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1.C.38 The Pore-forming Equinatoxin (Equinatoxin) Family

Sea anemones such as Actinia equina, Heteractis magnifica, and Stichodactyla helianthus produce a variety of sequence related toxins (called actinoporins) including Equinatoxins 1A,-1D, II, III, IV, V, etc. They have been given alternative designations such as Tenebrosin C (for Equinatoxin II), cytolysin, and hemolytic toxin. These cardiac stimulatory hemolysins penetrate membranes forming ion permeable, cation-selective pores, also permeable to small neutral solutes. They cause a variety of phenotypes in mammals including platelet aggregation, cytotoxicity of a variety of animal cells, lysis of some cells, and vasospasm of coronary vessels. They are of 175-179 aas in length and form tetrameric pore-forming structures in membranes. The 3-dimensional structure of the soluble form of equinatoxin II has been solved (Anastasiadis et al., 2001). The radius of the Sticholysin pore has been shown to be about 1.2 nm (diameter, ~2 nm). Pore size is independent of toxin concentration and is the same in biological and artificial membranes (Tejuca et al., 2001). Pore formation requires a flexible N-terminal region and a stable β-sandwich (Kristan et al., 2004).

Equinatoxin II inserts into the membrane via a two-step membrane-binding process involving an exposed cluster of aromatic residues (step 1) and a flexible N-terminal amphipathic α-helix (step 2) (Hong et al., 2002). The first step is similar to that of the evolutionarily distant cholesterol-dependent cytolysins. Interaction is dependent on sphingomyelin, and lipid phase coexistence favors membrane insertion (Barlic et al., 2004; Biserka et al., 2008; Schoen et al., 2008).

Equinatoxin II (EqtII) from Actinia equina and Sticholysin II (StnII) from Stichodactyla helianthus are the actinoporins that have been studied in greatest detail. Both proteins display a beta-sandwich fold composed of 10 β-strands flanked on each side by two short alpha-helices. Two-dimensional crystallization on lipid monolayers has allowed the determination of low-resolution models of tetrameric structures distinct from the pore. Wild-type EqtII and StnII, as well as a nice collection of natural and artificially made variants of both proteins, have been produced in Escherichia coli and purified. Four regions of the actinoporin structure seem to play an important role. The phosphatidyl choline or sphingomyelin-binding site and a cluster of exposed aromatic residues, together with a basic region, may be involved in the initial interaction with the membrane, whereas the amphipathic N-terminal region is essential for oligomerization and pore formation (Alegre-Cebollaba et al., 2007). Pore formation proceeds in at least four steps: Monomer binding to the membrane interface, assembly of four monomers, and at least two distinct conformational changes driving to the final formation of the functional pore. Sticholysin I is almost identical to sticholysin II. Conformational flexibility at the N-terminus of the protein does not provide higher affinity for the membrane, although it is necessary for correct pore formation (Alegre-Cebollada et al., 2008). An AF domain superfamily (abbreviated from actinoporin-like proteins and fungal fruit-body lectins) has been defined. It contains members from at least three animal and two plant phyla. On the basis of functional properties of some members, Crnigoj Kristan et al., 2009 hypothesised that AF domains mediate peripheral membrane interactions.

Fragaceatoxin C (FraC) is an α-barrel pore-forming toxin (PFT). The crystal structures of FraC at four different stages of the lytic mechanism have been determined at 3.1Å resolution, namely the water-soluble state, the monomeric lipid-bound form, an assembly intermediate and the fully assembled transmembrane pore (Tanaka et al. 2015). The structure of the transmembrane pore exhibits a unique architecture composed of both protein and lipids, with some of the lipids lining the pore wall, acting as assembly cofactors. The pore exhibits lateral fenestrations that expose the hydrophobic core of the membrane to the aqueous environment. The incorporation of lipids from the target membrane within the structure of the pore provides a membrane-specific trigger for the activation of this haemolytic toxin.

The generalized transport reaction catalyzed by members of the equinatoxin family is:

Small molecule (in) small molecule (out)


References associated with 1.C.38 family:

Alegre-Cebollada, J., M. Cunietti, E. Herrero-Galán, J.G. Gavilanes, and A. Martínez-del-Pozo. (2008). Calorimetric scrutiny of lipid binding by sticholysin II toxin mutants. J. Mol. Biol. 382: 920-930. 18687335
Alegre-Cebollada, J., M. Oñaderra, J.G. Gavilanes, and A.M. del Pozo. (2007). Sea anemone actinoporins: the transition from a folded soluble state to a functionally active membrane-bound oligomeric pore. Curr. Protein. Pept. Sci. 8: 558-572. 18220843
Alvarez C., Mancheno JM., Martinez D., Tejuca M., Pazos F. and Lanio ME. (2009). Sticholysins, two pore-forming toxins produced by the Caribbean Sea anemone Stichodactyla helianthus: their interaction with membranes. Toxicon. 54(8):1135-47. 19268489
Anastasiadis, A., G. Anderluh, P. Maeek, and D. Turk. (2001). Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina. Structure 9: 341-346. 11525171
Bakrac, B., A. Kladnik, P. Macek, G. McHaffie, A. Werner, J.H. Lakey, and G. Anderluh. (2010). A toxin-based probe reveals cytoplasmic exposure of Golgi sphingomyelin. J. Biol. Chem. 285: 22186-22195. 20463009
Bakrac, B., I. Gutiérrez-Aguirre, Z. Podlesek, A.F. Sonnen, R.J. Gilbert, P. Macek, J.H. Lakey, and G. Anderluh. (2008). Molecular determinants of sphingomyelin specificity of a eukaryotic pore-forming toxin. J. Biol. Chem. 283: 18665-18677. 18442982
Barlic, A., I. Gutiérrez-Aguirre, J.M.M. Caaveiro, A. Cruz, M.-B. Ruiz-Argüello, J. Pérez-Gil, and J.M. González-Mañas. (2004). Lipid phase coexistence favors membrane insertion of equinatoxin-II, a pore-forming toxin from Actinia equina. J. Biol. Chem. 279: 34209-34216. 15175339
Belmonte, G., G. Menestrina, C. Pederzolli, I. Kriaj, F. Gubensek, T. Turk, and P. Macek. (1994). Primary and secondary structure of a pore-forming toxin from the sea anemone, Actinia equina L, and its association with lipid vesicles. Biochim. Biophys. Acta 1192: 197-204. 7912550
Frazão, B., V. Vasconcelos, and A. Antunes. (2012). Sea anemone (Cnidaria, Anthozoa, Actiniaria) toxins: an overview. Mar Drugs 10: 1812-1851. 23015776
Hong, Q. I. Gutiérrez-Aguirre, A. Barlic, P. Malovrh, K. Kristan, Z. Podlesek, P. Macek, D. Turk, J.M. Gonzáles-Mañas, J.H. Lakey, and G. Anderluh. (2002). Two-step membrane binding by equinatoxin II, a pore-forming toxin from the sea anemone, involves an exposed aromatic cluster and a flexible helix. J. Biol. Chem. 277: 41916-41924. 12198118
Kawashima Y., H. Nagai, M. Ishida, Y. Nagashima, K. Shiomi. (2003). Primary structure of echotoxin 2, an actinoporin-like hemolytic toxin from the salivary gland of the marine gastropod Monoplex echo. Toxicon. 42: 491-497. 14529730
Kristan KC., Viero G., Dalla Serra M., Macek P. and Anderluh G. (2009). Molecular mechanism of pore formation by actinoporins. Toxicon. 54(8):1125-34. 19268680
Kristan, K., Z. Podlesek, V. Hojnik, I. Gutiérrez-Aguirre, G. Guncar, D. Turk, J.M. González-Mañas, J.H. Lakey, P. Macek, and G. Anderluh. (2004). Pore formation by equinatoxin, a eukaryotic pore-forming toxin, requires a flexible N-terminal region and a stable β-sandwich. J. Biol. Chem. 279: 46509-46517. 15322132
Mebs D., Langeluddeke T. (1992). European viper venoms: haemorrhagic and myotoxic activities. Toxicon. 30: 1303-1306. 1440635
Mechaly, A.E., A. Bellomio, D. Gil-Cartón, K. Morante, M. Valle, J.M. González-Mañas, and D.M. Guérin. (2011). Structural insights into the oligomerization and architecture of eukaryotic membrane pore-forming toxins. Structure 19: 181-191. 21300287
Poklar, N., J. Fritz, P. Macek, G. Vesnaver, and T.V. Chalikian. (1999). Interaction of the pore-forming protein equinatoxin II with model lipid membranes: a calorimetric and spectroscopic study. Biochemistry 38: 14999-15008. 10555982
Schön, P., A.J. García-Sáez, P. Malovrh, K. Bacia, G. Anderluh, and P. Schwille. (2008). Equinatoxin II permeabilizing activity depends on the presence of sphingomyelin and lipid phase coexistence. Biophys. J. 95: 691-698. 18390598
Shiomi K., Y. Kawashima, M. Mizukami, Y. Nagashima. (2002). Properties of proteinaceous toxins in the salivary gland of the marine gastropod (Monoplex echo). Toxicon. 40: 563-71. 11821129
Simpson, R.J., G.E. Reid, R.L. Maritz, C. Morton, and R.S. Norton. (1990). Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actina tenebrosa. Eur. J. Biochem. 190: 319-328. 1973096
Tanaka, K., J.M. Caaveiro, K. Morante, J.M. González-Mañas, and K. Tsumoto. (2015). Structural basis for self-assembly of a cytolytic pore lined by protein and lipid. Nat Commun 6: 6337. 25716479
Tejuca, M., S.M. Dalla, C. Potrich, C. Alvarez, and G. Menestrina. (2001). Sizing and radius of the pore formed in erythrocytes and lipid vesicles by the toxin sticholysin I from the sea anemone Stichodactyla helianthus. J. Membr. Biol. 183: 125-135. 11562794