Search

Abstract Background Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin. Results In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64− cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64− cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability. Conclusions HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting.

Background: Immunotoxin is a hybrid protein consisting of a toxin moiety that is linked to a targeting moiety for the purpose of specific elimination of target cells. Toxins used in traditional immunotoxins are practically difficult to be produced in large amount, have poor tissue penetration and a complex internalization process. We hypothesized that the smaller HALT-1, a cytolysin derived from Hydra magnipapillata, can be used as the toxin moiety in construction of a recombinant immunotoxin. Results: In this study, pro-inflammatory macrophage was selected as the target cell due to its major roles in numerous inflammatory and autoimmune disorders. We aimed to construct macrophage-targeted recombinant immunotoxins by combining HALT-1 with anti-CD64-scFv in two orientations, and to assess whether their cytotoxic activity and binding capability could be preserved upon molecular fusion. The recombinant immunotoxins, HALT-1-scFv and scFv-HALT-1, were successfully constructed and expressed in Escherichia coli (E. coli). Our data showed that HALT-1 still exhibited significant cytotoxicity against CD64+ and CD64− cell lines upon fusion with anti-CD64 scFv, although it had half cytotoxic activity as compared to HALT-1 alone. As positioning HALT-1 at N- or C-terminus did not affect its potency, the two constructs demonstrated comparable cytotoxic activities with IC50 lower in CD64+ cell line than in CD64− cell line. In contrast, the location of targeting moieties anti-CD64 scFv at C-terminal end was crucial in maintaining the scFv binding capability. Conclusions: HALT-1 could be fused with anti-CD64-scFv via a fsexible polypeptide linker. Upon the successful production of this recombinant HALT-1 scFv fusion protein, HALT-1 was proven effective for killing two human cell lines. Hence, this preliminary study strongly suggested that HALT-1 holds potential as the toxin moiety in therapeutic cell targeting. [ABSTRACT FROM AUTHOR]

Cnidarians are one of the oldest known animal lineages (ca. 700 million years), with a unique envenomation apparatus to deliver a potent mixture of peptides and proteins. Some peptide toxins from cnidarian venom have proven therapeutic potential. Here, we use a transcriptomic/proteomic strategy to identify sequences with similarity to known venom protein families in the tentacles of the endemic Australian 'speckled anemone' (Oulactis sp.). Illumina RNASeq data were assembled de novo. Annotated sequences in the library were verified by cross-referencing individuals' transcriptomes or protein expression evidence from LC-MS/MS data. Sequences include pore-forming toxins, phospholipases, peptidases, neurotoxins (sodium and potassium channel modulators), cysteine-rich secretory proteins and defensins (antimicrobial peptides). Fewer than 4% of the sequences in the library occurred across the three individuals examined, demonstrating high sequence variability of an individual's arsenal. We searched for actinoporins in Oulactis sp. to assess sequence similarity to the only described toxins (OR-A and -G) for this genus and examined the domain architecture of venom-related peptides and proteins. The novel putative actinoporin of Oulactis sp. has a greater similarity to other species in the Actiniidae family than to O. orientalis. Venom-related sequences have an architecture that occurs in single, repeat or multi-domain combinations of venom-related (e.g. ShK-like) and non-venom (e.g. whey acid protein) domains. This study has produced the first transcriptomes for an endemic Australian sea anemone species and the genus Oulactis, while identifying nearly 400 novel venom-related peptides and proteins for future structural and functional analyses and venom evolution studies. [ABSTRACT FROM AUTHOR]

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.

Abstract Sticholysin I and II (Sts: St I and St II) are proteins of biomedical interest that form pores upon the insertion of their N-terminus in the plasma membrane. Peptides spanning the N-terminal residues of StI (StI 1-31) or StII (StII 1-30) can mimic the permeabilizing ability of these toxins, emerging as candidates to rationalize their potential biomedical applications. These peptides have different activities that correlate with their hydrophobicity. However, it is not clear how this property contributes to peptide folding in solution or upon binding to membranes. Here we compared the conformational properties of these peptides and shorter versions lacking the hydrophobic segment 1–11 of StI (StI 12-31) or 1–10 of StII (StII 11-30). Folding of peptides was assessed in solution and in membrane mimetic systems and related with their ability to bind to membranes and to permeabilize lipid vesicles. Our results suggest that the differences in activity among peptides could be ascribed to their different folding propensity and different membrane binding properties. In solution, StII 1-30 tends to acquire α-helical conformation coexisting with self-associated structures, while StI 1-31 remains structureless. Both peptides fold as α-helix in membrane; but StII 1-30 also self-associates in the lipid environment, a process that is favored by its higher affinity for membrane. We stress the contribution of the non-polar/polar balance of the 1–10 amino acid sequence of the peptides as a determining factor for different self-association capabilities. Such difference in hydrophobicity seems to determine the molecular path of peptides folding upon binding to membranes, with an impact in their permeabilizing activity. This study contributes to a better understanding of the molecular mechanisms underlying the permeabilizing activity of Sts N-terminal derived peptides, with connotation for the exploitation of these small molecules as alternative of the full-length toxins in clinical settings. Graphical abstract Image 1 Highlights • Hydrophobicity of peptides StI 1-31 and StII 1-30 determines their self-association capabilities. • StII 1-30 but not StI 1-31 self-associates in solution and in the lipid environment. • Folding of StI 1-31 and StII 1-30 peptides is strongly conditioned by the membrane environment. • The higher hydrophobicity of StII 1-30 facilitates its partition into the membrane. • The permeabilizing activity of StII 1-30 could be favored by pre-association in solution. [ABSTRACT FROM AUTHOR]

A bstract 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 α-helix, and 35% of random structure. Moreover, binding experiments to lipidic monolayers and to liposomes, as well as permeabilization 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. Highlights • Nigrelysin (Ng) is a novel 20 kDa actinoporin produced by sea anemone A. nigrescens. • Ng efficiently permeabilizes cells and model membranes containing sphingomyelin. • Ng´s high permeabilizing activity is similar to that of StII with C 50 values ∼2 nM. [ABSTRACT FROM AUTHOR]

Actinoporins are the most abundant group of sea anemone cytolytic toxins. Their membranolytic activity is of high interest for the development of novel anticancer drugs. However, to date the activity of actinoporins in malignant cells has been poorly studied. Here, we report on recombinant analog of Hct-S3 (rHct-S3), belonging to the combinatory library of Heteractis crispa actinoporins. rHct-S3 exhibited cytotoxic activity against breast MDA-MB-231 (IC50 = 7.3 µM), colorectal HT-29 (IC50 = 6.8 µM), and melanoma SK-MEL-28 (IC50 = 8.3 µM) cancer cells. The actinoporin effectively prevented epidermal growth factor -induced neoplastic transformation of JB6 Cl41 cells by 34% ± 0.2 and decreased colony formation of HT-29 cells by 47% ± 0.9, MDA-MB-231 cells by 37% ± 1.2, and SK-MEL-28 cells by 34% ± 3.6. Moreover, rHct-S3 decreased proliferation and suppressed migration of colorectal carcinoma cells by 31% ± 5.0 and 99% ± 6.4, respectively. The potent anti-migratory activity was proposed to mediate by decreased matrix metalloproteinases-2 and -9 expression. In addition, rHct-S3 induced programmed cell death by cleavage of caspase-3 and poly (ADP-ribose) polymerase, as well as regulation of Bax and Bcl-2. Our results indicate rHct-S3 to be a promising anticancer drug with a high anti-migratory potential.

Actinoporins (APs) are a family of pore-forming toxins (PFTs) from sea anemones. These biomolecules exhibit the ability to exist as soluble monomers within an aqueous medium or as constitutively open oligomers in biological membranes. Through their conformational plasticity, actinoporins are considered good candidate molecules to be included for the rational design of molecular tools, such as immunotoxins directed against tumor cells and stochastic biosensors based on nanopores to analyze unique DNA or protein molecules. Additionally, the ability of these proteins to bind to sphingomyelin (SM) facilitates their use for the design of molecular probes to identify SM in the cells. The immunomodulatory activity of actinoporins in liposomal formulations for vaccine development has also been evaluated. In this review, we describe the potential of actinoporins for use in the development of molecular tools that could be used for possible medical and biotechnological applications.

Actinoporins are α-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol.

Transcriptomic profiling of venom producing tissues from different animals is an effective approach for discovering new toxins useful in biotechnological and pharmaceutical applications, as well in evolutionary comparative studies of venomous animals. Stichodactyla helianthus is a Caribbean sea anemone which produces actinoporins as part of its toxic venom. This family of pore forming toxins is multigenic and at least two different isoforms, encoded by separate genes, are produced by S. helianthus . These isoforms, sticholysins I and II, share 93% amino acid identity but differ in their pore forming activity and act synergistically. This observation suggests that other actinoporin isoforms, if present in the venomous mixture, could offer an advantageous strategy to modulate whole venom activity. Using high-throughput sequencing we generated a de novo transcriptome of S. helianthus and determined the relative expression of assembled transcripts using RNA-Seq to better characterize components of this species' venom, focusing on actinoporin diversity. Applying this approach, we have discovered at least one new actinoporin variant from S. helianthus in addition to several other putative venom components. [ABSTRACT FROM AUTHOR]

Actinoporins are pore-forming proteins found in sea anemones. Although we now have a large collection of data on actinoporins, our knowledge is based heavily on those identified in shallow-water anemones. Because the deep sea differs considerably from shallow waters in hydrostatic pressures, temperatures, and the prey composition, the deep-sea actinoporin may have evolved in unique ways. This study, therefore, aimed to obtain new actinoporins in the deep-sea anemone Cribrinopis japonica (Actiniaria, Actiniidae). An actinoporin-like sequence was identified from the previously established C. japonica RNA-Seq database, and the complete length (663 bp) of the deep-sea actinoporin gene, Cjtox I , was obtained. In addition, a similar gene, Cjtox II (666 bp), was also identified from RNA of actinopharynx. CJTOX I and CJTOX II were similar in their primary structures, but CJTOX I lacked one residue in the middle of the protein. There was also a difference in the gene expression in live animals, where only Cjtox I was expressed in tentacles of C. japonica . In the heterologous expression where BL21 (DE3) strain was retransformed with the plasmid containing either Cjtox I or Cjtox II gene, the supernatants of both cell lysates showed hemolytic activity on the equine erythrocytes. Preincubation of the supernatants with sphingomyelin caused reduced activity, implying that the CJTOX I and II would target sphingomyelin as with other actinoporins. Because of the structures similarity to the known actinoporins and the sphingomyelin-inhibitable hemolytic activity, both CJTOX I and II were concluded to be new actinoporins, which were identified for the first time from a deep-sea anemone. [ABSTRACT FROM AUTHOR]

Animal venoms are complex mixtures of highly specialized toxic molecules. Cnidarians and arachnids produce pore-forming proteins (PFPs) directed against the plasma membrane of their target cells. Among PFPs from cnidarians, actinoporins stand out for their small size and molecular simplicity. While native actinoporins require only sphingomyelin for membrane binding, engineered chimeras containing a recognition antibody-derived domain fused to an actinoporin isoform can nonetheless serve as highly specific immunotoxins. Examples of such constructs targeted against malignant cells have been already reported. However, PFPs from arachnid venoms are less well-studied from a structural and functional point of view. Spiders from the Latrodectus genus are professional insect hunters that, as part of their toxic arsenal, produce large PFPs known as latrotoxins. Interestingly, some latrotoxins have been identified as potent and highly-specific insecticides. Given the proteinaceous nature of these toxins, their promising future use as efficient bioinsecticides is discussed throughout this Perspective. Protein engineering and large-scale recombinant production are critical steps for the use of these PFPs as tools to control agriculturally important insect pests. In summary, both families of PFPs, from Cnidaria and Arachnida, appear to be molecules with promising biotechnological applications.

Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus that belongs to the actinoporin protein family. The high affinity of actinoporins for sphingomyelin (SM)-containing membranes has been well documented. However, the molecular determinants that define this affinity have not been fully clarified. Here, we have examined the binding and permeabilizing activity of StII to different single and mixed lipidic systems by combining lipid monolayers, liposomes, and permeabilizing assays. This study characterizes the contribution of ceramide-derived compounds for StII-membrane interaction. Molecular dynamics simulations revealed a differential binding mode of StII with the polar head group of SM and PC. The electrostatic interaction energies were the major energetic contributors to the better affinity of StII for SM compared to PC, while the van der Waals interaction energies were the major driving forces of the better affinity of StII for SM respect to Cer. Furthermore, the presence of sugar residues in glycosphingolipids modulated binding and pore-formation by actinoporins probably by hindering StII to reach relevant structural motifs in membrane for binding or inducing a non-competent adsorption to membrane. Our results demonstrate that StII-membrane interaction, leading to pore formation, may critically respond to changes in lipid head group properties, and the access to SM interfacial structural motif. [ABSTRACT FROM AUTHOR]

Hydra actinoporin-like toxin-1 (HALT-1) is a 20.8 kDa pore-forming toxin isolated from Hydra magnipapillata . HALT-1 shares structural similarity with actinoporins, a family that is well known for its haemolytic and cytolytic activity. However, the precise pore-forming mechanism of HALT-1 remains an open question since little is known about the specific target binding for HALT-1. For this reason, a comprehensive proteomic analysis was performed using affinity purification and SILAC-based mass spectrometry to identify potential protein-protein interactions between mammalian HeLa cell surface proteins and HALT-1. A total of 4 mammalian proteins was identified, of which only folate receptor alpha was further verified by ELISA. Our preliminary results highlight an alternative-binding mode of HALT-1 to the human plasma membrane. This is the first evidence showing that HALT-1, an actinoporin-like protein, binds to a membrane protein, the folate receptor alpha. This study would advance our understanding of the molecular basis of toxicity of pore-forming toxins and provide new insights in the production of more potent inhibitors for the toxin-membrane receptor interactions. [ABSTRACT FROM AUTHOR]

A group of stable, water-soluble and membrane-bound proteins constitute the pore forming toxins (PFTs) in cnidarians. They interact with membranes to physically alter the membrane structure and permeability, resulting in the formation of pores. These lesions on the plasma membrane causes an imbalance of cellular ionic gradients, resulting in swelling of the cell and eventually its rupture. Of all cnidarian PFTs, actinoporins are by far the best studied subgroup with established knowledge of their molecular structure and their mode of pore-forming action. However, the current view of necrotic action by actinoporins may not be the only mechanism that induces cell death since there is increasing evidence showing that pore-forming toxins can induce either necrosis or apoptosis in a cell-type, receptor and dose-dependent manner. In this review, we focus on the response of the cellular immune system to the cnidarian pore-forming toxins and the signaling pathways that might be involved in these cellular responses. Since PFTs represent potential candidates for targeted toxin therapy for the treatment of numerous cancers, we also address the challenge to overcoming the immunogenicity of these toxins when used as therapeutics.

Pore-forming toxins (PFTs) represent a unique class of highly specific lipid-binding proteins. The cytotoxicity of these compounds has been overcome through crystallographic structure and mutation studies, facilitating the development of non-toxic lipid probes. As a consequence, non-toxic PFTs have been utilized as highly specific probes to visualize the diversity and dynamics of lipid nanostructures in living and fixed cells. This review is focused on the application of PFTs and their non-toxic analogs as tools to visualize sphingomyelin and ceramide phosphoethanolamine, two major phosphosphingolipids in mammalian and insect cells, respectively. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Mauro Dalla Serra and Franco Gambale. [ABSTRACT FROM AUTHOR]

Actinoporins (APs) from sea anemones are ~ 20 kDa pore forming toxins with a β-sandwich structure flanked by two α-helices. The molecular mechanism of APs pore formation is composed of several well-defined steps. APs bind to membrane by interfacial binding site composed of several aromatic amino acid residues that allow binding to phosphatidylcholine and specific recognition of sphingomyelin. Subsequently, the N-terminal α-helix from the β-sandwich has to be inserted into the lipid/water interphase in order to form a functional pore. Functional studies and single molecule imaging revealed that only several monomers, 3–4, oligomerise to form a functional pore. In this model the α-helices and surrounding lipid molecules build toroidal pore. In agreement, AP pores are transient and electrically heterogeneous. On the contrary, crystallized oligomers of actinoporin fragaceatoxin C were found to be composed of eight monomers with no lipids present between the adjacent α-helices. This article is part of a Special Issue entitled: Pore-Forming Toxins edited by Maur Dalla Serra and Franco Gambale. [ABSTRACT FROM AUTHOR]

Actinoporins are pore-forming toxins (PFT) produced by sea anemones with molecular mass around 20 kDa and high affinity for sphingomyelin. The most studied atinoporins are sticholysins I and II (StI/StII) from Stichodactyla helianthus , equinatoxin II (EqtII) from Actinia equina , and fragaceatoxin C (FraC) from Actinia fragacea . Their N-terminal sequences encompassing residues 1–30 seem to be the best candidates for pore formation. This segment comprises an amphipathic α-helix preceded by a more or less hydrophobic segment, depending on the toxin, of around 10 amino acid residues. Although it is clear that the N-terminal is the most variable sequence in this protein family, the role of their hydrophobic segment in not fully understood. Here we show a comparison of StI, StII, EqtII, and FraC activities with that of their respective N-terminal synthetic peptides. The hemolytic and permeabilizing activity of the peptides reproduce qualitatively the behavior of their respective parental proteins and are particularly related to the hydrophobicity of the corresponding 1–10 segment. Furthermore, the dendrogram analysis of actinoporins' N-terminal sequence allows relating differences in alignment with differences in activity among the four toxins. We have also evaluated the penetration depth of the N-terminal segment of StI and StII by using Trp-containing peptide-analogs. Our data suggest that the N-terminus of StII is more deeply buried into the hydrophobic core of the bilayer than that of StI. We hypothesize that the highest activity of StII could be ascribed to a larger hydrophobic continuum , an uninterrupted sequence of non-charged mainly hydrophobic amino acid residues, of its N-terminus promoting a highest ability to partially insert in the membrane core. Moreover, as we show for four related peptides that a higher hydrophobicity contributes to increase the activity, we reinforce the notion that this property must be taken into account to design new potent membranotropic agents. [ABSTRACT FROM AUTHOR]

Actinoporins are a-pore forming proteins with therapeutic potential, produced by sea anemones. Sticholysin II (StnII) from Stichodactyla helianthus is one of its most extensively characterized members. These proteins remain stably folded in water, but upon interaction with lipid bilayers, they oligomerize to form a pore. This event is triggered by the presence of sphingomyelin (SM), but cholesterol (Chol) facilitates pore formation. Membrane attachment and pore formation require changes involving long-distance rearrangements of residues located at the protein-membrane interface. The influence of Chol on membrane recognition, oligomerization, and/or pore formation is now studied using StnII variants, which are characterized in terms of their ability to interact with model membranes in the presence or absence of Chol. The results obtained frame Chol not only as an important partner for SM for functional membrane recognition but also as a molecule which significantly reduces the structural requirements for the mentioned conformational rearrangements to occur. However, given that the DOPC:SM:Chol vesicles employed display phase coexistence and have domain boundaries, the observed effects could be also due to the presence of these different phases on the membrane. In addition, it is also shown that the Arg51 guanidinium group is strictly required for membrane recognition, independently of the presence of Chol. [ABSTRACT FROM AUTHOR]

Abstract: Sticholysin I (St I) is a pore-forming toxin (PFT) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin protein family, a unique class of eukaryotic PFT exclusively found in sea anemones. As for actinoporins, it has been proposed that the presence of sphingomyelin (SM) and the coexistence of lipid phases increase binding to the target membrane. However, little is known about the role of membrane structure and dynamics (phase state, fluidity, presence of lipid domains) on actinoporins' activity or which regions of the membrane are the most favorable platforms for protein insertion. To gain insight into the role of SM on the interaction of St I to lipid membranes we studied their binding to monolayers of phosphatidylcholine (PC) and SM in different proportions. Additionally, the effect of acyl chain length and unsaturation, two features related to membrane fluidity, was evaluated on St I binding to monolayers. This study revealed that St I binds and penetrates preferentially and with a faster kinetic to liquid-expanded films with high lateral mobility and moderately enriched in SM. A high content of SM induces a lower lateral diffusion and/or liquid-condensed phases, which hinder St I binding and penetration to the lipid monolayer. Furthermore, the presence of lipid domain borders does not appear as an important factor for St I binding to the lipid monolayer. [Copyright &y& Elsevier]

Sticholysin I (StI), an actinoporin expressed as a water-soluble protein by the sea anemone Stichodactyla helianthus, binds to natural and model membranes, forming oligomeric pores. It is proposed that the first event of a multistep pore formation mechanism consists of the monomeric protein attachment to the lipid bilayer. To date there is no high-resolution structure of the actinoporin pore or other membrane-bound form available. Here we evaluated StI:micelle complexes of variable lipid composition to look for a suitable model for NMR studies. Micelles of pure or mixed lysophospholipids and of dihexanoyl phosphatidylcholine (DHPC) were examined. The StI:DHPC micelle was found to be the best system, yielding a stable sample and good quality spectra. A comprehensive chemical shift perturbation analysis was performed to map the StI membrane recognition site in the presence of DHPC micelles. The region mapped (residues F51, R52, S53 in loop 3; F107, D108, Y109, W111, Y112, W115 in loop 7; Q129, Y132, D134, M135, Y136, Y137, G138 in helix-α2) is in agreement with previously reported data, but additional residues were found to interact, especially residues V81, A82, T83, G84 in loop 5, and A85, A87 in strand-β5. Backbone dynamics measurements of StI free in solution and bound to micelles highlighted the relevance of protein flexibility for membrane binding and suggested that a conformer selection process may take place during protein-membrane interaction. We conclude that the StI:DHPC micelles system is a suitable model for further characterization of an actinoporin membrane-bound form by solution NMR. Proteins 2014; 82:1022-1034. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Sea anemone actinoporins constitute an optimum model to investigate mechanisms of membrane pore formation. All actinoporins of known structure show a general fold of a β-sandwich motif flanked by two α-helices. The crucial structure for pore formation seems to be the helix located at the N-terminal end. The role of several other protein regions in membrane attachment is also well established. However, not much is known about the protein residues involved in the oligomerization required for pore formation. Previous detailed analysis of the soluble three-dimensional structures of different wild-type and mutant actinoporins from Stychodactyla helianthus suggested residues which could be involved in this oligomerization. One of these stretches contains a conserved sequence compatible with an integrin-binding RGD motif. The results presented now deal with mutants affecting this motif in the well-characterized actinoporin sticholysin II. Small modifications along this three-residue sequence had profound effects on its solubility. Just a single methyl group yielded an RAD mutant version with a highly diminished haemolytic activity and altered oligomerization behaviour. The results obtained are discussed in terms of a key role for the RGD motif in maintaining the actinoporins' pore-competent state of protein oligomerization. Structured digital abstract and by (, ) [ABSTRACT FROM AUTHOR]

Actinoporins are highly hemolytic pore-forming proteins with a molecular mass of around 20kDa 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. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Pore-forming proteins perforate lipid membranes and consequently affect their integrity and cell fitness. Therefore, it is not surprising that many of these proteins from bacteria, fungi, or certain animals act as toxins. While pore-forming proteins have also been found in plants, there is little information about their molecular structure and mode of action. Bryoporin is a protein from the moss Physcomitrium patens, and its corresponding gene was found to be upregulated by various abiotic stresses, especially dehydration, as well as upon fungal infection. Based on the amino acid sequence, it was suggested that bryoporin was related to the actinoporin family of pore-forming proteins, originally discovered in sea anemones. Here, we provide the first detailed structural and functional analysis of this plant cytolysin. The crystal structure of monomeric bryoporin is highly similar to those of actinoporins. Our cryo-EM analysis of its pores showed an actinoporin-like octameric structure, thereby revealing a close kinship of proteins from evolutionarily distant organisms. This was further confirmed by our observation of bryoporin's preferential binding to and formation of pores in membranes containing animal sphingolipids, such as sphingomyelin and ceramide phosphoethanolamine; however, its binding affinity was weaker than that of actinoporin equinatoxin II. We determined bryoporin did not bind to major sphingolipids found in fungi or plants, and its membrane-binding and pore-forming activity was enhanced by various sterols. Our results suggest that bryoporin could represent a part of the moss defense arsenal, acting as a pore-forming toxin against membranes of potential animal pathogens, parasites, or predators., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abstract: Actinoporins are water-soluble proteins with the ability to form pores upon insertion into biological membranes. They constitute a family of proteins with high degree of sequence identities but different hemolytic activities, suggesting that minor conformational arrangements result in major functional changes. A good example of this situation is the sea anemone Stichodactyla helianthus which produces two very similar actinoporins, sticholysins I (StnI) and II (StnII), but of very different hemolytic efficiency. Within this idea, given that the high resolution three-dimensional structure of StnII is already known, we have now solved that one corresponding to StnI in order to analyze the influence of particular residues on the conformation and activity of these proteins. In addition, random mutagenesis has been also used to produce five less hemolytic variants of StnI. All these mutations map to functionally relevant regions because they are probably involved in conformational changes associated with pore formation, which take place after membrane binding, and involve long-distance rearrangements of the polypeptide chain of actinoporins. [Copyright &y& Elsevier]

The sea anemone Stichodactyla helianthus produces two pore-forming proteins, sticholysins I and II (St I and St II). Despite their high identity (93%), these toxins exhibit differences in hemolytic activity that can be related to those found in their N-terminal. To clarify the contribution of the N-terminal amino acid residues to the activity of the toxins, we synthesized peptides spanning residues 1-31 of St I (StI) or 1-30 of St II (StII) and demonstrated that StII promotes erythrocyte lysis to a higher extent than StI. For a better understanding of the molecular mechanism underlying the peptide activity, here we studied their binding to lipid monolayers and pemeabilizing activity in liposomes. For this, we examined the effect on peptide membranotropic activity of including phospatidic acid and cholesterol in a lipid mixture of phosphatidylcholine and sphingomyelin. The results suggest the importance of continuity of the 1-10 hydrophobic sequence in StII for displaying higher binding and activity, in spite of both peptides' abilities to form pores in giant unilamellar vesicles. Thus, the different peptide membranotropic action is explained in terms of the differences in hydrophobic and electrostatic peptide properties as well as the enhancing role of membrane inhomogeneities. [ABSTRACT FROM AUTHOR]

A new actinoporin Hct-S4 (molecular mass 19,414 ± 10 Da) belonging to the sphingomyelin-inhibited α-pore forming toxin (α-PFT) family was isolated from the tropical sea anemone Heteractis crispa (also called Radianthus macrodactylus) and purified by methods of protein chemistry. The N-terminal nucleotide sequence (encoding 20 amino acid residues) of actinoporin Hct-S4 was determined. Genes encoding 18 new isoforms of H. crispa actinoporins were cloned and sequenced. These genes form a multigene Hct-S family characterized by presence of N-terminal serine in the mature proteins. Highly conserved residues comprising the aromatic phosphorylcholine-binding site and significant structure-function changes in the N-terminal segment (10-27 amino acid residues) of actinoporins were established. Two expressed recombinant actinoporins (rHct-S5 and rHct-S6) were one order less hemolytically active than native actinoporins. [ABSTRACT FROM AUTHOR]

Abstract: Actinoporins constitute a group of small and basic α-pore forming toxins produced by sea anemones. They display high sequence identity and appear as multigene families. They show a singular behaviour at the water–membrane interface: In aqueous solution, actinoporins remain stably folded but, upon interaction with lipid bilayers, become integral membrane structures. These membranes contain sphingomyelin, display phase coexistence, or both. The water soluble structures of the actinoporins equinatoxin II (EqtII) and sticholysin II (StnII) are known in detail. The crystalline structure of a fragaceatoxin C (FraC) nonamer has been also determined. The three proteins fold as a β-sandwich motif flanked by two α-helices, one of them at the N-terminal end. Four regions seem to be especially important: A cluster of aromatic residues, a phosphocholine binding site, an array of basic amino acids, and the N-terminal α-helix. Initial binding of the soluble monomers to the membrane is accomplished by the cluster of aromatic amino acids, the array of basic residues, and the phosphocholine binding site. Then, the N-terminal α-helix detaches from the β-sandwich, extends, and lies parallel to the membrane. Simultaneously, oligomerization occurs. Finally, the extended N-terminal α-helix penetrates the membrane to build a toroidal pore. This model has been however recently challenged by the cryo-EM reconstruction of FraC bound to phospholipid vesicles. Actinoporins structural fold appears across all eukaryotic kingdoms in other functionally unrelated proteins. Many of these proteins neither bind to lipid membranes nor induce cell lysis. Finally, studies focusing on the therapeutic potential of actinoporins also abound. [Copyright &y& Elsevier]

Abstract: Experimental evidence shows that the mechanism of pore formation by actinoporins is a multistep process, involving binding of the water-soluble monomer to the membrane and subsequent oligomerization on the membrane surface, leading to the formation of a functional pore. However, as for other eukaryotic pore-forming toxins, the molecular details of the mechanism of membrane insertion and oligomerization are not clear. In order to obtain further insight with regard to the structure-function relationship in sticholysins, we designed and produced three cysteine mutants of recombinant sticholysin I (rStI) in relevant functional regions for membrane interaction: StI E2C and StI F15C (in the N-terminal region) and StI R52C (in the membrane binding site). The conformational characterization derived from fluorescence and CD spectroscopic studies of StI E2C, StI F15C and StI R52C suggests that replacement of these residues by Cys in rStI did not noticeably change the conformation of the protein. The substitution by Cys of Arg52 in the phosphocholine-binding site, provoked noticeable changes in rStI permeabilizing activity; however, the substitutions in the N-terminal region (Glu2, Phe15) did not modify the toxin’s permeabilizing ability. The presence of a dimerized population stabilized by a disulfide bond in the StI E2C mutant showed higher pore-forming activity than when the protein is in the monomeric state, suggesting that sticholysins pre-ensembled at the N-terminal region could facilitate pore formation. [Copyright &y& Elsevier]

The use of pore-forming toxins from sea anemones (actinoporins) in the construction of immunotoxins (ITs) against tumour cells is an alternative for cancer therapy. However, the main disadvantage of actinoporin-based ITs obtained so far has been the poor cellular specificity associated with the toxin's ability to bind and exert its activity in almost any cell membrane. Our final goal is the construction of tumour proteinase-activated ITs using a cysteine mutant at the membrane binding region of sticholysin-I (StI), a cytolysin isolated from the sea anemone Stichodactyla helianthus. The mutant and the ligand moiety would be linked by proteinase-sensitive peptides through the StI cysteine residue blocking the toxin binding region and hence the IT non-specific killing activity. To accomplish this objective the first step was to obtain the mutant StI W111C, and to evaluate the impact of mutating tryptophan 111 by cysteine on the toxin pore-forming capacity. After proteolysis of the cleavage sequence, a short peptide would remain attached to the toxin. The next step was to evaluate whether this mutant is able to form pores even with a residual peptide linked to cysteine 111. In this work we demonstrated that (i) StI W111C shows pore-forming capacity in a nanomolar range, although it is 8-fold less active than the wild-type recombinant StI, corroborating the previously reported importance of residue 111 for the binding of StI to membranes, and (ii) the mutant is able to form pores even with a residual seven-residue peptide linked to cysteine 111. In addition, it was demonstrated that binding of a large molecule to cysteine 111 renders an inactive toxin that is no longer able to bind to the membrane. These results validate the mutant StI W111C for its use in the construction of tumour proteinase-activated ITs. [ABSTRACT FROM AUTHOR]

Stichodactyla heliantus II (St II) is a haemolytic toxin whose activity depends of the characteristics of red blood cells (RBC). Among the factors that may tune the response of the RBC to the toxin activity stand the oxidative status of the cell. This study investigates how pre-oxidation of RBC modifies St II activity employing two oxidants, peroxynitrite and hypochlorous acid. Results show that peroxynitrite-treated RBC are more resistant to St II activity. On the other hand, hypochlorous acid-treated RBC become more susceptible to St II. This contrasting behaviour of both oxidants is related to the modifications elicited in RBC by both oxidant agents. Peroxynitrite does not modify RBC osmotic fragility but reduces anion transport through band 3 protein. This effect, together with an increase in K++ efflux, can explain the increased resistance to the toxin activity. On the other hand, results obtained with hypochlorous acid can be explained in terms of a disruption of the membrane organization without the compensating effect of a reduction in band 3-mediated anion transport. The present results, obtained employing the effect of a model haemolytic toxin on RBC, emphasize the specificity of the RBC response to different endogenous oxidative agents. [ABSTRACT FROM AUTHOR]

The mechanism of pore formation by actinoporins is a multistep process, involving binding of water soluble monomer to membrane and subsequent oligomerization of monomers on the membrane surface, forming a functional pore. However , molecular details of membrane insertion mechanism and oligomerization are not clear . A phosphocholine-binding site and a surface cluster of aromatic rings, together with a basic region, are important to the initial interaction with membrane and the N-terminal region is relevant in the pore formation. Aiming to deepen into the structure-function relationship in sticholysins, we designed and produced two Cys mutants of recombinant sticholysin I (rSt I) in relevant functional regions for membrane interaction: St I E2C (in the N-terminal region) and St I R52C (in the membrane binding site). Conformational studies suggested that the replacement of Glu-2 and Arg-52 by a Cys residue in rSt I not noticeably changes protein conformation as assessed by fluorescence and CD spectroscopy, the first change not affecting toxin's permeabilizing ability. The relative decrease in the pore forming capacity of St I R52C is not related with a smaller binding capacity of this mutant to membrane. In summary, St I E2C and St I R52C retain the main conformational properties of the wild type and show similar binding to liposomal vesicles while differing in their permeabilizing activity. St I E2C and St I R52C constitute good tools to study those steps of the permeabilizing mechanism of sticholysins that take place after binding to membrane, using thiol-specific probes such as fluorescent and spin labels. [ABSTRACT FROM AUTHOR]

Abstract: We recently identified the existence of new isoforms of Avt-I (from sea anemone Actineria villosa) and Pstx20 (from sea anemone Phyllodiscus semoni) hemolytic toxins, and named them Avt-II and Pst-I. Avt-II and Pst-I differ in length by 14 and 7 bp, respectively, as compared to their corresponding isoform genes. Both newly found isoform genes have the coding regions with the identical length of 1033 bp. The restriction fragment length polymorphism analysis with endonuclease HphI was able to clearly distinguish between the two Avt isoforms, but not Pstx isoforms, and based on the densitometric analysis of DNA bands, it indicated that relative expression levels of Avt-I and Avt-II genes were 18.3% and 81.7%, respectively. PCR amplification of the two Avt isoform genes using the genomic DNA as template indicated the existence of two introns within each toxin isoform gene. The first intron with the identical 242 bp in length for both Avt isoform was found within the 5′-untranslated region, and the second intron with lengths of 654 bp and 661 bp in Avt-I and Avt-II isoforms, respectively, was found within the signal sequence coding region. This is for the first time to identify the existence of introns within hemolysin genes of sea anemone. Having several unique characteristics that have identified only for a new member of actinoporin family of A. villosa and P. semoni, e.g., strong toxicity and genes with introns, it is plausible to speculate that these toxins have a unique genetic evolutionary linage differed from that for other sea anemone hemolytic toxins. [ABSTRACT FROM AUTHOR]

Abstract: Actinoporins Or-A and Or-G from the northern sea anemone Oulactis orientalis and actinoporins RTX-A and RTX-SII from the tropical sea anemone Radianthus macrodactylus (=Heteractis crispa) were compared with each other and with some known actinoporins. In this work the complete amino acid sequence of RTX-SII was determined by molecular biology methods. The following differences were revealed in functionally significant regions of Radianthus, Oulactis, and some other actinoporins: (i) tryptophan is substituted for leucine in the position equivalent to Trp112 in the POC binding site of EqtII; (ii) 13 and 5 residues are truncated in N-terminal regions of Or-A and Or-G, respectively. A possible role of these structural differences in specific regions of the actinoporin sequence was analyzed. Some differences in hydrophobicity parameters, distribution of charged residues, and length of actinoporins’ N-terminus apparently cause considerable differences in their hemolytic activities. Homology models of Radianthus and Oulactis actinoporin monomers were generated using crystal structures of equinatoxin II from Actinia equina and sticholysin II from Stichodactyla helianthus as templates. The current data on actinoporin structures and activities, coupled with results of our earlier differential scanning calorimetric and electrophoretic experiments with RTX-A-modified erythrocyte ghosts (), suggests that the exposed RGD motif located near the POC binding site can interact with membrane integrin(s). [ABSTRACT FROM AUTHOR]

Echotoxins are hemolytic and lethal proteinaceous toxins of about 25 kDa that are contained in the salivary gland of marine gastropod Monoplex echo. In this study, complementary DNAs (cDNAs) encoding four echotoxins (2, A, B1 and B2) were isolated from the cDNA library constructed from the M. echo salivary gland and completely sequenced. Although the amino acid sequence identity between the four echotoxins and actinoporins (20 kDa hemolysins from sea anemones) was very low (12-16%), some amino acid residues important for the biological activity of actinoporins were well conserved in the echotoxins. In the case of echotoxin 2, the genomic DNA corresponding to the coding region was amplified. Sequencing data revealed that the echotoxin 2 gene is devoid of introns within the coding region as reported for the actinoporin genes. These results suggest that echotoxins have evolved from actinoporins or both toxins have evolved from the same ancestor. [ABSTRACT FROM AUTHOR]

Abstract: Four isoforms of actinoporins were isolated in 2002–2004 from the tropical sea anemone Heteractis crispa (=Radianthus macrodactylus). Their potent hemolytic activities and effects on Ehrlich ascites carcinoma bearing mice were also studied. In this study, the individual actinoporin (RTX-A) demonstrated potential cancer-preventive activity at extremely low and non-cytotoxic concentrations. The substance suppressed the malignant transformation of mouse JB6 P+ Cl41 cells stimulated by epidermal growth factor (EGF) in soft agar with the inhibition of number of the colonies C50 (INCC50)=0.034nM. Actinoporin RTX-A also was shown to inhibit the phenotype expression of HeLa human cancer cells with an INCC50 =0.03nM. The cytotoxic effect of RTX-A against JB6 P+ Cl41 cells and HeLa, THP-1, MDA-MB-231, and SNU-C4 human tumor cell lines was high (IC50 =0.57, 2.26, 1.11, 30.0 and 4.66nM), but significantly less than their capacity to suppress tumor cell colony formation or phenotype expression. RTX-A also induced apoptosis and inhibited basal AP-1, NF-κB, and p53-dependent transcriptional activity in JB6 Cl41 cells. These results confirmed that actinoporin RTX-A from H. crispa, at least partially, might exhibit cancer-preventive and anticancer cytotoxic properties through the induction of p53-independent apoptosis and inhibition of the oncogenic AP-1 and NF-κB nuclear factors activity. [Copyright &y& Elsevier]

Sticholysin II is an actinoporin of 175 amino acids produced by the sea anemone Stichodactyla helianthus. Several studies with different mutants have been performed to characterize its molecular properties and activity. As a first step towards a 3D structural characterization and its interaction with membrane models at a residue level, herein we report the nearly complete NMR 15N, 13C and 1H chemical shifts assignments of the Y111N variant at pH 4.0 and 25°C (BMRB No. 16630). The assignment is complete for the biologically relevant residues, specially for those implicated in membrane interactions. [ABSTRACT FROM AUTHOR]

Abstract: The use of membrane active toxins as toxic moieties in the construction of immunotoxins (ITs) is an attractive alternative to overcome some of the problems of classical ITs since these new conjugates are based in the use of a different mechanism of killing undesired cells. Pore-forming cytolysins from sea anemones were used in the construction of ITs targeted to different cell types including tumour cell lines and the parasite Giardia duodenalis. The results obtained support the feasibility of directing these cytolysins to the surface of the cancer cells or the parasite through their conjugation to monoclonal antibodies recognizing tumour-associated or parasite antigens, respectively. However the main problem with the IT constructed in this fashion is the lack of specificity associated with the toxin moiety. An approach designed to overcome this limitation was the construction of inactive cytolysin with built-in biological “trigger” that renders the toxin active in the presence of tumour-specific proteinases. This construction is considered as a proof of concept to demonstrate the feasibility of such activation systems in the construction of ITs based on pore-forming cytolysins from sea anemones with reduced unspecific activity. The future prospects of the use of the N-terminal region of actinoporins for construction of IT is also described. [Copyright &y& Elsevier]

Abstract: Cnidarians (Coelenterates), a very old and diverse animal phylum, possess a wide variety of biologically active substances that can be considered as toxins. Anthozoan toxins can be classified into two chemically very different groups, namely polypeptide toxins isolated from sea anemones and diterpenes isolated from octocorals. Cubozoan and scyphozoan protein toxins have been the most elusive cnidarian toxins to investigate – despite a tremendous effort in the past few decades, very few of these large, relatively unstable protein toxins were isolated, but recently this has been achieved for cubozoan venoms. Hydrozoans mainly contain large proteins with physiological mechanisms of action similar to the sea anemone and jellyfish pore-forming toxins. This article will focus on the in vivo physiological effects of cnidarian toxins and venoms; their actions at the cellular level will only be considered to understand their actions at the organ and whole animal levels. An understanding of mechanisms underlying the in vivo toxic effects will facilitate the development of more effective treatments of cnidarian envenomations. [Copyright &y& Elsevier]

Abstract: 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. As for the rest of the members of this family, Sts are cysteine-less proteins, with molecular weights around 20kDa, high isoelectric points (>9.5), and a preference for sphingomyelin-containing membranes. A three-dimensional structure of StII, solved by X-ray crystallography, showed that it is composed of a hydrophobic β-sandwich core flanked on the opposite sides by two α helices comprising residues 14–23 and 128–135. A variety of experimental results indicate that the first thirty N-terminal residues, which include one of the helices, are directly involved in pore formation. This region contains an amphipathic stretch, well conserved in all actinoporins, which is the only portion of the molecule that can change conformation without perturbing the general protein fold; in fact, binding to model membranes only produces a slight increase in the regular secondary structure content of Sts. Sts are produced in soluble form but they readily bind to different cell and model membrane systems such as lipidic monolayers, micelles, and lipid vesicles. Remarkably, both the binding and pore-formation steps are critically dependent on the physico-chemical nature of the membrane. In fact, a large population of toxin irreversibly binds with high affinity in membranes containing sphingomyelin whereas binding in membranes lacking this sphingolipid is relatively low and reversible. The joint presence of SM and cholesterol largely promotes binding and pore formation. Minor amounts of lipids favoring a non-lamellar organization also augment the efficiency of pore formation. The functional pore formed in cellular and model membranes has a diameter of ∼2.0nm and is presumably formed by the N-terminal α helices of four monomers tilted 31° in relation to the bilayer normal. Experimental evidence supports the hypothesis that sticholysins, as well as equinatoxin II, another actinoporin, form a toroidal pore in membranes in which the polypeptide chains as well as the polar head groups of phospholipids are involved. [Copyright &y& Elsevier]

Abstract: Cnidarians are the largest phylum of generally toxic animals, yet their toxins and venoms have not received as much scientific attention as those of many terrestrial (snakes, scorpions, spiders, etc.) and even some marine animals (i.e. cone snails). Approximately 13,000 living cnidarian species have been described by systematists. A major rationale for their study in the past, besides scientific curiousity, was to better treat victims of their envenomation. While that goal remains a high priority, it is now appreciated that the toxins of these mostly marine animals can be very useful molecular probes for the analysis of ion channels involved in electrical signaling, immune responses and other signal transduction processes of biomedical interest. For instance, anaphylaxis was discovered by during experiments with sea anemone and hydrozoan tentacular extracts. Similarly, it has recently been shown that a toxin from another sea anemone is able to potently inhibit T-lymphocyte proliferation in models of certain autoimmune diseases. Thus, these natural substances continue to be of relevance for understanding and treating human diseases. In addition to introducing phylum Cnidaria (Coelenterata), we provide a short history of early (until about 1990) research on cnidarian toxins and venoms, to provide a perspective for appreciating the scientific advances of the past two decades that are summarized in the ensuing 19 papers in this special Toxicon issue. [Copyright &y& Elsevier]

Abstract: Sticholysin II (St II) is a toxin from the sea anemona Stichodactyla helianthus that produces erythrocytes lysis at low concentration and its activity depends on the presence of calcium. Calcium may act modifying toxin interaction with erythrocyte membranes or activating cellular processes which may result in a modified St II lytic action. In this study we are reporting that, in the presence of external K+, extracellular calcium decreased St II activity on erythrocytes. On the other hand an increase of intracellular calcium promotes Sty II lytic activity. The effect of intracellular calcium was specifically studied in relation to membrane lipid translocation elicited by scramblases and how this action influence St II lytic activity on erythrocytes. We used 0.5mmol/L calcium and 10mmol/L A23187, as calcium ionophore, for scramblases activation and found increased St II activity associated to increase of intracellular calcium. N-ethyl maleimide (activator) and 4,4′-diisothiocyanatostilbene-2,2′-disulfonate (inhibitor) were used as scramblases modulators in the assays which produced an increase and a decrease of the calcium effect, respectively. Results reported suggest an improved St II membrane pore-forming capacity promoted by intracellular calcium associated to membrane phospholipids translocation. [Copyright &y& Elsevier]

• Modern land plants arose from a green algae-like ancestor c. 480 million years ago. While several novel morphological features were critical for survival in the aerial environment, physiological innovation undoubtedly played a key role in the colonization of terrestrial habitats. Recently, actinoporin genes, a small group of pore-forming toxins from sea anemones, have been found in the bryophyte and lycophyte lineages of land plants where they are upregulated in water-stressed tissues. • The bryoporin gene in the moss Physcomitrella patens ( PpBP) was functionally characterized by RNA blot analyses and overexpression in P. patens. In order to examine functional homology between PpBP and sea anemone actinoporins, the recombinant PpBP was subjected to hemolytic analysis of pig blood cells, which is one of the specific activities of actinoporins. • PpBP was upregulated by various abiotic stresses, in particular most strongly by dehydration stress. Overexpression of the bryoporin gene heightens drought tolerance in P. patens significantly. In addition, PpBP shared the highest structural homology with actinoporins in a three-dimensional structural database and showed hemolytic activity. • These results suggest that this phylogenetic distribution may have resulted from an ancient horizontal gene transfer and actinoporins may have played an important role in early land plants. [ABSTRACT FROM AUTHOR]

A 20-kDa protein toxin (bandaporin) was isolated from the sea anemone Anthopleura asiatica (Actiniidae) using the precipitation of ammonium sulfate saturation, and ion-exchange and gel-permeation chromatographies. Bandaporin showed potent hemolytic activity (EC50 value: 8.8 ng/ml to 0.8% suspension of sheep red blood cells) and lethal toxicity to crayfish (LD100 value: 0.58 mg/kg). The hemolytic activity of bandaporin was inhibited specifically by sphingomyelin. Eight other tested membrane lipids, including cholesterol, and gangliosides GM1 and GM2, did not inhibit bandaporin hemolytic activity. Antimicrobial and phospholipase A2 (PLA2) activities were not detected in bandaporin. The complementary DNA (cDNA) encoding bandaporin was sequenced and the deduced amino acid sequence of bandaporin revealed that this actinoporin had not been previously characterized. Actinoporins are cytolytic peptide toxins that are widely distributed in Actiniidae and Stichodactylidae sea anemones. This study was the first characterization of an actinoporin from a sea anemone of the genus Anthopleura. [ABSTRACT FROM AUTHOR]

Abstract: The mechanisms by which pore-forming toxins are able to insert into lipid membranes are a subject of the highest interest in the field of lipid–protein interaction. Eight mutants affecting different regions of sticholysin II, a member of the pore-forming actinoporin family, have been produced, and their hemolytic and lipid-binding properties were compared to those of the wild-type protein. A thermodynamic approach to the mechanism of pore formation is also presented. Isothermal titration calorimetry experiments show that pore formation by sticholysin II is an enthalpy-driven process that occurs with a high affinity constant (1.7×108 M−1). Results suggest that 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. Membrane binding is achieved through two separate mechanisms, that is, recognition of the lipid–water interface by a cluster of aromatic residues and additional specific interactions that include a phosphocholine-binding site. Thermodynamic parameters derived from titration experiments are discussed in terms of a putative model for pore formation. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]