Isolation, characterization, and amino acid sequence of a polypeptide neurotoxin occurring in the sea anemone Stichodactyla helianthus

An aqueous exudate collected from frozen and thawed bodies of a Caribbean sea anemone, Stichodactyla (formerly Stoichactis) helianthus, contained a polypeptide neurotoxin (Sh I) selectively toxic to crustaceans. The polypeptide was purified by G-50 Sephadex, phosphocellulose, and sulfopropyl-Sephadex chromatography and shown to have a molecular size of 5200 daltons and a pI of 8.3. The amino acid sequence determined by automatic Edman degradations of whole RCM Sh I and of its clostripain, staphylococcal protease, and cyanogen bromide digest peptides is A1ACKC5DDEGP10DIRTA15PLTGT20VDLGS25CNAGW30EKCAS35YYTII40ADCCR45KKK . Only 33% of this sequence is identical with the sequence of Anemonia sulcata toxin II, a sea anemone toxin isolated from the taxonomic family Actiniidae. The six half-cystines are located in equivalent positions to those of the actiniid toxins and account for nearly half of the residues common to all of the toxins. However, 69% of the Sh I sequence is identical with that of toxin II from Heteractis paumotensis, another sea anemone belonging to the family Stichodactylidae. Stichodactylid toxins lack the initial N-terminal residue of actiniid toxins and possess three consecutive acidic residues at positions 6-8, a single tryptophan at position 30, and four consecutive basic residues at positions 45-48 (C-terminus). A rabbit IgG prepared by Sh I immunization bound Sh I with a K0.5 of 4.7 nM but failed to bind homologous actiniid (Anemonia sulcata II, Condylactis gigantea III) or bolocerid (Bolocera tuedae II) polypeptide neurotoxins.(ABSTRACT TRUNCATED AT 250 WORDS)     

CgNa, a type I toxin from the giant Caribbean sea anemone Condylactis gigantea shows structural similarities to both type I and II toxins, as well as distinctive structural and functional properties(1)

CgNa (Condylactis gigantea neurotoxin) is a 47-amino-acid- residue toxin from the giant Caribbean sea anemone Condylactis gigantea. The structure of CgNa, which was solved by 1H-NMR spectroscopy, is somewhat atypical and displays significant homology with both type I and II anemone toxins. CgNa also displays a considerable number of exceptions to the canonical structural elements that are thought to be essential for the activity of this group of toxins. Furthermore, unique residues in CgNa define a characteristic structure with strong negatively charged surface patches. These patches disrupt a surface-exposed cluster of hydrophobic residues present in all anemone-derived toxins described to date. A thorough characterization by patch-clamp analysis using rat DRG (dorsal root ganglion) neurons indicated that CgNa preferentially binds to TTX-S (tetrodotoxin-sensitive) voltage-gated sodium channels in the resting state. This association increased the inactivation time constant and the rate of recovery from inactivation, inducing a significant shift in the steady state of inactivation curve to the left. The specific structural features of CgNa may explain its weaker inhibitory capacity when compared with the other type I and II anemone toxins.     

Effects of several sea anemone and scorpion toxins on excitability and ionic currents in the giant axon of the cockroach

The membrane effects of 4 sea anemone and 6 scorpion toxins have been studied under current clamp and voltage clamp conditions. Micromolar concentrations of the purified toxins were applied externally on single giant axons of the american cockroach. Periplaneta americana in a double oil-gap arrangement and the effects on the resting potential, action potential and underlying currents analysed. The 4 sea anemone toxins (Condylactis toxin, Anemonia toxin 2, Anthopleurin toxin A and Parasicyonis toxin) were found to considerably prolong the action potential. This effect is frequency dependent and long plateau spikes (100-500 ms in duration) are consistently seen for frequencies lower than 0.2 Hz. This effect is due to a considerable delay in the turning-off of the sodium current during square membrane depolarizations associated, for large concentrations, with a decrease in the potassium conductance. Toxin effects on the sodium current are not prevented by pretreatment with STX. From the 4 purified toxins extracted from the venom of the scorpion, Androctonus australis Hector, 3 (Mammal toxins 1 and 2 and crustacean toxin) were found to have sea anemone toxin like effects and to induce long duration plateau action potentials. As for sea anemone toxins, this effect is due to a lengthening of the falling phase of the sodium current associated with a small decrease in the potassium conductance. The 4th toxin (insect toxin or ITAaH) depolarizes the membrane and induces repetitive firing of short action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calitoxin, a neurotoxic peptide from the sea anemone Calliactis parasitica: amino acid sequence and electrophysiological properties

We have isolated a new toxin, calitoxin (CLX), from the sea anemone Calliactis parasitica whose amino acid sequence differs greatly from that of other sea anemone toxins. The polypeptide chain contains 46 amino acid residues, with a molecular mass of 4886 Da and an isoelectric point at pH 5.4. The amino acid sequence determined by Edman degradation of the reduced, S-carboxymethylated polypeptide chain and tryptic and chymotryptic peptides is Ile-Glu-Cys-Lys-Cys-Glu-Gly-Asp-Ala-Pro-Asp-Leu-Ser-His-Met-Thr-Gly-Thr- Val-Tyr - Phe-Ser-Cys-Lys-Gly-Gly-Asp-Gly-Ser-Trp-Ser-Lys-Cys-Asn-Thr-Tyr-Thr-Ala- Val-Ala - Asp-Cys-Cys-His-Glu-Ala. No cysteine residues were present in the peptide. Similarly to other sea anemone toxins, calitoxin interacts, in crustacean nerve muscle preparations, with axonal and not with muscle membranes, inducing a massive release of neurotransmitter that causes a strong muscle contraction. The low homology of CLX with RP II and ATX II toxins has implications regarding the role played by particular amino acid residues.     

Purification, sequence, and pharmacological properties of sea anemone toxins from Radianthus paumotensis. A new class of sea anemone toxins acting on the sodium channel

Four new toxins have been isolated from the sea anemone Radianthus paumotensis: RpI, RpII, RpIII, and RpIV. They are polypeptides comprised of 48 or 49 amino acids; the sequence of RpII has been determined. Toxicities of these toxins in mice and crabs are similar to those of the other known sea anemone toxins, but they fall into a different immunochemically defined class. The sequence of RpII shows close similarities with the N-terminal end (up to residue 20) of the previously sequenced long sea anemone toxins, but most of the remaining part of the molecule is completely different. Like the other sea anemone toxins, Radianthus toxins are active on sodium channels; they slow down the inactivation process. Through their Na+ channel action, Radianthus toxins stimulate Na+ influx into tetrodotoxin-sensitive neuroblastoma cells and tetrodotoxin-resistant rat skeletal myoblasts. The efficiency of the toxins is similar in the two cellular systems. In that respect, Radianthus toxins behave much more like scorpion neurotoxins than sea anemone toxins from Anemonia sulcata or Anthopleura xanthogrammica. In binding experiments to synaptosomal Na+ channels, Radianthus toxins compete with toxin II from the scorpion Androctonus australis but not with toxins II and V from Anemonia sulcata.     

Isolation of the two components of Condylactis toxin.

The two toxic components from the sea anemone Condylactis gigantea have been isolated and purified using gel filtration, ion-exchange chromatography, and preparative isoelectric focusing. The molecular weight of both components is estimated by gel filtration to be 4500. The isoelectric points of the two toxins were determined to be 4.8 and 5.8 with LD₅₀ values of 19 and 58 μg/kg, respectively, when injected into Armadillidium vulgare, a terrestrial crustacean. Low-level labeling with ¹²⁵I using lactoperoxidase was effected for the purpose of estimating homogeneity.     

Purification and pharmacological properties of eight sea anemone toxins from Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum

Eight different polypeptide toxins from sea anemones of four different origins (Anemonia sulcata, Anthopleura xanthogrammica, Stoichactis giganteus, and Actinodendron plumosum) have been studied. Three of these toxins are new; the purification procedure for the five other ones has been improved. Sea anemone toxins were assayed (i) for their toxicity to crabs and mice, (ii) for their affinity for the specific sea anemone toxin receptor situated on the Na+ channels of rat brain synaptosomes, and (iii) for their capacity to increase, in synergy with veratridine, the rate of 22Na+ entry into neuroblastoma cells via the Na+ channel. Some of the toxins are more active on crustaceans, whereas others are more toxic to mammals. A very good correlation exists between the toxic activity to mice, the affinity of the toxin for the Na+ channel in rat brain synaptosomes, and the stimulating effect on 22 Na+ uptake by neuroblastoma cells. The observation has also been made that the most cationic toxins are also the most active on mammals and the least active on crustaceans. Toxicities (LD50) to mice of the most active sea anemone toxins and of the most active scorpion toxins are similar, and sea anemone toxins at high enough concentrations prevent binding of scorpion toxins to their receptor. However, scorpion toxins have affinities for the Na+ channel which are approximately 60 times higher than those found for the most active sea anemone toxins. Three sea anemone toxins appear to be more interesting than toxin II from A. sulcata (the "classical" sea anemone toxin) for studies of the Na+ channel structure and mechanism when the source of the channel is of a mammalian origin. Two of these three toxins can be radiolabeled with iodine while retaining their toxic activity; they appear to be useful tools for future biochemical studies of the Na+ channel.     

Primary structure of a potassium channel toxin from the sea anemone Actinia equina

A potassium channel toxin (AeK) was isolated from the sea anemone Actinia equina by gel filtration on Sephadex G-50 and reverse-phase HPLC on TSKgel ODS-120T. AeK and α-dendrotoxin inhibited the binding of ¹²⁵I-α-dendrotoxin to rat synaptosomal membranes with IC₅₀ of 22 and 0.34 nM, respectively, indicating that AeK is about sixty-five times less toxic than α-dendrotoxin. The complete amino acid sequence of AeK was elucidated; it is composed of 36 amino acid residues including six half-Cys residues. The determined sequence showed that AeK is analogous to the three potassium channel toxins from sea anemones (BgK from Bunodosoma granulifera, ShK from Stichodactyla helianthus and AsKS from Anemonia sulcata), with an especially high sequence homology (86%) with AsKS.     

Amino acid sequence of neurotoxins IV and V from the sea anemone Radianthus macrodactylus

Amino acid sequences of neurotoxins RTX-IV and RTX-V isolated from the sea anemone Radianthus macrodactylus were determined by the automated Edman degradation; their polypeptide chains consist of 48 and 47 amino acid residues, respectively. For identification of tryptophan-30 in toxin RTX-IV, its trypsin and chymotrypsin digests were investigated. Amino acid sequences of the above toxins show that they belong to a new structural class and that C-terminal positive charge and tyrosine-25 are important for toxic activity of sea anemone polypeptides.     

Amino-acid sequence of toxin I from Anemonia sulcata.

Toxin I from Anemonia sulcata, a major component of the sea anemone venom, consists of 46 amino acid residues which are linked by three disulfide bridges. The [14C]carboxymethylated polypeptide was sequenced to position 29 by automated Edman degradation. The remaining sequence was determined from cyanogen bromide peptides and from tryptic peptides of the citraconylated [14C]carboxymethylated toxin. Toxin I is homologous to toxin II from Anemonia sulcata and to anthopleurin A, a toxin from the sea anemone Anthopleura xanthogrammica. These toxins constitute a new class of polypeptide toxins. No significant homologies exist with toxin III from Anemonia sulcata nor with known sequences of neurotoxins or cardiotoxins of various origin.     

The amino acid sequence of toxin RpIII from the sea anemone, Radianthus paumotensis

The amino acid sequence of the sodium channel toxin RpIII from the sea anemone Radianthus paumotensis has been determined. The protein is homologous with five analogous toxins from three anemone species, and is most similar to a less toxic protein, RpII, from the same organism. Twelve residues are conserved in all six toxins, one of which is an arginine residue thought to be essential for toxicity. The others (Cys, Gly, Pro and Trp) tend to be conserved in other sets of homologous proteins to maintain functional folds. Comparisons of the sequences suggest the existence of two separate but related classes of toxins cumon the three species of anemone.     

Amino acid sequences of two novel long-chain neurotoxins from the venom of the sea snake Laticauda colubrina.

From the venom of a population of the sea snake Laticauda colubrina from the Solomon Islands, a neurotoxic component, Laticauda colubrina a (toxin Lc a), was isolated in 16.6% (A280) yield. Similarly, from the venom of a population of L. colubrina from the Philippines, a neurotoxic component, Laticauda colubrina b (toxin Lc b), was obtained in 10.0% (A280) yield. The LD50 values of these toxins were 0.12 microgram/g body wt. on intramuscular injection in mice. Toxins Lc a and Lc b were each composed of molecules containing 69 amino acid residues with eight half-cystine residues. The complete amino acid sequences of these two toxins were elucidated. Toxins Lc a and Lc b are different from each other at five positions of their sequences, namely at positions 31 (Phe/Ser), 32 (Leu/Ile), 33 (Lys/Arg), 50 (Pro/Arg) and 53 (Asp/His) (residues in parentheses give the residues in toxins Lc a and Lc b respectively). Toxins Lc a and Lc b have a novel structure in that they have only four disulphide bridges, although the whole amino acid sequences are homologous to those of other known long-chain neurotoxins. It is remarkable that toxins Lc a and Lc b are not coexistent at the detection error of 6% of the other toxin. Populations of Laticauda colubrina from the Solomon Islands and from the Philippines have either toxin Lc a or toxin Lc b and not both of them.     

Tetrodotoxin-insensitive sodium channels. Binding of polypeptide neurotoxins in primary cultures of rat muscle cells

The binding of 125I-labeled derivatives of scorpion toxin and sea anemone toxin to tetrodotoxin-insensitive sodium channels in cultured rat muscle cells has been studied. Specific binding of 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin was each blocked by either native scorpion toxin or native sea anemone toxin. K0.5 for block of binding by several polypeptide toxins was closely correlated with K0.5 for enhancement of sodium channel activation in rat muscle cells. These results directly demonstrate binding of sea anemone toxin and scorpion toxin to a common receptor site on the sodium channel. Binding of both 125I-labeled toxin derivatives is enhanced by the alkaloids aconitine and batrachotoxin due to a decrease in KD for polypeptide toxin. Enhancement of polypeptide toxin binding by aconitine and batrachotoxin is precisely correlated with persistent activation of sodium channels by the alkaloid toxins consistent with the conclusion that there is allosteric coupling between receptor sites for alkaloid and polypeptide toxins on the sodium channel. The binding of both 125I-labeled scorpion toxin and 125I-labeled sea anemone toxin is reduced by depolarization due to a voltage-dependent increase in KD. Scorpion toxin binding is more voltage-sensitive than sea anemone toxin binding. Our results directly demonstrate voltage-dependent binding of both scorpion toxin and sea anemone toxin to a common receptor site on the sodium channel and introduce the 125I-labeled polypeptide toxin derivatives as specific binding probes of tetrodotoxin-insensitive sodium channels in cultured muscle cells.     

Purification and properties of a toxin from the sea anemone Condylactis gigantea

A cytolytic toxin from the sea anemone Condylactis gigantea was isolated and characterized as a thermolabile basic protein (pI 8.9) having a molecular weight of 18,300. It lacks methionine but contains relatively large amounts of glycine, serine, tryptophan, and half-cystine. Its hemolytic action is inhibited by sphingomyelin. It is lytic for rabbit blood platelets, is lethal in low concentration for crayfish (LD₅₀ = 0.06 μg), and may be identical with a neurotoxic protein isolated earlier from the same species. It broadly resembles the toxin of Stoichactis helianthus but differs from it in amino acid composition and in minor respects.     

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.     

Expression and mutagenesis of the sea anemone toxin Av2 reveals key amino acid residues important for activity on voltage-gated sodium channels

Type I sea anemone toxins are highly potent modulators of voltage-gated Na-channels (Na(v)s) and compete with the structurally dissimilar scorpion alpha-toxins on binding to receptor site-3. Although these features provide two structurally different probes for studying receptor site-3 and channel fast inactivation, the bioactive surface of sea anemone toxins has not been fully resolved. We established an efficient expression system for Av2 (known as ATX II), a highly insecticidal sea anemone toxin from Anemonia viridis (previously named A. sulcata), and mutagenized it throughout. Each toxin mutant was analyzed in toxicity and binding assays as well as by circular dichroism spectroscopy to discern the effects derived from structural perturbation from those related to bioactivity. Six residues were found to constitute the anti-insect bioactive surface of Av2 (Val-2, Leu-5, Asn-16, Leu-18, and Ile-41). Further analysis of nine Av2 mutants on the human heart channel Na(v)1.5 expressed in Xenopus oocytes indicated that the bioactive surfaces toward insects and mammals practically coincide but differ from the bioactive surface of a structurally similar sea anemone toxin, Anthopleurin B, from Anthopleura xanthogrammica. Hence, our results not only demonstrate clear differences in the bioactive surfaces of Av2 and scorpion alpha-toxins but also indicate that despite the general conservation in structure and importance of the Arg-14 loop and its flanking residues Gly-10 and Gly-20 for function, the surface of interaction between different sea anemone toxins and Na(v)s varies.     

Immunochemical study of neurotoxins from Radianthus macrodactylus actinin]

Polyclonal antibodies to neurotoxin Rm-III from sea anemone Radianthus macrodactylus have been obtained. Constants of inhibition of the Rm-III binding to its antibodies by the homologous toxins have been determined. Antigenic activity of the second type toxins is shown to depend not only on the degree of homology but also no the type of substitution in the amino acid sequence. As shown by the calculation methods, the antigenic determinants of all the homologues have similar positions. Amino acid residues at positions 2, 11, 20, 28, and 46-48 seem to be included into the antigenic sites of the toxins studied.     

海葵神经毒素研究进展

过去30多年的研究表明,海葵毒液中富含多肽或蛋白类生物毒素活性物质,分子量从3000到80000Da不等。这些毒素可以特异地与某些离子通道或细胞膜受体相结合,从而影响生物的某些生理功能。按照它们功能的不同,可以将海葵毒素大致分为两大类:神经毒素和溶细胞素。由于海葵神经毒素对它们的作用位点具有高度的特异性和亲和性,使得它们成为神经生理学和药理学研究的一种重要工具。就海葵毒素的类型、结构特征、生物活性、应用状况及开发前景的新进展进行综述,以期对同类研究些微启迪。     

The sodium channel in non-impulsive cells. Interaction with specific neurotoxins.

The cell line C9 used in this paper has a resting potential of --50 mV (+/- 10 mV) but is unable to generate an action potential upon electrical stimulation. The cell membrane has receptors for the selectivity filter toxin tetrodotoxin as well as for the gating system toxins, veratridine, scorpion toxin and sea anemone toxin. The Na+ channel which remains silent to electrical stimulation in the absence of toxins can be chemically activated by the gating system toxins. This has been demonstarted by electrophysiological techniques and by 22Na+ flux studies. The electrophysiological approach has shown that the sea anemone toxin is able to induce a spontaneous slow-wave activity inhibited by tetrodotoxin. 22Na+ influx analyses have shown that veratridine and the sea anemone toxin produce an important increase of the initial rate of 22Na+ influx into the C9 cell. The stimulation of 22Na+ entry by these gating system toxins is similar to that found using spiking neuroblastoma cells. Veratridine and the sea anemone toxin on one hand as well as veratridine and the scorpion toxin on the other hand are synergistic in their action to stabilize an open and highly permeable form of the sodium channel. Stimulation of 22Na+ entry into the cell through the sodium channel maintained open by the gating system neurotoxins is completely suppressed by tetrodotoxin.