A new polypeptide toxin from the nematocyst venom of an Okinawan sea anemone Phyllodiscus semoni (Japanese name "unbachi-isoginchaku")

The venomous sea anemone Phyllodiscus semoni causes cases of severe stinging. We isolated Phyllodiscus semoni toxin 20A (PsTX-20A), a hemolytic and lethal polypeptide (20 kDa), from the nematocyst venom of this species for the first time. Furthermore, we sequenced the cDNA encoding PsTX-20A. The deduced amino acid sequence of PsTX-20A showed that this toxin was a new member of the actinoporin family, which consists of several cytolytic polypeptides originating from sea anemones. PsTX-20A showed lethal toxicity to the shrimp Palaemon paucidens when administered via intraperitoneal injection (LD50, 50 microg/kg) and hemolytic activity toward 0.8% sheep red blood cells (ED50, 80 ng/ml).     

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: Purification and biological characterization

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA₂ activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA₂ activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED₅₀ of 0.270 μg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.     

Cytotoxicity of the nematocyst venom from the sea anemone Aiptasia mutabilis

Crude extracts of the coelenterate Aiptasia mutabilis (Anthozoa, Aiptasiidae) nematocysts have been tested for their cytotoxicity of Vero and HEp-2 cells monolayers. The results indicate that the nematocyte venom contains one or more toxins with an extremely powerful cytolytic activity. An extract containing the equivalent of as little as 0.6 nematocysts/microL is sufficient to induce significant cellular necrosis, and IC50 can be estimated to be ca. 2 nematocysts/microL on Vero cells. These values are 1-2 orders of magnitude lower than those reported so far for other sea anemone venoms. The extreme potency is accompanied by poor stability of the venom that is readily inactivated by moderate heat and by buffers at non-neutral pH values. The extract is unstable even when kept for short times at 4 degrees C, or after storage at -20 degrees C. Separation of crude venom by affinity chromatography on ConA-Sepharose allowed us to identify two main components with molecular masses of 95 and 31 kDa, respectively, as responsible for the cytolytic properties of A. mutabilis nematocyst extract.     

Molecular cloning and functional expression of hemolysin from the sea anemone Actineria villosa

The full-length cDNA that encodes the hemolytic toxin Avt-I, with 226 amino acids, from the venomous sea anemone Actineria villosa has been cloned using the oligo-capping method. The cDNA contains 681bp open reading frame and its predicted amino acid sequences revealed that Avt-I was basic polypeptides without cysteine residues and Arg-Gly-Asp (RGD) motif sequence. The mature Avt-I has a predicted molecular weight of 19.6 kDa and its theoretical isoelectric point is 9.3. The Avt-I revealed 99, 61, 57, and 57% amino acid similarity with hemolytic toxins Pstx20, EqtII, StII, and HmT from Phyllodiscus semoni, Actinia Equina, Stichodactyla helianthus, and Heteractis magnifica, respectively. The characteristic amphiphilic alpha-helix structure was found at the N-terminal region of the mature Avt-I. Recombinant Avt-I (rAvt-I) was expressed in Escherichia coli BL21 (DE3) strain as a biologically active form and purified rAvt-I caused 50% hemolytic activity against 1% sheep erythrocytes at a concentration of 6.3 ng/ml (0.32 nM). M9Y medium led to more than 2-fold increase in rAvt-I yield than cultivation in Luria-Bertani medium.     

Vitamin B1 thiazole derivative reduces transmembrane current through ionic channels formed by toxins from black widow spider venom and sea anemone in planar phospholipid membranes

The vitamin B₁ (thiamine) structural analogue 3-decyloxycarbonylmethyl-4-methyl-5-(β-hydroxyethyl) thiazole chloride (DMHT) (0.1 mM) reversibly reduced transmembrane currents in CaCl₂ and KCl solutions via ionic channels produced by latrotoxins (α-latrotoxin (α-LT) and α-latroinsectotoxin (α-LIT)) from black widow spider venom and sea anemone toxin (RTX) in the bilayer lipid membranes (BLMs). Introduction of DMHT from the cis-side of BLM bathed in 10 mM CaCl₂ inhibited transmembrane current by 31.6 ± 3% and by 61.8 ± 3% from the trans-side of BLM for α-LT channels. Application of DMHT in the solution of 10 mM CaCl₂ to the cis-side of BLM decreased the current through the α-LIT and RTX channels by 52 ± 4% and 50 ± 5%, respectively. Addition of Cd²⁺ (1 mM) to the cis- or trans-side of the membrane after the DMHT-induced depression of Ca²⁺-current across the α-LT channels caused its further decrease by 85 ± 5% that coincides favorably with the intensity of Cd²⁺ blocking in control experiments without DMHT. These data suggest that DMHT inhibiting is not specific for latrotoxin channels only and DMHT may exert its action on α-LT channels without considerable influence on the ionogenic groups of Ca²⁺-selective site inside the channel cavity. The binding kinetics of DMHT with the α-LT channel shows no cooperativity and allows to expect that the DMHT binding site of the toxin is formed by one ionogenic group as the slopes of inhibition rate determined in log-log coordinates are 1.25 on the trans-side and 0.68 on the cis-side. Similar pK of binding (5.4 on the trans-side and 5.7 on the cis-side) also suggest that DMHT may interact with the same high affinity site of α-LT channel on either side of the BLM. The comparative analysis of effective radii measured for α-LT, α-LIT and RTX channels on the cis-side (0.9 nm, 0.53 nm and 0.55 nm, correspondingly) and for α-LT channel on the trans-side (0.28 ± 0.18 nm) with the intensity of DMHT inhibitory action obtained on these channels allowed to conclude that the potency of DMHT inhibition increased on toxin pores of smaller lumen.     

Membrane damage by a toxin from the sea anemone Stoichactis helianthus. I. Formation of transmembrane channels in lipid bilayers

The addition of nanomolar amounts of a toxin preparation derived from the sea anemone Stoichactis helianthus to black lipid membranes increases their electrical conductance by one million-fold. In addition, the membranes become permeable predominantly to monovalent cations. The elevated bilayer conductance is voltage-dependent, and the current-voltage curves of these bilayers display rectification as well as a region of negative resistance. The membrane activity of the toxin is proportional to the third power of its concentration, and at very low concentrations the membrane conductance increases in discrete uniform steps. These observations indicate that the mechanism of toxin action involves the formation of transmembrane channels constructed by the aggregation of protein molecules which are inserted in the bilayer. The voltage-dependent membrane conductance arises from two distinct channel characteristics: (1) the unit conductance of individual channels is dependent on the polarity of applied voltage; (2) the number of ion-conducting channels is influenced by the polarity as well as the magnitude of applied potential. It is believed that these effects are due to the influence of an electric field on the insertion of toxin molecules into the bilayer or on their subsequent association with each other to produce channels. Partial chemical characterization of the toxin material has shown that the membrane active factor is a basic protein with a molecular weight of 17 500.     

Convenient nomenclature of cysteine-rich polypeptide toxins from sea anemones

Polypeptide toxins are the main constituents of natural venoms. Considerable progress in the study of these molecules has resulted in the determination of a large number of structurally related sequences. To classify newly discovered molecules, a rational nomenclature for naming peptide toxins was developed, which takes into account toxin biological activity, the species name, and structural peculiarities of the polypeptide. Herein, we suggest modifications to this nomenclature for cysteine-rich polypeptide toxins from sea anemones and describe 11 novel polypeptide structures deduced after common database revision.     

Isolation and characterization of a novel protein toxin from fire coral

Fire corals (Millepora spp.) cause severe pain and inflammatory effects in humans upon contact, and the organs responsible for these effects are called nematocysts. Here, we isolated an active cytotoxin of ca. 18 kDa (MCTx-1) from nematocysts of Millepora dichotoma var. tenera. MCTx-1 was potently cytotoxic (EC₅₀ value 79 ng/mL) towards L1210 mouse leukemia cells, hemagglutinated a 0.8% suspension of sheep erythrocytes (0.2 μg protein/mL) and was lethal in crayfish (LD₅₀, 106 μg/kg). We deduced the primary structure of MCTx-1 from the corresponding cDNA sequence and found that MCTx-1 is a novel dermatopontin that is an extracellular matrix protein in mammals. This is the first characterization of a proteinaceous toxin from fire coral.     

Ultrastructure of nematocyst discharge in catch tentacles of the sea anemone Haliplanella luciae (Cnidaria: Anthozoa)

The mature nematocyst lies just beneath the cnidodyte plasma membrane. A microtubule array surrounds the nematocyst capsule just beneath the capsule tip. We propose that the array helps to hold the capsule at the cnidocyte cell surface until discharge. The undischarged capsule tip is sealed by three apical flaps, joined together along complex radial seams. The seams are filled with subunits that appear to bind the flaps together. Upon discharge, the flaps separate along the radial seams to permit thread eversion. The everted thread is lined on both sides by subunits that are stained by antimonate, indicating that they bind calcium. We suggest that, together, the subunits hold the uneverted thread in its folded and coiled configuration. Thread eversion would follow subunit uncoupling. The capsule and thread interiors of partially discharged nematocysts are stained by antimonate. In contrast, the capsule and thread interiors of fully discharged nematocysts are not stained by antimonate. Thus, nematocyst calcium might be injected into the target tissue where it is presumed to act in conjunction with nematocyst venom to promote cell death.     

Functional expression and characterization of four novel neurotoxins from sea anemone Anthopleura sp

The genes of four novel neurotoxins, named Hk2a, Hk7a, Hk8a, and Hk16a, were obtained from sea anemone Anthopleura sp. All four neurotoxins were composed of 47 amino acid residues and the variable residues among them were found in positions 14, 22, 25, and 37. To study their activities, the four toxins fused to the Escherichia coli thioredoxin were overexpressed by BL21 (DE3), cleaved off from the fusion partner, purified, and characterized with MALDI-TOF and CD assays. Contractile force studies of isolated SD atria indicated that rHk2a had the strongest and rHk7a the longest heart stimulation effect. Consequently, the Arg14, a highly conserved residue in various sea anemone neurotoxins, can be inferred to contribute to the duration but not the intensity of contraction-stimulating activity. Our work renders useful information to studies of sea anemone neurotoxins, especially to the clarification of the function of the disputative Arg14.     

Enzymatic and structural characterization of a basic phospholipase A2 from the sea anemone Condylactis gigantea

This work aimed at the isolation and structural/functional characterization of a phospholipase A₂ (CgPLA₂) from the extract of the anemone Condylactis gigantea. CgPLA₂ was isolated with a high purity level through three chromatographic steps, showing pI ˜ 8.6 and molecular weights of 14,500 and 29,000 for the monomer and dimer, respectively. CgPLA₂ showed a high catalytic activity upon fluorescent phospholipids inducing no direct hemolytic activity. This enzyme, which is Ca²⁺-dependent, showed a lower stability against temperature and pH variations when compared with snake venom enzymes. The enzymatic activity was significantly reduced or completely abolished after chemical modification of CgPLA₂ with BPB. Its cDNA was then obtained, with 357 base pairs which codified for a mature protein of 119 amino acid residues. A comparative analysis of the primary structure of CgPLA₂ revealed 84%, 61%, 43% and 42% similarity to the PLA₂s from Adamsia carciniopados, Nematostella vectensis, Vipera russelli russelli and Bothrops jararacussu, respectively.     

Nature and role of newly described symbiotic associations between a sea anemone and gastropods at bathyal depths in the NW Atlantic

The hormathiid sea anemone Allantactis parasitica was found living as an epibiont on numerous species of gastropods at depths of 725-1100 m along the continental slope of eastern Canada. The proportion of bathyal gastropods hosting 1-6 sea anemones reached 72.5% in a single trawl. Although A. parasitica was occasionally found on other substrata (i.e. empty shells, pebbles), laboratory trials confirmed that they preferably associate with living gastropods. Settlement of planula larvae occurred significantly more often on the shells of live bathyal gastropods than on all other substrata present in the tanks. Juvenile sea anemones (∼ 1 mm diameter) readily moved from the mud or other inert substrata onto shells of burrowed bathyal gastropods. Conversely, larvae, juveniles and adults of A. parasitica never associated with any shallow-water gastropods when given the opportunity. Trials exposing predatory sea stars (Leptasterias polaris) from shallow and bathyal depths to bathyal gastropods (Buccinum undatum) with epibiotic A. parasitica, and to asymbiotic bathyal and shallow-water B. undatum, revealed adaptive behaviours in both prey and predator. Shallow-water gastropods (devoid of epibionts) reacted defensively to L. polaris, whereas bathyal gastropods relied mostly on their epibionts to protect them, thus falling prey to L. polaris when the epibionts were removed. L. polaris from bathyal depths typically ignored symbiotic gastropods, but they consistently preyed on asymbiotic ones, while L. polaris from shallow areas initially attempted to prey on all gastropods, but learned to avoid those harbouring sea anemones. Furthermore, living as epibionts afforded sea anemones a means to escape one of their own predators, the sea star Crossaster papposus. The mutualistic relationship between hormathiid sea anemones and bathyal gastropods from the NW Atlantic may have evolved in response to predation pressure.     

Autotrophy versus heterotrophy: The origin of carbon determines its fate in a symbiotic sea anemone

Cnidarians - corals and their relatives - dominate the shallow, illuminated sea-floor in tropical nutrient-poor seas, mainly because their food sources are both heterotrophic, by predation, and autotrophic, from their intracellular symbiotic microalgae, the zooxanthellae. This trophic flexibility is rare in the animal kingdom, and the cellular usage and biological lifetime of the organic food molecules derived from both sources have never been investigated. Here we show that autotrophically-derived carbon is stored mostly in fatty tissue and is quickly consumed by the organism, while predation-derived carbon is incorporated throughout the animal’s body and remains there for a much longer time. We hypothesize that the simple photosynthate molecules are used primarily for immediate energetic processes like respiration, whereas the more complex heterotrophic organic molecules are utilized to build cellular structures such as proteins and membranes. Our study reveals, for the first time, the different fates and roles of autotrophy and heterothrophy in zooxanthellate coelenterates.     

The annual gametogenic cycle of the sea anemone Metridium senile from the Gulf of Maine

A year-long study was conducted to quantify the reproductive cycle of the clonal sea anemone, Metridium senile, an important member of the benthic community in the Gulf of Maine (GOM). M.senile is an abundant sessile invertebrate that forms identifiable clonal aggregations that are maintained by pedal laceration but individuals can also reproduce sexually. Specimens were collected monthly from a shallow (< 10 m) subtidal site and the reproductive cycle of male and female anemones was described on a seasonal basis using histological preparation of the gonads and light microscopy. Gametogenesis was determined for both sexes and showed that females undergo spawning during the seasonal transition from summer to fall, while males have mature sperm within testicular cysts in the winter, spring, and summer. Environmental data recorded at the study site shows that the spawning period occurs during a period of peak water temperature (August-September), and rapid gametogenesis occurs during periods of high food availability (April). The low percentage of reproductively mature anemones and the dominance of asexual reproduction in this population are believed to be the result of the high flow regime at this site.     

Functional expression and characterization of an acidic actinoporin from sea anemone Sagartia rosea

Src I is the first reported acidic actinoporin from sea anemone Sagartia rosea with a pI value of 4.8 and comprises 13.9% alpha-helix, 65.1% beta-sheet, and 18.2% random coil. For structure-function studies, Src I was expressed in Escherichia coli as a cleavable fusion protein. Recombinant Src I exhibited obviously hemolytic activity, but the fusion protein Trx-Src I almost lost its hemolytic activity, suggesting the importance of the N-terminal amphiphilic alpha-helix for its functional activity. The cytotoxic effects of Src I depending on the toxin concentration and incubation time were also observed on cultured cells. Among five cell lines: NIH/3T3, U251, NSCLC, BEL-7402, and BGC-823, NSCLC was the most sensitive cells with ID(50) 2.8 microg/ml and BGC-823 was the least sensitive cells with ID(50) 7.4 microg/ml. After incubated with lipid SUVs, such as SM-SUVs and SM/PC-SUVs, the hemolytic activity of Src I was inhibited to some extent. When incubated with calcein-entrapped lipid LUVs, such as SM-LUVs, SM/PC-LUVs, and SM/PG-LUVs, Src I induced release of entrapped calcein. According to the interaction with lipid vesicles, we proposed that it was the membrane matrix made up of phospholipids, not a particular phospholipid that facilitates Src I to react properly.     

Evidence for the production of a novel proteinaceous hemolytic exotoxin by dinoflagellate Alexandrium taylori

We found that a whole cell suspension of Alexandrium taylori, which is toxic to Artemia, causes species-specific hemolysis against mammalian erythrocytes. Among the erythrocytes tested, rabbit and guinea-pig erythrocytes were highly sensitive, but human, sheep, and cattle erythrocytes were insensitive. The cell-free culture supernatant also showed potent hemolytic activity toward rabbit erythrocytes as seen in whole cell suspension. The hemolytic activity in the culture medium gradually increased with increase in cell number during exponential growth phase, and relatively high activity was maintained even after reaching the death phase. These results suggest that the hemolytic substance is actively released into the medium from A. taylori cells rather than simple leakage from ruptured or dead cells, and a part of them are steadily accumulated in the medium during the algal growth. Chemical characterization with ultrafiltration and trypsin-treatment suggested that the hemolytic substance released into the medium is protein-like compound with molecular weight more than 10,000 Da. The ammonium sulfate precipitated fraction obtained from the cell-free supernatant of A. taylori showed cytotoxic effect on HeLa cells as well as the hemolytic activity in a similar concentration range on a protein content basis. Our results suggest that A. taylori produces a novel proteinaceous hemolytic exotoxin.     

Purification,properties and cDNA cloning of neoverrucotoxin (neoVTX), a hemolytic lethal factor from the stonefish Synanceia verrucosa venom

A proteinaceous toxin with hemolytic and lethal activities, named neoverrucotoxin (neoVTX), was purified from the venom fluid of stonefish Synanceia verrucosa and its primary structure was elucidated by a cDNA cloning technique. NeoVTX is a dimeric 166 kDa protein composed of α-subunit (702 amino acid residues) and β-subunit (699 amino acid residues) and lacks carbohydrate moieties. Its hemolytic activity is inhibited by anionic lipids, especially potently by cardiolipin. These properties are comparable to those of stonustoxin (SNTX) previously purified from S. horrida. Alignment of the amino acid sequences also reveals that the neoVTX α- and β-subunits share as high as 87 and 95% sequence identity with the SNTX α- and β-subunits, respectively. The distinct differences between neoVTX and SNTX are recognized only in the numbers of Cys residues (18 for neoVTX and 15 for SNTX) and free thiol groups (10 for neoVTX and 5 for SNTX). In contrast, neoVTX considerably differs from verrucotoxin (VTX), a tetrameric 322 kDa glycoprotein, previously purified from S. verrucosa. In addition, the sequence identity of the neoVTX β-subunit with the reported VTX β-subunit is 90%, being lower than that with the SNTX β-subunit.