Toxinology DOI 10.1007/978-94-007-6650-1_1-1 # Springer Science+Business Media Dordrecht 2014 Equinatoxins: A Review Dušan Šuput* Faculty of Medicine, Institute of Pathophysiology and Centre for Clinical Physiology, University of Ljubljana, Ljubljana, Slovenia Abstract Equinatoxins are basic pore forming proteins isolated from the sea anemone Actinia equina. Pore formation is the underlying mechanism of their hemolytic and cytolytic effect. Equinatoxin con- centrations required for pore formation are higher than those causing significant effects in heart and skeletal muscle. This means that other mechanisms must also be involved in the toxic and lethal effects of equinatoxins. Effects of equinatoxins have been studied on lipid bilayers, several cells and cell lines, on isolated organs and in vivo. Different cells have distinct susceptibilities to the toxin, ranging from <1pMupto>100 nM. The cells are swollen after a prolonged treatment with low concentrations of equinatoxin II, or rapidly when 100 nM or higher concentrations of the toxin are used. Equinatoxins increase cation-specific membrane conductance and leakage current, affect the function of potassium and sodium channels in nerve, muscle and erythrocytes, increase intracellular Ca2+ activity, and cause a significant increase of cell volume. In smooth muscle cells and in neuroblastoma NG108-15 cells, an increase in intracellular Ca2+ activity is observed after exposure to 100 nM equinatoxin II. The large difference in toxin concentrations needed for the pore formation and other effects suggest that equinatoxins exert their effects through at least two different mech- anisms. It is well known that lipid environment is important for the proper functioning of membrane channels and other membrane proteins. It is possible that toxin monomers disturb local conditions around ionic channels and/or receptors by binding in the vicinity of those structures, thus altering their function. Introduction Synthesis of toxins by toxic live organisms is energetically demanding and it is reasonable to assume that toxins must provide a kind of benefit for survival. Some toxic substances have regulatory roles in the life cycle of an organism, such as cyanotoxins in cyanobacteria. Other toxins provide either defense against predators or serve as a “weapon” in hunting a prey. Some toxins, such as cytolytic toxins, may serve several roles – they can help catch and digest the pray, or they serve as defense chemicals and substances that help secure their territory in interspecies competition. Anthozoans and Their Toxins Cnidarians comprise four classes: Anthozoa, Cubozoa, Hydrozoa and Scyphozoa. Tentacles of all cnidarians contain specialized cells, nematocytes, organized in nematocysts. This is an organelle capable of producing and delivering their toxins into a prey. Several Anthozoans can also release their toxins from the digestive cavity. Although all cnidarians produce powerful toxins and all of *Email: [email protected] Page 1 of 17 Toxinology DOI 10.1007/978-94-007-6650-1_1-1 # Springer Science+Business Media Dordrecht 2014 Fig. 1 Actinia equina in its natural environment on a rock in shallow water. Adriatic sea, island Vis, July 2013 them possess nematocysts not all of them are harmful to humans. Nematocysts of several Antho- zoans, including Actinia equina (Fig. 1), are not capable of penetrating human skin. A Brief History on Equinatoxin Research In the early seventies of the previous century a toxic protein, equinatoxin has been found in the tentacle extract from the sea anemone Actinia equina. The toxin has been purified and characterized by Lebez and Ferlan a year later; in 1973. The isolated and purified toxin was a polypeptide with isoelectric point (pI) of 12.5 and molecular weight (m.w.) of roughly 20 kDa. A remarkable property of the toxin was the fact that it contained no cysteine; therefore there were no disulphide bonds in that protein (Ferlan and Lebez 1974). At that time this was the only sea anemone toxin with the m.w. of 20 kDa; other coelenterate toxins were either larger (>70 kDa) or smaller (<15 kDa). The toxin was lethal to rats with the LD50 ¼ 33.3 mg/kg. The animals died because of cardio- respiratory arrest (Sket et al. 1974), but animals that have survived the intoxication showed tacyphylaxis – a larger dose was needed to cause apnea and cardiac arrest. On the contrary, it has been shown that equinatoxin has a cardio-stimulatory effect on guinea-pig atrium. These contro- versial findings might be explained by higher resistance of the atrial myocytes to the effects of equinatoxin, and less so to different concentrations of the toxin used in experiments. In fact, minute concentrations of equinatoxin can be cardiostimulatory due to the entry of calcium into a cell. However, the initially elevated arterial blood pressure (aBP) after injection of equinatoxin is always followed by a profound fall of aBP within a few minutes. Respiratory arrest caused by equinatoxin is poorly understood, but efferent impulses from the respiratory centre were absent after application of the toxin. Direct stimulation of the phrenic nerve still caused contractions of diaphragm. A detailed study of the effects of equinatoxin on isolated lungs showed that the vascular resistance increases and lungs become edematous when perfused with 50–100 nM equinatoxin (Lafranconi et al. 1984). This may explain breathing disturbances such as restriction of ventilation and decreased alveolar ventilation, but not necessarily also the complete cessation of breathing. Hemolytic activity was the most evident and documented action of equinatoxin. A very small concentration of the toxin, only 0.1 nM, was needed for complete hemolysis of erythrocytes (Macek and Lebez 1981). Hemolysis was increased in the presence of calcium ions. An important finding was that the action of equinatoxin on erythrocytes is inhibited in the presence of sphingomyelin. The importance of reactions between some pore forming toxins from sea anemones and sphingomyelin has been described by Bernheimer and Avigad who demonstrated that pre-incubation of a cytolysin Page 2 of 17 Toxinology DOI 10.1007/978-94-007-6650-1_1-1 # Springer Science+Business Media Dordrecht 2014 Table 1 Basic characteristics of equinatoxins EqT I EqT II EqT III Isoelectric point (pI) 9.80 10.50 10.50 LD50 mice (mg/kg) 23.00 35.00 83.00 LD50 window Æ30 % of LD50 Æ30 % of LD50 Æ30 % of LD50 Time to death 5 min 5 min 5 min to 12 h Time to half hemolysis (min) 1.6 0.8 0.5 Time to half hemolysis relative to EqT I (%) 100 49.38 30.86 from Stichodactyla helianthus with sphingomyelin abolishes its hemolytic activity. This lead them to the conclusion that sphingomyelin is the acceptor for the toxin in the erythrocyte membrane (Bernheimer et al. 1982). This finding is also important for the present research of membrane rafts, the sphingomyelin and cholesterol rich cell domains, as labeled equinatoxins can be used for tagging these membrane domains. In 1988 Kem named the whole class of sea anemone toxins “actinoporins” due to their ability to bind to sphingomyelin and to porate cell membranes (Kem and Dunn 1988). Potential use of equinatoxin as anti-neoplastic agent has been studied on mice bearing the ascitic form of Ehrlich carcinoma. Equinatoxin increased the survival time of those mice. In vitro it was cytotoxic to tumor cell lines in 0.1 nM concentrations, but the resistance of the cells increased after several administrations of the toxin. Lung macrophages were resistant to the action of equinatoxin. Toxic effects of equinatoxin and very steep dose/response curve made equinatoxin a poor candidate for clinical use (Giraldi et al. 1976). “Equinatoxin” Comprises a Group of Three Isotoxins with Similar Action Soon after the isolation of equinatoxin a number of basic polypeptide toxins from several sea anemones have been isolated. All of them were cytolytic, and their action was inhibited by sphingomyelin. Reinvestigation of the venom from the sea anemone Actinia equina yielded three isotoxins with similar structure and function (Macek and Lebez 1988). All of them were proteins devoid of cysteine with m.w. roughly 20 kDa and pI 10. They are stable proteins although their structure is not fixed with disulphide bonds. The major constituent of the hemolytic fraction of the venom was equinatoxin II (EqT II), and two smaller fractions were named equinatoxin I and III (EqT I and EqT III respectively). One gram of wet weight of the sea anemone yielded 61.3 mg of EqT I, 288 mg of EqT II, and 108.9 mg of EqT III (Macek and Lebez 1988). The most toxic isotoxin is EqT I with LD50 23 mg/kg in mice, followed by EqT II (LD50 35 mg/kg) and EqT III (LD50 83 mg/kg). Contrary to its toxicity, EqT III is roughly three times more efficient in producing hemolysis of sheep erythrocytes than EqT I and nearly two times more efficient than EqT II (see Fig. 1 and Table 1). EqT II is present in the tentacles and the body of the sea anemone while EqT I and EqT III seem to be present only in the body of the animal. Equinatoxin has been shown to increase leakage current before the lysis of skeletal muscle cells in sub-nanomolar concentrations (Šuput 1986). Page 3 of 17 Toxinology DOI 10.1007/978-94-007-6650-1_1-1 # Springer Science+Business Media Dordrecht 2014 Cloning, Sequencing and Expression of Equinatoxins Soon after isolation of three isotoxins from Actinia equina the most abundant isotoxin, EqT II, was cloned and expressed in the cytoplasm of Escherichia coli (Anderluh et al. 1996). The cloned and expressed EqT II was a 20 kDa protein that reacted with polyclonal antibodies against the wild type EqT II. It had identical hemolytic activity as the natural EqT II. Like EqT II, the cloned and expressed EqT II consists of 179 amino acids (a.a.).