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Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs’ intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.

Objective To evaluate the antimicrobial and antifungal activities of the aqueous and partitioned extract of sea anemone Anthopleura nigrescens (A. nigrescens). Methods The sea anemone A. nigrescens was collected, minced, homogenized, lyophilized and then further partitioned with diethyl ether, acetone, ethanol and water. These fractions were evaluated for antimicrobial activity against bacterial and fungal pathogens. Results Acetone extract was found to produce a pronounced inhibition of 7.0 mm against Proteus vulgaris and diethyl ether extract inhibited Pseudomonas aeruginosa with an inhibition zone of 6.5 mm. In antifungal activity, ethanol extract showed good activity against Botrytis cinerea, Trichoderma harzianum and Rhizopus oryzae compared with other strains. Acetone and ethanol extract of A. nigrescens showed activity against all of pathogens tested. Slight activity was observed in the water extract with inhibition zone of 1.5 mm. Conclusions The present study revealed that sea anemone A. nigrescens may also contain some biologically active agents which have potential activity against pathogenic microorganisms.

Actinoporins constitute a unique class of pore-forming toxins found in sea anemones that being secreted as soluble monomers are able to bind and permeabilize membranes leading to cell death. The interest in these proteins has risen due to their high cytotoxicity that can be properly used to design immunotoxins against tumor cells and antigen-releasing systems to cell cytosol. In this work we describe a novel actinoporin produced by Anthopleura nigrescens, an anemone found in the Central American Pacific Ocean. Here we report the amino acid sequence of an actinoporin as deduced from cDNA obtained from total body RNA. The synthetic DNA sequence encoding for one cytolysin variant was expressed in BL21 Star (DE3) Escherichia coli and the protein purified by chromatography on CM Sephadex C-25 with more than 97% homogeneity as verified by MS-MS and HPLC analyses. This actinoporin comprises 179 amino acid residues, consistent with its observed isotope-averaged molecular mass of 19 661 Da. The toxin lacks Cys and readily permeabilizes erythrocytes, as well as L1210 cells. CD spectroscopy revealed that its secondary structure is dominated by beta structure (58.5%) with 5.5% of a-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as per meabilization studies in vesicles, revealed that the affinity of this toxin for sphingomyelin-containing membranes is quite similar to sticholysin II (StII). Comparison by spectroscopic techniques and modeling the three-dimensional structure of nigrelysin (Ng) showed a high homology with StII but several differences were also detectable. Taken together, these results reinforce the notion that Ng is a novel member of the actinoporin pore-forming toxin (PFT) family with a HA as high as that of StII, the most potent actinoporin so far described, but with peculiar structural characteristics contributing to expand the understanding of the structure-function relationship in this protein family. Las actinoporinas constituyen una clase única de toxinas formadoras de poros que se encuentran en las anémonas de mar y que, al ser secretadas como monómeros solubles, pueden unirse y permeabilizar las membranas que conducen a la muerte celular. El interés en estas proteínas ha aumentado debido a su alta citotoxicidad que puede usarse adecuadamente para diseñar inmunotoxinas contra células tumorales y sistemas de liberación de antígenos para el citosol celular. En este trabajo describimos una nueva actinoporina producida por Anthopleura nigrescens, una anémona que se encuentra en el Océano Pacífico centroamericano. Aquí presentamos la secuencia de aminoácidos de una actinoporina deducida del ADNc obtenido del ARN corporal total. La secuencia de ADN sintético que codifica una variante de citolisina se expresó en BL21 Star (DE3) Escherichia coli y la proteína se purificó mediante cromatografía en CM Sephadex C-25 con más del 97% de homogeneidad, como se verificó mediante análisis de MS-MS y HPLC. Esta actinoporina comprende 179 residuos de aminoácidos, de acuerdo con su masa molecular promedio de isótopos observada de 19 661 Da. La toxina carece de Cys y permeabiliza fácilmente los eritrocitos, así como las células L1210. La espectroscopia de CD reveló que su estructura secundaria está dominada por una estructura beta (58,5%) con un 5,5% de hélice a y un 35% de estructura aleatoria. Además, los experimentos de unión a monocapas lipídicas y a los liposomas, así como los estudios de permeabilización en vesículas, revelaron que la afinidad de esta toxina por las membranas que contienen esfingomielina es bastante similar a la esticholisina II (StII). La comparación mediante técnicas espectroscópicas y el modelado de la estructura tridimensional de nigrelysin (Ng) mostró una alta homología con StII, pero también fueron detectables varias diferencias. En conjunto, estos resultados refuerzan la noción de que Ng es un miembro nuevo de la familia de toxinas formadoras de poros de actinoporina (PFT) con un HA tan alto como el de StII, la actinoporina más potente descrita hasta ahora, pero con características estructurales peculiares que contribuyen a ampliar la comprensión de la relación estructura-función en esta familia de proteínas. Universidad Nacional, Costa Rica Universidad de La Habana, Cuba University of Brasilia, Brazil Universidad de Costa Rica, Costa Rica Escuela de Ciencias Biológicas Escuela de Química

Resumen El filo Cnidaria incluye organismos considerados dentro de los más venenosos en la naturaleza. Más específicamente, alrededor de 32 especies de anémonas marinas han sido reportadas como productoras de péptidos y proteínas citolíticas. Este estudio fue llevado a cabo para evaluar la actividad hemolítica del extracto crudo de la anémona marina Aiptasia pallida en Costa Rica. El material biológico fue colectado, triturado, homogenizado y posteriormente centrifugado para la obtención del extracto crudo. El mismo fue caracterizado a través de una evaluación de su actividad hemolítica (HA) con glóbulos rojos de sangre humana (RBC). Interesantemente, los resultados de HA no mostraron rompimiento de los eritrocitos, sin embargo hubo un incremento de la absorbancia en función del tiempo. El extracto crudo reveló la existencia de una banda de proteína alrededor de 30 kDa por SDS-PAGE, no se encontró banda características de actinoporinas en A. pallida. Estos resultados podrían estar asociados con una concentración sub-lítica de otra familia de proteínas las cuales alteran el equilibrio osmótico y cambian el volumen de las células, sin un efecto letal a las concentraciones ensayadas. Abstract The Cnidaria phylum includes organisms that are among the most venomous animals. More than 32 species of sea anemones have been reported to produce lethal cytolytic peptides and proteins. This study was designed to evaluate the hemolytic activity on the crude extract of the sea anemone Aiptasia pallida. The anemone was collected, minced, homogenized and then centrifuged. Functional characterization of the crude extract was evaluated by hemolytic activity (HA) against human red blood cells. Interestingly, the HA results showed no lysis of the cells, but absorbance increased in function of time. The crude extract revealed the existence of proteins band around 30 kDa by SDS-PAGE, no actinoporin band was found in A. pallida. These results could be associated with a sub-lytic concentration of other protein family which is altering osmotic equilibrium and changing cells volume, with no lethal effect at the concentrations tested.

Actinoporins are basic pore-forming proteins produced by sea anemones, with molecular weight around 20 kDa showing high affinity for sphingomyelin-containing membranes. Most sea anemones produce more than one actinoporin isoform differing in isoelectric point, molecular weigth and cytolytic activity. Examples of sea anemones with actinoporin isoforms are: Actinia equina with at least five isoform genes; Actinia tenebrosa, three isoforms; Actinia fragacea, five isoforms; Actineria villosa, Phyllodiscus semoni, Stichodactyla helianthus and Oulactis orientalis, with two isoforms each one, and Heteractis crispa with twenty-four isoforms. Additionally, thirty-four different amino acid sequences were deduced from fifty-two nucleotide sequences of Heteractis magnifica toxins suggesting the presence of a large number of isoforms or allelic variants. Many amino acidic changes in the isoforms are located in important regions for pore formation. The genetic structure of actinoporins comprises a pre-propeptide and a mature toxin region; therefore, actinoporins could be synthetized in the Golgi apparatus as precursor forms. The subsequent maturation of the toxins involves a proteolytic processing during secretion. Here we hypothesize that sea anemones could have suffered duplication, conversion and mutation of genes that produced multigene families as an efficient response to evolutionary pressure, leading to successful strategies of predatory and defensive function.