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Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.

Sticholysin II (Stn-II) is a pore-forming cytolysin. Stn-II interacts with several supports for size exclusion chromatography, which results in an abnormal retardation precluding molecular mass calculations. Sedimentation equilibrium analysis has revealed that the protein is an associating system at neutral pH. The obtained data fit a monomer-tetramer equilibrium with an association constant Kc4 of 109 M−3. The electrophoretic pattern of Stn-II treated with different cross-linking reagents, in a wide range of protein concentrations, corroborates the existence of tetrameric forms in solution. A planar configuration of the four monomers, C4 or D2 symmetry, is proposed from modelling of the cross-linking data.

Actinoporins are highly hemolytic pore-forming proteins with a molecular mass of around 20 kDa and high affinity for sphingomyelin-containing membranes. On the crude extract of the sea anemone Anthopleura nigrescens, the hemolytic activity (HA) was detected. In order to identify the presence of pore-forming proteins similar to actinoporins in this anemone, the fractionation and analysis of its crude extract was carried out. The aqueous extract of the whole body was subjected to gel filtration chromatography on Sephadex G50 medium rendering three resolved peaks (P-I, P-II, and P-III) as measured by their absorbance at 280 nm. Functional characterization of the crude extract and chromatographic fractions was evaluated by HA against human red blood cells. The crude extract and two peaks (P-II and P-III) showed HA. Interestingly, the HA of the crude extract and P-II were specifically inhibited by small unilamellar vesicles of phosphatidylcholine: sphingomyelin (1:1). Both the crude extract and P-II revealed the existence of at least one protein band around 20 kDa by SDS-PAGE. The inhibition by sphingomyelin of the whole body extract HA and the localization of this property in P-II that could be associated with a protein of around 20 kDa suggest the presence of at least one novel actinoporin in A. nigrescens. Furthermore, this is the first report of a biochemical activity for this sea anemone. Work is in progress in order to purify and characterize the molecular entities responsible for this HA inhibited by sphingomyelin. Las actinoporinas son proteínas formadoras de poros altamente hemolíticas con una masa molecular de alrededor de 20 kDa y alta afinidad por las membranas que contienen esfingomielina. Sobre el extracto crudo de la anémona de mar Anthopleura nigrescens, se detectó la actividad hemolítica (HA). Para identificar la presencia de proteínas formadoras de poros similares a las actinoporinas en esta anémona, se realizó el fraccionamiento y análisis de su extracto crudo. El extracto acuoso de todo el cuerpo se sometió a cromatografía de filtración en gel en medio Sephadex G50 dando tres picos resueltos (P-I, P-II y P-III) medidos por su absorbancia a 280 nm. La caracterización funcional del extracto crudo y las fracciones cromatográficas se evaluó mediante HA frente a glóbulos rojos humanos. El extracto crudo y dos picos (P-II y P-III) mostraron HA. Curiosamente, el HA del extracto crudo y P-II fueron inhibidos específicamente por pequeñas vesículas unilaminares de fosfatidilcolina: esfingomielina (1: 1). Tanto el extracto crudo como el P-II revelaron la existencia de al menos una banda de proteína alrededor de 20 kDa por SDS-PAGE. La inhibición por esfingomielina del extracto de HA de todo el cuerpo y la localización de esta propiedad en P-II que podría estar asociada con una proteína de alrededor de 20 kDa sugieren la presencia de al menos una actinoporina nueva en A. nigrescens. Además, este es el primer informe de una actividad bioquímica de esta anémona de mar. Se está trabajando para purificar y caracterizar las entidades moleculares responsables de este HA inhibido por la esfingomielina. Universidad Nacional, Costa Rica Escuela de Ciencias Biológicas

Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore-forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review.