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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.

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 (IC 50 = 7.3 µM), colorectal HT-29 (IC 50 = 6.8 µM), and melanoma SK-MEL-28 (IC 50 = 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.

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.

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

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., (© 2013.)

A new Cytolysin, termed as Gigantoxin-4, was isolated from the sea anemone Stichodactyla gigantea and found to be highly homologous with Cytolysin-3 (HMg III) from Heteractis magnifica, RTX-A from Radianthus macrodactylus, and Sticholysin-1 (St I) and Sticholysin-2 (St II) from Stichodactyla helianthus (homology 82%, 86%, 82% and 86% respectively). Its 20 N-terminal residues were identified and the full-length cDNA sequence was obtained by reverse transcription-polymerase chain reaction (RT-PCR). Multiple sequence alignments with other Cytolysins of the actinoporin family clearly indicated that Gigantoxin-4 belongs to this protein family. SDS-PAGE electrophoresis showed that this new actinoporin had a molecular mass of about 19 kDa, and possessed a high hemolytic activity to human erythrocytes (HA(50)= 40 ng/ml), which was inhibited by pre-incubation with sphingomyelin (SM) or SM-cholesterol mixtures. Our in vivo experiments showed that Gigantoxin-4 had wide toxicity to the rat cardiovascular system and the respiratory system. A concentration of 30 μg/kg Gigantoxin-4, i.v. produced a positive inotropic effect on the rat heart although final cardiovascular failure was inevitable, and 60 μg/kg Gigantoxin-4 caused respiratory arrest rapidly resulting in rat death. HE staining indicated pathological changes in various organs and tissues after i.v. administration of Gigantoxin-4.

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 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]

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]

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]

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]

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]

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: 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]

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: 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: 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]

Equinatoxin II is a cytolytic protein isolated from the sea anemone Actinia equina. It is a member of the actinoporins, a family of eukaryotic pore-forming toxins with a unique mechanism of pore formation. Equinatoxin II is a 20 kDa cysteineless protein, with sphingomyelin-dependent activity. Recent studies showed that the N-terminal region of the molecule requires conformational flexibility during pore formation. An understanding of the N-terminal position in the final pore and its role in membrane insertion and pore stability is essential to define the precise molecular mechanism of pore formation. The formation of pores and their electrophysiologic characteristics were studied with planar lipid membranes. We show that amino acids at positions 1 and 3 of equinatoxin II are exposed to the lumen of the pore. Moreover, sulfhydryl reagents and a hexa-histidine tag attached to the N-terminus revealed that the N-terminus of the toxin extends through the pore to the other ( trans) side of the membrane and that negatively charged residues inside the pore are crucial to define the electrophysiologic characteristics of the channel. Finally, we detected a new, less stable, state with a lower conductance by using a deletion mutant in which the first five N-terminal amino acids were removed. We propose that the first five amino acids help to anchor the amphipathic helix on the trans side of the membrane and consequently stabilize the final transmembrane pore. [ABSTRACT FROM AUTHOR]

Abstract: Wild-type actinoporins StnI and StnII from the sea anemone Stichodactyla helianthus, as well as their NH2-terminal six-His tagged versions, have been overproduced in Escherichia coli. Overproduction of both wild-type proteins was only possible after introducing silent mutations within the 5′-end of their original cDNA sequences. These mutations would prevent the formation of RNA secondary structures blocking the ribosome-binding site and the initiation codon. The four recombinant proteins were purified to homogeneity in milligrams amount and characterized from spectroscopic and functional points of view. All the isolated proteins behaved as the corresponding natural ones although the six-His tagged variants exhibited a decreased lytic activity. The strategy described will be useful to allow the production of mutant variants of these proteins and probably of other actinoporins. [Copyright &y& Elsevier]

Sticholysin II (St II) is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus with a potent hemolytic activity. For an insight into the role of different St II sequence fragments in their interaction with membranes, we carried out protein digestion with cyanogen bromide. This treatment rendered three peptides, one of them (P3) containing the St II N-terminal sequence (amino acids 1 to 92). Neither P2 nor P1 (amino acids 102 to 130, and 131 to 157, respectively) exhibit measurable hemolytic activity or interfere with the native St II function. On the other hand, P3 is able to inhibit toxin hemolytic activity. These differences are discussed in terms of their relative capacity to interact with lipidic membranes. [ABSTRACT FROM AUTHOR]

Abstract: The amino acid sequence of actinoporin RTX-A (175 aa) from the sea anemone Radianthus macrodactylus was determined by sequencing of clones obtained via amplification of cDNA. It was established that RTX-A possessed high homology with HmgIII from Heteractis magnifica (87%) and StI, StII from Stichodactyla helianthus (84 and 87%, respectively). The analysis of structural and functional relationships within RTX-A was carried out. The some disagreement concerning to significant role of several amino acid residues for actinoporins exhibition of hemolytic activity was found. [Copyright &y& Elsevier]

The spatial organization of actinoporin RTX-SII from the sea anemone Radianthus macrodactylus on the level of tertiary and secondary structures was studied by UV and CD spectroscopy and intrinsic protein fluorescence. The specific and molar extinction coefficients of RTX-SII were determined. The percentages of canonical secondary structures of actinoporin were calculated. The tertiary structure of the polypeptide is well developed and its secondary structure is highly ordered and contains about 50% antiparallel folded β-sheets. The irreversible thermal denaturation of RTX-SII was studied by CD spectroscopy; a conformational transition occurs at 53°C. Above this temperature irreversible conformational changes are observed in the secondary and tertiary structures. This is accompanied by redistribution of the content of regular and distorted forms of β-sheet and also by increase in the content of an unordered form. It is suggested that an intermediate is formed in the process of thermal denaturation. Acid-base titration of RTX-SII results in irreversible conformational changes at pH below 2.0 and above 12.0. As shown by intrinsic protein fluorescence, tyrosine residues of RTX-SII make a fundamental contribution to emission, and the total fluorescence depends more on temperature and ionic strength of the solution than tryptophan fluorescence. The data on conformational stability of actinoporin are correlated with data on its hemolytic activity. Activity of RTX-SII significantly decreases at increased temperature and slightly decreases at low pH. Hemolytic activity drastically increases at high pH. Increase in the actinoporin activity at pH above 10 seems to be caused by ionization of the molecule. [ABSTRACT FROM AUTHOR]

A rapid screening method for haemolytic activity, using blood agar plates, has been developed to analyze randomly produced mutant variants of the pore-forming protein sticholysin II (Stn II). Those exhibiting a reduced activity were selected and the DNA corresponding to each Stn II variant sequenced. Once the mutation produced was determined, protein variants were isolated and characterized in terms of structure (circular dichroism spectra and thermal stability) and haemolytic activity. Three single mutation protein variants, at residues K19, F106 and Y111, showed a significantly decreased haemolytic activity while their thermostability was identical to that of the wild-type protein. Considering the obtained data and based on the three-dimensional structure of the protein, the role of these residues on the mechanism of haemolysis has been analyzed. [Copyright &y& Elsevier]

Sticholysin I (StI), a potent cytolysin isolated from the sea anemone Stichodactyla helianthus, was linked to the monoclonal antibody (mAb) ior C5. StI acts by forming hydrophilic pores in the membrane of the attacked cells leading to osmotic lysis. ior C5 is a murine IgG1, which recognizes the tumor associated antigen (TAA) ior C2. The cytolysin and the mAb were coupled by using the heterobifunctional cross-linking reagent sulfosuccinimidyl 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (SMCC). Two hybrid molecules composed by one ior C5 and one or two StI molecules were obtained (named conjugated I and II, respectively). The purified conjugates were evaluated by a binding affinity assay against an ior C2-positive colon cancer cell line (SW948). Both molecules were able to recognize the antigen (Ag) in the same way that unconjugated ior C5 does. The activity of both conjugates against human erythrocytes and SW948 cells was assessed. They lost most of their hemolytic activity but their residual activity was very similar. Nevertheless, when their cytotoxicity was studied on the SW948 cell line, only conjugate II killed efficiently the cells, indicating a specific mAb-Ag interaction. In this chimeric molecule the ratio between the cytotoxic and the hemolytic activity was larger than that of the free cytolysin. This fact indicates an increase of the specificity of the toxic effect toward the SW948 cell line and consequently an increase of the difference between its hemolytic and cytotoxic doses. The results herein support the feasibility of directing StI to the surface of cancer cells expressing ior C2 Ag via the mAb ior C5. [Copyright &y& Elsevier]

The radius of the pore formed by sticholysin I and II (StI, StII) in erythrocytes and sticholysin I in lipid vesicles was investigated. The rate of colloid osmotic lysis of human erythrocytes, exposed to one of the toxins in the presence of sugars of different size, was measured. The relative permeability of each sugar was derived and the pore radius estimated with the Renkin equation. The radius was similar for sticholysin I and II and was independent of the reference sugar chosen and of the toxin concentration applied. It was also the same when erythrocytes were pretreated with different toxin doses in the presence of a polyethylene glycol (PEG) large enough to prevent lysis and thereafter transferred to solutions containing oligosaccharides of different size where they did lyse at different rates. The osmometric behavior of large unilamellar vesicles (LUV) was thereafter used to estimate the toxin lesion radius in a model system. LUV transferred to a hyperosmotic solution with a certain sugar immediately shrank and then re-swelled at a rate dependent on the bilayer permeability to water and sugar. When LUV were previously permeabilized with StI, only a fraction of them, namely those not carrying pores, continued to behave as osmometers. By increasing the size of the added sugar and approaching the pore radius, the fraction of osmometric LUV increased. Relative permeabilities were derived and used to estimate a channel radius around 1.2 nm, both for sugars and for PEGs. In conclusion the sticholysin pore has a constant size independent of toxin concentration and similar in natural and artificial membranes, suggesting it has a fixed predominant structure. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]