Advances in the characterization of the Scorpaena plumieri cytolytic toxin (Sp-CTx)

Abstract

Proteins that account for the hemolytic activity found in scorpaeniform fish venoms are responsible for the majority of the effects observed upon envenomation, for instance, neurotoxic, cardiotoxic and inflammatory effects. These multifunctional toxins, described as protein lethal factors and referred to as cytolysins, are known to be extremely labile molecules. In the present work, we endeavored to overcome this constraint by determining optimal storage conditions for Sp-CTx, the major bioactive component from the scorpionfish Scorpaena plumieri venom. This cardiotoxic hemolytic cytolysin is a large dimeric glycoprotein (subunits of ≈65 kDa) with pore-forming ability. We were able to establish storage conditions that allowed us to keep the toxin partially active for up to 60 days. Stability was achieved by storing Sp-CTx at -80 and -196 °C in the presence of glycerol 10% in a pH 7.4 solution. It was demonstrated that the hemolytic activity of Sp-CTx is calcium dependent, being abolished by EDTA and zinc ions. Furthermore, the toxin exhibited its maximal hemolytic activity at pH between 8 and 9, displaying typical N- and O- linked glycoconjugated residues (galactose (1-4) N-acetylglucosamine and sialic acid (2-3) galactose in N- and/or O-glycan complexes). The hemolytic activity of Sp-CTx was inhibited by phosphatidylglycerol and phosphatidylethanolamine, suggesting a direct electrostatic interaction lipid - toxin in the pore-formation mechanism of action of this toxin. In addition, we observed that the hemolytic activity was inhibited by increasing doses of cholesterol. Finally, we were able to show, for first time, that Sp-CTx is at least partially responsible for the pain and inflammation observed upon envenomation. However, while the edema induced by Sp-CTx was reduced by pre-treatment with aprotinin and HOE-140, pointing to the involvement of the kallikrein-kinin system in this response, these drugs had no significant effect in the toxin-induced nociception. Taken together, our results could suggest that, as has been already reported for other fish cytolysins, Sp-CTx acts mostly through lipid-dependent pore formation not only in erythrocytes but also in other cell types, which could account for the pain observed upon envenomation. We believe that the present work paves the way towards the complete characterization of fish cytolysins.

Evolutionary Ecology of Fish Venom: Adaptations and Consequences of Evolving a Venom System

“…The proteomic analysis of fish venom is challenging due to the difficulty of collecting them as well as their lability and the problems of avoiding contamination of the venom samples with mucus from the body surface [18,19]. Therefore, efforts have been made to develop better methods of proteomic analysis of these delicate venom molecules for characterisation and functional analysis [20,21].…”

Fish Cytolysins in All Their Complexity

“…A molecular mass of 121 kDa was estimated for the native Sp-CTx, while a mass of 65,251 Da m/z was revealed by its mass spectrum, pointing to it having a dimeric nature with subunits of very similar molecular masses, much like SNTX and TLY [41]. However, unlike TLY and SNTX, Sp-CTx is a glycoprotein, displaying typical N-and O-linked glycoconjugate residues [41,43]. Three years later, the same group optimized the purification protocol of Sp-CTx and this new method increased the final yield by 13-fold when compared to the previous one [42].…”

“…Sp-CTx *-S. plumieri cytolytic toxin Lethal [15] Hemolytic [41,42] ardiovascular [15,41,42] Edematogenic [43] Nociceptive [43…”

“…By the end of the 20th century, when some of the difficulties imposed by the aforesaid lability of fish cytolysins were overcome through the establishment of proper purification and storage conditions [13,25,41,43], the biochemical and pharmacological characterization of these toxins was greatly accelerated. In addition, the combination of different methods, including liquid chromatography, cDNA cloning, automated Edman degradation, mass spectrometry (MS), and X-ray crystallography resulted in significant advances in the biochemical characterization of these molecules.…”

Getting stoned: Characterisation of the coagulotoxic and neurotoxic effects of reef stonefish (Synanceia verrucosa) venom

“…but on fish venom in general (Harris and Jenner, 2019). This is likely in part due to the extreme labile nature of fish venoms (Barnett et al, 2017;Carlson et al, 1971;Malacarne et al, 2018;Shiomi et al, 1989). The venom toxins can be extremely sensitive to denaturing with slight changes in pH, temperature, lyophilisation and even fractionation processes causing the crude venom/toxins to decrease or even abolish toxic activities.…”