Characterization of PsTX-60B, a new membrane-attack complex/perforin (MACPF) family toxin, from the venomous sea anemone Phyllodiscus semoni

The sea anemone Phyllodiscus semoni has been known as one of the most venomous sea anemones. Previously, we reported the isolation of the major lethal protein toxins, PsTX-60A and PsTX-60B, from P. semoni and the primary structure of PsTX-60A. In this paper, we report the complete sequence of cDNA (1600 base pairs) encoding PsTX-60B and the deduced primary structure (488 amino acids) of PsTX-60B. The amino acid sequence of PsTX-60B showed the homology with those of PsTX-60A and Actineria villosa toxin, AvTX-60A. The results showed that PsTX-60B is a new member of the membrane-attack complex/perforin (MACPF) family toxin.     

Isolation and molecular cloning of novel peptide toxins from the sea anemone Antheopsis maculata.

Three peptide toxins (Am I-III) with crab toxicity were isolated from the sea anemone Anthopleura maculata by gel filtration and reverse-phase HPLC. Am I was weakly lethal to crabs (LD50 830 microg/kg) and Am III was potently lethal (LD50 70 microg/kg), while Am II was only paralytic (ED50 420 microg/kg). The complete amino acid sequences of the three toxins were determined by cDNA cloning based on 3'-Race and 5'-Race. Although Am III (47 residues) is an analogue of the well-known type 1 sea anemone sodium channel toxins, both Am I (27 residues) and II (46 residues) are structurally novel peptide toxins. Am I is a new toxin having no sequence homologies with any toxins. Am II shares 28-39% identity with the recently characterized sea anemone toxins inhibiting specialized ion channels, BDS-I and II from Anemonia sulcata and APETx1 and 2 from Anthopleura elegantissima. The precursor proteins of the three toxins are commonly composed of a signal peptide, a propart with a pair of basic residues (Lys-Arg) at the end and the remaining portion. Very interestingly, the Am I precursor protein contains as many as six copies of Am I.     

Separation and characterization of four different amino acid sequence variants of a sea anemone (Stichodactyla helianthus) protein cytolysin

A basic protein cytolysin previously isolated from the Caribbean sea anemone Stichodactyla helianthus was shown by CM cellulose chromatography to consist of four isotoxins possessing different N-terminal amino acid sequences. These are designated as toxins I-IV in order of increasing isoelectric point. The estimated molecular sizes (17,400-18,200) of toxins I-III were very similar; toxins I and II posses one additional amino acid at their amino terminus relative to toxin III. Under denaturing conditions, toxin IV behaved as a significantly larger (19,600) polypeptide; Edman sequencing established that it possesses a seven residue extension at the N-terminal end relative to toxin III. None of the variants contained half-cystines or reducing sugars. Toxin III contributed 83% of the total purified cytolytic (hemolytic) activity, toxin II 14%, and the relatively insoluble toxins I and IV together only contributed about 3% of the total cytolytic activity. Cytolysin III lysed Ehrlich ascitic tumour cells, but when administered intraperitoneally in nonlethal doses to mice already inoculated with this tumour, it failed to protect the mice against the tumour. Comparison of the partial amino acid sequence of equinatoxin, another sea anemone protein cytolysin, with that of Stichodactyla cytolysin III indicates they are highly homologous. Many other cytolytic proteins isolated from sea anemones share these properties with Stichodactyla cytolysins: (1) selective inhibition of hemolytic activity by preincubation with sphingomyelin, (2) a molecular size of 10,000-20,000, and (3) an isoelectric point of 9 or above.     

A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation.

A new peptide toxin exhibiting a molecular weight of 5043Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD(50) approx. 1microg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11kg of wet starting material, approximately 1g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique (n=60). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253-259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67-77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365-1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693-702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.     

Biochemical and physiological analyses of a hemolytic toxin isolated from a sea anemone Actineria villosa.

A species of venomous sea anemone Actineria villosa was recently found inhabiting the coastal areas of Okinawa, Japan. This marine animal produces various proteinous toxins, so that a local health organization was called for medical treatment for those who had accidental contact with this animal. In this study we analyzed the biochemical and physiological properties of hemolytic protein from A. villosa. The toxin purified from the tentacles of the animals was found to be a protein with a molecular weight of approximately 19 kDa. We named this newly found hemolytic toxin of A. villosa, Avt-I. Incubation of the toxin with sphingomyelin inhibited hemolytic activity by up to 85%, showing that Avt-I may target sphingomyelin on the erythrocyte membrane. The hemolytic activity was stably maintained at temperatures below 45 degrees C, however, a sharp linear decrease in heat stability was observed within the range of 45-55 degrees C. Our results provide the first evidence that A. villosa produces a toxin with strong hemolytic activity similar in biochemical and physiological properties to other members of actinoporin family previously isolated from related species of sea anemones.     

Isolation and chemical characterization of a toxin isolated from the venom of the sea snake, Hydrophis torquatus aagardi

Sea snakes (family: Hydrophiidae) are serpents found in the coastal areas of the Indian and Pacific Oceans. There are two subfamilies in Hydrophiidae: Hydrophiinae and Laticaudinae. A toxin, aagardi toxin, was isolated from the venom of the Hydrophiinae snake, Hydrophis torquatus aagardi and its chemical properties such as molecular weight, isoelectric point, importance of disulfide bonds, lack of enzymatic activity and amino acid sequence were determined. The amino acid sequence indicated a close relationship to the primary structure of other Hydrophiinae toxins and a significant difference from Laticaudinae toxins, confirming that primary toxin structure is closely related to sea snake phylogenecity.     

Isolation and cDNA cloning of a potassium channel peptide toxin from the sea anemone Anemonia erythraea.

A potassium channel peptide toxin (AETX K) was isolated from the sea anemone Anemonia erythraea by gel filtration on Sephadex G-50, reverse-phase HPLC on TSKgel ODS-120T and anion-exchange HPLC on Mono Q. AETX K inhibited the binding of (125)I-alpha-dendrotoxin to rat synaptosomal membranes, although much less potently than alpha-dendrotoxin. Based on the determined N-terminal amino acid sequence, the nucleotide sequence of the full-length cDNA (609bp) encoding AETX K was elucidated by a combination of degenerate RT-PCR, 3'RACE and 5'RACE. The precursor protein of AETX K is composed of a signal peptide (22 residues), a propart (27 residues) ended with a pair of basic residues (Lys-Arg) and a mature peptide (34 residues). AETX K is the sixth member of the type 1 potassium channel toxins from sea anemones, showing especially high sequence identities with HmK from Heteractis magnifica and ShK from Stichodactyla helianthus. It has six Cys residues at the same position as the known type 1 toxins. In addition, the dyad comprising Lys and Tyr, which is considered to be essential for the binding of the known type 1 toxins to potassium channels, is also conserved in AETX K.     

A Trial of Mass Spectrometric Characterization of Femto‐Molar Amount from Subtropical Islands

Many kinds of venomous principles modulate physiological responses of mammalian signal transduction systems, on which they act selectively as enhancers, inhibitors or some other kind of effectors. These toxins have become useful tools for physiological research. We have characterized paralyzing toxins from the venom of spiders, scorpions, insects, jellyfishes and sea anemones in the subtropical region including the Ryukyu Islands. Venom profiles are screened by MALDI‐TOF fingerprinting analysis prior to purification of the venomous components, then marked target toxins of small molecular mass (1000–5000) are characterized directly by means of mass spectrometric techniques such as Frit‐FAB MS/MS, PSD/CID‐TOF MS, Capil. ‐HPLC/Q‐TOF MS/MS etc. The proteinous toxins of jellyfish or sea anemone are characterized by RT‐PCR technique by the information of the cleaved peptides after the protein was hydrolyzed by appropriate peptidase and the sequence of the cleaved peptide was determined by conventional methods. The venom of Araneid spider is mainly composed of a mixture of closely related acylpolyamines. More than 90 polyamine toxins were identified from one venom sac of the Madagascan spider, Nephilengys borbonica, by Frit‐Fab MS/MS employing charge remote fragmentation technique. A novel polyamine toxin was also found from the rare wondering spider, Macrothele gigas from Iriomote Island. The structure of the toxin is an analog of polyamine toxin found in trapdoor spiders. Many kinds of cystine‐rich peptides showing various types of ion channel antagonism have been isolated from spiders. A series of toxins possessing the same mode of cystine knots was recently isolated from the saliva of assassin bugs, Peirates turpis, Isyndus obscurus, Agriophodrus dohrni. These toxins act as calcium channel blocker. Most of the scorpion toxins reported are from scorpions hazardous to humans, and they belong to the major super family Buthoidea. Among them are the well‐known genera, such as Buthus, Androctonus, Centruroides, Leiurus, or Tytius. We have investigated the minor group of scorpions from the super family Chactoidea (Scorpionidae, Ishnuridae). The venoms of these scorpions, involving the genera Heterometrus, Pandinus, Opisthacanthus, and Isometrus, contain different kinds of peptide toxins. Fingerprinting peptide analysis of the toxin profiles for these scorpions showed some difference from the profiles of Buthoidea scorpions. These venoms contain linear pore‐forming peptides and 2‐cystine‐bridged toxins in addition to 4‐cystine‐bridged toxins. The most hazardous jellyfish in Okinawa, Chiropsalmus quadrigatus, and the related box jellyfishes, Carybdea rastoni, C. alata, contain quite labile proteinaceous toxins, CqTX, CrTX and CaTX, respectively. The toxins were inactivated by adding an organic solvent such as methanol or acetonitrile, by changing the pH of the toxin solution, dialyzing the toxin solution, storing the toxin in a refrigerator, or by lyophilizing the toxin solution. However, the toxic activity was retained in the presence of sodium chloride. We purified the jellyfish toxins by adding sodium chloride through all steps of the purification procedure and finally obtained the whole primary amino acid sequence of the toxin by RT‐PCR method. The toxic protein CqTX is homologous to the other box jelly fish toxin, CrTX and CaTX. These toxins belong to a new class of proteins since they show no homology to known proteins. Another notorious and dangerous specimen in the Ryukyu Islands is Phyllodiscus semori. The venom is composed of three kinds of proteins (PsTX‐20A, PsTX‐60A, PsTX‐60B). PsTX‐20A shows homology to the proteinaceous toxin actinoporin, a cytolytic protein isolated from the genus Actinia, but PsTX‐60s has no homology to any ever cloned proteins. Further elucidation of the mechanism of toxic action of these Coelenterates is in progress.     

An epidermal growth factor-like toxin and two sodium channel toxins from the sea anemone Stichodactyla gigantea.

Three peptide toxins (gigantoxins I-III) with crab toxicity were isolated from the sea anemone Stichodactyla gigantea by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T and their complete amino acid sequences were determined. Gigantoxins II (44 residues) and III (48 residues) have LD(50) (against crabs) of 70 and 120 microg/kg, respectively, and are analogous to the known type 1 and 2 sea anemone sodium channel toxins, respectively. On the other hand, gigantoxin I (48 residues) is potently paralytic to crabs (ED(50) 215 microg/kg), although its lethality is very weak (LD(50)>1000 microg/kg). Interestingly, gigantoxin I has 31-33% homologies with mammalian epidermal growth factors (EGFs), with the same location of six cysteine residues. In accordance with the sequence similarity, gigantoxin I exhibits EGF activity as evidenced by rounding of A431 cells and tyrosine phosphorylation of the EGF receptor in the cells, although much less potently than human EGF. Gigantoxin I is the first example of EGF-like toxins of natural origin.     

Novel peptide toxins from acrorhagi, aggressive organs of the sea anemone Actinia equina.

Two peptide toxins, acrorhagin I (50 residues) and II (44 residues), were isolated from special aggressive organs (acrorhagi) of the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. The LD50 against crabs of acrorhagin I and II were estimated to be 520 and 80 microg/kg, respectively. 3'- and 5'-RACE established the amino acid sequences of the acrorhagin precursors. The precursor of acrorhagin I is composed of both signal and mature peptides and that of acrorhagin II has an additional sequence (propart) between signal and mature peptides. Acrorhagin I has no sequence homologies with any toxins, while acrorhagin II is somewhat similar to spider neurotoxins (hainantoxin-I from Selenocosmia hainana and Tx 3-2 from Phoneutria nigriventer) and cone snail neurotoxin (omega-conotoxin MVIIB from Conus magus). In addition, analogous peptides (acrorhagin Ia and IIa) were also cloned during RT-PCR experiments performed to confirm the nucleotide sequences of acrorhagins. This is the first to demonstrate the existence of novel peptide toxins in the sea anemone acrorhagi.     

A new cytolytic protein from the sea anemone Urticina crassicornis that binds to cholesterol- and sphingomyelin-rich membranes

A new pore-forming cytolytic protein was isolated from the Northern red sea anemone, Urticina crassicornis. Its biochemical properties were characterized and partial N-terminal amino acid sequence was determined. The cytolysin, named UcI, has a molecular mass of around 30 kDa and lacks phospholipase A₂ activity. UcI lyses bovine erythrocytes at nanomolar concentrations. Hemolysis is a result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores and can be prevented by osmotic protectants of size >600 Da. The functional radius of an average pore was estimated to be about 0.66 nm. A more detailed study of the cytolytic activity of UcI was performed with lipid vesicles and monolayers. The toxin binds to monolayers and efficiently permeabilizes small lipid vesicles composed of sphingomyelin and cholesterol. However, the cytolytic activity is not prevented by preincubation with either pure cholesterol or sphingomyelin dispersions. We conclude that the presence of both sphingomyelin and cholesterol, key components of lipid rafts, greatly enhances toxin binding to membranes and probably facilitates pore formation. Alignment of the toxin partial amino acid sequence with sequences of cytolysins belonging to the actinoporin family reveals no sequence homology. We conclude that partial sequence of UcI resembles only the N-terminal part of UpI, a cytolytic protein isolated from a related sea anemone species, Urticina piscivora. The two proteins most probably belong to a separate family of sea anemone cytolysins that are worthy of further characterization.     

Amino acid sequence studies on cytolytic toxins from sea anemone Heteractis magnifica, Entacmaea quadricolor and Stichodactyla mertensii (Anthozoa).

The complete amino acid sequence of a cytolytic toxin, HmT, isolated from sea anemone Heteractis magnifica was determined. It is composed of 177 amino acid residues and lacks half-cystines. Partial N-terminal sequences of three other cytolysins from Entacmaea quadricolor (EnT) and Stichodactyla mertensii (SmT-1 and SmT-2) were also determined. Comparing these sequences with those of other sea anemone cytolysins, a high degree of homology was observed.     

Development of a rational nomenclature for naming peptide and protein toxins from sea anemones

Sea anemone toxins are predominantly peptide and proteins that act mainly on sodium and potassium channels, as well as in a variety of target cells causing lysis. Over recent years, the number of sea anemone peptide toxins as well as cytolytic pore-forming proteins and phospholipase A₂ sequences submitted to databases has been rapidly increasing due to the developments in DNA sequencing technology and proteomic approaches. However, the lack of a systematic nomenclature has resulted in multiple names being assigned to the same toxins, toxins from unrelated species being designated by the same name, and ambiguous name designations. Therefore, in this work we propose a systematic nomenclature in which we adopted specific criteria, based on order of discovery and phylogenetic analysis, in order to avoid redundant sea anemone toxin names. Implementation of the nomenclature proposed here not only allowed us to rename the already published 191 anemone toxins without ambiguities, but it can be used to unambiguously name newly discovered toxins whether or not they are related to previously published sea anemone sequences. In the new nomenclature each toxin name contains information about the toxin's biological activity, origin and relationship to known isoforms. Ongoing increases in the speed of DNA sequencing will raise significantly the number of sea anemone toxin sequences in the literature. This will represent a constant challenge in their clear identification and logical classification, which could be solved using the proposed nomenclature.     

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom.

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC(50) 0.689 mg/50mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin (P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice (P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity (P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.     

Properties of a cytolytic toxin from the sea anemone, Stoichactis kenti

A cytolytic toxin (kentin) from the Indo-Pacific sea anemone, Stoichactis kenti, was purified to near homogeneity. The toxin is a basic polypeptide of molecular weight approximately 18,000. It broadly resembles cytotoxins from Stoichactis helianthus (helianthin), as well as similar toxins from a number of other anemones, namely Condylactis, Epiactis, Actinia, Pseudactinia, Tealia, Anthopleura, Radianthus and Gyrostoma. The amino acid composition of kentin shows considerable resemblance to that of helianthin, but there are also several significant differences. Neutralization tests indicate that kentin and helianthin are immunologically related but distinguishable. In contrast, no immunological relatedness was found between helianthin and cytolytic toxins from Condylactis gigantea and Epiactis prolifera.     

Occurrence of type 3 sodium channel peptide toxins in two species of sea anemones (Dofleinia armata and Entacmaea ramsayi).

From two species of sea anemones, Dofleinia armata and Entacmaea ramsayi, three peptide toxins (two from the former and one from the latter) with crab toxicity were purified and completely sequenced. The three toxins (30-32 residues) are highly homologous to each other and also to PaTX from Entacmaea actinostoloides, a type 3 sea anemone sodium channel toxin. This study reveals that there is a family of PaTX-like toxins in sea anemones.     

Complete amino acid sequences of two cardiotoxin-like analogues from Bungarus fasciatus (banded krait) snake venom

Three cardiotoxin-like proteins have been isolated from Bungarus fasciatus venom and the amino acid sequence of the major toxin (toxin VI) have been determined. The amino acid sequences of two other analogues (toxins V-2 and V-3) were investigated. The reduced and S-carboxymethylated toxins were digested with trypsin-TPCK and the resulting tryptic peptides were isolated by fingerprinting technique on paper. The amino acid compositions, N-terminal residues and partial amino acid sequences of some of the tryptic peptides were determined. Thirteen tryptic peptides were aligned by following the order of corresponding fragments of toxin VI. Toxins V-2 and V-3 contained 118 and 117 amino acid residues, respectively, in a single polypeptide chain cross-linked with six pairs of intramolecular disulphide bonds. There are only four (for toxin V-2) and five (for toxin V-3) places of differences in their primary structures when compared with that of the major toxin (toxin VI) of Bungarus fasciatus venom.     

Amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus

The amino acid sequence of pelamitoxin a, the main neurotoxin of the sea snake, Pelamis platurus, has been completed. Analysis was done principally by means of Edman degradation of constituent tryptic peptides; chymotryptic and thermolytic peptides were also studied to assist in the sequence alignment. The results indicate that pelamitoxin a is identical to schistosa toxin 5 in amino acid sequence. These results indicate the feasibility of producing a common antivenin against toxins from some of the most common sea snakes.     

Characterization of two Bunodosoma granulifera toxins active on cardiac sodium channels.

1. Two sodium channel toxins, BgII and BgIII, have been isolated and purified from the sea anemone Bunodosoma granulifera. Combining different techniques, we have investigated the electrophysiological properties of these toxins. 2. We examined the effect of BgII and BgIII on rat ventricular strips. These toxins prolong action potentials with EC50 values of 60 and 660 nM and modify the resting potentials. 3. The effect on Na+ currents in rat cardiomyocytes was studied using the patch-clamp technique. BgII and BgIII slow the rapid inactivation process and increase the current density with EC50 values of 58 and 78 nM, respectively. 4. On the cloned hH1 cardiac Na+ channel expressed in Xenopus laevis oocytes, BgII and BgIII slow the inactivation process of Na+ currents (respective EC50 values of 0.38 and 7.8 microM), shift the steady-state activation and inactivation parameters to more positive potentials and the reversal potential to more negative potentials. 5. The amino acid sequences of these toxins are almost identical except for an asparagine at position 16 in BgII which is replaced by an aspartic acid in BgIII. In all experiments, BgII was more potent than BgIII suggesting that this conservative residue is important for the toxicity of sea anemone toxins. 6. We conclude that BgII and BgIII, generally known as neurotoxins, are also cardiotoxic and combine the classical effects of sea anemone Na+ channels toxins (slowing of inactivation kinetics, shift of steady-state activation and inactivation parameters) with a striking decrease on the ionic selectivity of Na+ channels.