 10A) when Serpentes protein FASTA databases (DB) were used in the analysis; (C) Distribution of identified proteins for all fractions (0, 3A, 5A, 8A, 9A and 10A) when Vipera protein FASTA databases (DB) were used in the analysis; (D) Distribution of identified proteins for all fractions (0, 3A, 5A, 8A, 9A and 10A) when V. ammodytes protein FASTA databases (DB) were used in the analysis; (E) Distribution of identified proteins for all fractions (0, 3A, 5A, 8A, 9A and 10A) when different protein FASTA databases were used in the analysis. Next to each fraction, the number of proteins identified in this fraction for all three databases used is given in brackets.

Figure 3

Relative distribution of protein groups (%) in the Vaa venom determined by nano-liquid chromatography–tandem mass spectrometry-based proteomics: (A) DB V. ammodytes; (B) DB Vipera.

Figure 4

Representative recording of contractions of a neuromuscular preparation of the diaphragm (NPD) induced by indirect EFS (·····) in the absence of venom. C₁ and C₂—control contractions; panc 1 μM—contractions under the influence of 1 μM pancuronium; W₁ and W₂—contractions after the washout of pancuronium; 10 “packages” of contractions in the function of time.

Figure 5

Representative recording of contractions of a neuromuscular preparation of the diaphragm (NPD) induced by indirect EFS (·····) and direct EFS (-----) under the influence of venom. C₁ and C₂—control contractions; panc 3 μM—contractions under the influence of 3 μM pancuronium; W₁ and W₂—contractions after the washout of pancuronium; 12 “packages” of contractions induced by indirect EFS; 2 “packages” of contractions induced by direct EFS.

Figure 6

Sigmoidal curves of the reduction in contractions of the neuromuscular preparation of the diaphragm (NPD) in a logarithmic function of time under the influence of venom and venom/antivenom mixtures at ratios of 1:2; 1:10 and 1:20 (w/w).

Figure 7

Comparison of ET₅₀ (minutes) after the administration of venom and a venom/antivenom mixture at the ratios of 1:2; 1:10 and 1:20 (w/w) (mean ± SD, ^# p < 0.05, ^### p < 0.001 vs. venom; ⁺⁺⁺ p < 0.001 between different mass ratios of venom/antivenom).

Figure 8

Representative recording of contraction peaks of the neuromuscular preparations of the diaphragm (NPD) induced by indirect EFS: (A) Control contractions; (B) Contractions under the influence of pancuronium (tetanic fade); (C) Contractions under the influence of venom; (D) Contractions under the influence of a mixture of venom/antivenom at a ratio of 1:20 (w/w) (white arrows show a facilitated release of neurotransmitters; black arrows show a reduced release of neurotransmitters—tetanic fade).

Figure 9

AChE activity (U/mg P) in the neuromuscular preparations of the diaphragm (NPD) without the presence of venom (control), under the influence of venom and for the mixture of venom/antivenom at a ratio of 1:2, 1:10 and 1:20 (w/w) (mean ± SD, p > 0.05).

Figure 10

Na⁺/K⁺-ATPase activity (U/mg P) in the neuromuscular preparations of the diaphragm (NPD) without the presence of venom (control), under the influence of venom and under the influence of a mixture of venom and antivenom at the ratios of 1:2; 1:10 and 1:20 (w/w) (mean ± SD, *** p < 0.001 vs. control; ^## p < 0.01, ^### p < 0.001 vs. venom; ⁺⁺ p<0.01 between different mass ratios of venom/antivenom).

Figure 11

(A) Activity of the PLA2 in increasing concentrations of the Vaa venom (mg/mL); (B) Inhibition of the PLA2 activity in 1 mg/mL of the Vaa venom by increasing concentrations of the antivenom (mg/mL).