Molecular and biochemical characterization of a cytolysin from the Scorpaena plumieri (scorpionfish) venom: Evidence of pore formation on erythrocyte cell membrane

Abstract

Previously, a potent hemolytic toxin (Sp-CTx - 121 kDa) was isolated from Atlantic Scorpionfish Scorpaena plumieri venom. In the present work, we aimed to elucidate the action mechanisms involved in the hemolytic activity induced by this toxin, but to achieve our goal we faced the need to optimize its purification procedure in order to improve its activity and protein recovery. In this new method, Sp-CTx was purified to homogeneity through a combination of sequential ammonium sulfate precipitation and two chromatographic steps: hydrophobic interaction (Butyl HP) and anion exchange (Synchropak SAX 300). Orbitrap mass spectrometry analysis revealed that the amino acids sequences determined to Sp-CTx peptides are shared by other hemolytic toxins from fish venoms. The hemolytic activity of Sp-CTx upon rabbit erythrocytes was attenuated in the presence of osmotic protectants (polyethylene glycol polymers), and molecules larger than 6 nm in diameter inhibited cell lysis. This result strongly suggests that Sp-CTx may be a pore-forming protein, since it lacks phospholipase A2 activity. All these results contribute to the better understanding of Sp-CTx molecular/cellular actions in envenomation caused by S. plumieri. The results are also in agreement with previous reports of structural and functional similarities among piscine hemolytic toxins.

Identification of C-type isolectins in the venom of the scorpionfish Scorpaena plumieri

“…The venom of this species is highly hemolytic for rabbit erythrocytes (Carrijo et al, 2005), a consequence of the action of a potent pore-forming toxin, Sp-CTx (Gomes et al, 2013). Besides cell membrane disruption activity, the fractionation of this venom by gel filtration chromatography also revealed a hemagglutinating fraction (unpublished data).…”

Expressed sequence tags in venomous tissue of Scorpaena plumieri (Scorpaeniformes: Scorpaenidae)

“…Further, the protein is dimeric comprising subunits of approximately 75 kDa each, similar to what has been described in other stonefish species (Gomes et al, 2013). Sp-CTx displays potent hemolytic activity on washed rabbit erythrocytes (EC 50 0.46 nM); its effect is antagonized by antivenom raised against stonefish venom -Synanceia trachynis (SFAV).…”

Avaliação da citotoxicidade do extrato do ferrão de arraia Potamotrygon falkneri (Myliobatiformes:Potamotrygonidae)

“…Scorpaena guttata, entretanto, apresenta atividade hemolítica sobre eritrócitos ovinos e Notesthes robusta mostrou-se ativa sobre eritrócitos humanos (CHURCH e HODGSON, 2002). A espécie Scorpaena plumieri (peixe escorpião) apresentou alta atividade hemolítica em eritrócitos de coelhos, sendo que a maior atividade foi com a peçonha recém-extraída (CARRIJO et al, 2005;GOMES et al, 2013), fator que pode influenciar nos resultados obtidos nos estudos mencionados, sendo difícil estabelecer comparações entre os ensaios citados, sendo que diversos fatores como temperatura do ambiente e do ensaio, tempo de exposição à toxina entre outros podem ser responsáveis pelas alterações relatas (ANDRICH et al, 2010).…”

“…Geralmente estas funções são resultantes da lise celular enzimática oriunda das fosfolipases A 2 e C, enzimas comuns presentes em diversos organismos como répteis, anfíbios e cnidários (GOMES et al, 2013). Componentes com atividades citolíticas como a atividade de fosfolipase foram relatadas em arraias de ambos os habitats tanto dulcícola como marinhas (BARBARO et al, 2007).…”

“…No presente trabalho, foi observada tal atividade na peçonha bruta, corroborando com BARBARO et al (2007). O extrato de P. falkneri apresentou maior atividade na ausência de cálcio, entretanto a maior parte dos grupos de fosfolipases são Ca + dependentes (STÁBELI et al, 2012 (GWEE et al, 1994;GARNIER et al, 1995;GOMES et al, 2013 horas após a incubação com a peçonha (SIVAN et al, 2007).…”