Pioneering Study on Rhopalurus crassicauda Scorpion Venom: Isolation and Characterization of the Major Toxin and Hyaluronidase

Abstract

Scorpionism is responsible for most accidents involving venomous animals in Brazil, which leads to severe symptoms that can evolve to death. Scorpion venoms consist of complexes cocktails, including peptides, proteins, and non-protein compounds, making separation and purification procedures extremely difficult and time-consuming. Scorpion toxins target different biological systems and can be used in basic science, for clinical, and biotechnological applications. This study is the first to explore the venom content of the unexplored scorpion species Rhopalurus crassicauda, which inhabits exclusively the northernmost state of Brazil, named Roraima, and southern region of Guyana. Here, we pioneer the fractionation of the R. crassicauda venom and isolated and characterized a novel scorpion beta-neurotoxin, designated Rc1, and a monomeric hyaluronidase. R. crassicauda venom and Rc1 (6,882 Da) demonstrated pro-inflammatory activities in vitro and a nociceptive response in vivo. Moreover, Rc1 toxin showed specificity for activating Na_v1.4, Na_v1.6, and BgNa_v1 voltage-gated ion channels. This study also represents a new perspective for the treatment of envenomings in Roraima, since the Brazilian scorpion and arachnid antivenoms were not able to recognize R. crassicauda venom and its fractions (with exception of hyaluronidase). Our work provides useful insights for the first understanding of the painful sting and pro-inflammatory effects associated with R. crassicauda envenomings.

Keywords: Rhopalurus crassicauda; electrophysiology; neurotoxin; nociception; pro-inflammatory toxin; scorpion venom; toxin.

FIGURE 1

Rhopalurus crassicauda: collection and morphology. Left panel shows Roraima, the northernmost state of Brazil. The yellow star indicates the capital city of the state, Boa Vista, where R. crassicauda scorpions were collected. Zoom view in the right panel shows the R. crassicauda species (scorpion length – 10 cm). Photo taken of a specimen kept in the scorpion vivarium of the research group.

FIGURE 2

Chromatographic profiles of R. crassicauda venom using RP-FPLC system. The protein elution was carried out in a segmented concentration gradient from 0 to 100% of solution B (80% ACN in 0.1% TFA) and absorbance was monitored at 214 nm. (A) R. crassicauda venom (2 mg) was eluted using 5 concentration gradient steps on a C18 column (250 × 10 mm, 300 Å, and 5 μm particles), at a flow rate of 5 mL/min. (B) P8 (40 μg) was re-chromatographed using 4 concentration gradient steps on a C18 column (250 × 2.1 mm, 300 Å, and 5 μm particles), at a flow rate of 0.5 mL/min.

FIGURE 3

Electrophoretical profile of R. crassicauda venom and fractions. Tris-tricine-SDS-PAGE (16.5%) under reducing (Lanes 1–15) and non-reducing (Lanes 16–22) conditions. Lanes 16–22 were incorporated with hyaluronan before gel polymerization. Lanes 1, 16, and 22: T. serrulatus venom (18, 16, and 5 μg, respectively). Lanes 2, 17, and 21: R. crassicauda venom (18, 5, and 5 μg, respectively). Lanes 3 and 11: molecular mass markers (Sigma M3546). Lanes 15 and 19: molecular mass markers (GE Healthcare 17-0446-01). Lanes 4 and 5: Ts1 and Rc1 (P8), respectively (2 μg). Lanes: 6: P1; 7: P2; 8: P3; 9: P4; 10: P5; 12: P6; 13: P7 (all 2 μg). Lanes 14, 18, and 20: P9 (2 μg). Lanes 1–19 were stained with PlusOne Coomassie Blue PhastGel^® R-350. Lanes 20–22 were stained with Stains-all for evaluation of the hyaluronidase activity.

FIGURE 4

(A) Mass spectrum of Rc1 obtained by MALDI-TOF mass spectrometry in a positive linear ionization mode using DHB matrix. (B) Sequence alignments of the Rc1 partial sequence (B3EWP2) with other beta-neurotoxins. Css9 (Centruroides suffusus, F1CGT6), CsEv5 (C. sculpturatus, P58779), Ts4 (T. serrulatus, P45669), Ts1 (T. serrulatus, P15226), RjAa2f (R. junceus, E7CLP6), and RjAa12 (R. junceus, E7CLN6). Conserved residues are in red and Cys residues are shaded in black. Alignment was generated by Clustal Omega server. Identity percentage considered among the aligned residues.

FIGURE 5

Immunorecognition of R. crassicauda venom by Brazilian scorpion and arachnid antivenoms. (A) Scorpion antivenom. (B) Arachnid antivenom. (C) Scorpion antivenom and R. crassicauda fractions. The 96-well plates were coated with 2 μg of R. crassicauda venom or fractions (1–9) diluted in a solution of 0.05 M carbonate-bicarbonate buffer (pH 9.6). Positive controls (+) were performed with wells coated with non-immune horse serum or TsV, and negative controls (−) were performed by replacing antivenoms with non-immune horse serum. Absorbance was measured at 490 nm. NHS: non-immune horse serum. TsV, T. serrulatus venom; ScA, scorpion antivenom; ArA, arachnid antivenom; and RcV, R. crassicauda venom. Results are presented as mean ± SD (n = 3), which were analyzed by ANOVA followed by Tukey’s multiple comparison test (*p < 0.05, when compared to the negative respective controls).

FIGURE 6

Effect of R. crassicauda venom and the major toxin on cytokine levels. J774.1 cells were stimulated with R. crassicauda venom (100 μg/mL) or Rc1 toxin (50 μg/mL) for 24 h. As negative control, stimuli with T. serrulatus venom (100 μg/mL), Ts1 (50 μg/mL), and unstimulated cells were used. (A) TNF-α. (B) IL-6. (C) IL-1β. TsV: T. serrulatus venom. RcV: R. crassicauda venom. Results are presented as mean ± SD (n = 4), which were analyzed by ANOVA followed by Tukey’s post hoc test (*p < 0.05 when compared to controls; **p < 0.001 when compared to Ts1).

FIGURE 7

Effect of R. crassicauda venom and the major toxin on the NF-kB signaling pathway. (A) Western blot of phospho-NF-kB p65 and NF-kB p65 proteins (GAPDH as an internal control). Lane 1 represents control (medium) and lanes 2–5 represent TsV, Ts1, RcV, and Rc1, respectively. (B) Percentage of expression of the target protein against the reference protein as quantified by band densitometry. TsV: T. serrulatus venom. RcV: R. crassicauda venom. Results are presented as mean ± SD (n = 4), which were analyzed by ANOVA followed by Tukey’s post hoc test (*p < 0.001 when compared to control).

FIGURE 8

Electrophysiological effects of the major toxin on voltage-gated sodium channels. (A) Effects of Rc1 on the voltage dependence of steady-state activation and inactivation curves under control conditions (black symbols) and after the addition of 1 μM of Rc1 (blue symbols), n = 4 cells ± SEM. (B) Current traces of Nav1.5 (non-effect) and BgNav channels in control (black) and in the presence of Rc1 channels (blue). (C) IV curves in control (black) and after application of Rc1 for Nav1.5 (non-effect) and BgNav1 channels.

FIGURE 9

Spontaneous nociception induced by R. crassicauda venom and the major toxin. Nociception was assessed by recording the time course of paw licking and lifting behavior after intraplanar (ipl) injections of RcV venom (A) or Rc1 (B) into C57BL/6 mice right hind paw. T. serrulatus venom and its main toxin (Ts1) were used as controls. TsV: T. serrulatus venom. RcV: R. crassicauda venom. Data are presented as the mean ± SD (n = 5), which were analyzed by Two-way ANOVA followed by Tukey’s post hoc test. Same and different letters represent, respectively, no and statistically significant differences between groups.