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.     

Amide proton exchange rates in cardioactive sea anemone polypeptides

Amide hydrogen exchange rates have been measured using high-resolution 1H nuclear magnetic resonance (NMR) spectroscopy at 300 MHz for three homologous cardioactive polypeptides, anthopleurin-A from Anthopleura xanthogrammica and Anemonia sulcata toxins I and II. There are approximately 15 slowly exchanging hydrogens in each polypeptide, but the slowest exchange rates are found in ATX II, with ATX I and AP-A having rates similar to one another. The exchange rates correlate with the thermal stability of these molecules, but not with the potency and species specificity of their biological activities. The data for AP-A are interpreted in terms of a recent structural model for this polypeptide.     

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)     

Amino acid sequence of toxin III from Anemonia sulcata.

Toxin III, the smallest toxin component of the poison of the sea anemone Anemonia sulcata, is a polypeptide with 27 amino acids. Its structure is stabilized by three disulfide bridges. The amino acid sequence was determined by solid-phase Edman degradation of the aminoethylated derivative. The peptide was coupled to the carrier, porous glass, by thiourea bridges between the alpha-amino group of arginine-1 and the epsilon-amino group of lysine-26 and the isothiocyanate groups of the carrier. Another fraction of the polypeptide was bound by an acid-amide condensation of the C-terminal valine-27 with the aminopropyl group of the carrier. The sequence of toxin III has no regions homologous to the 47-residue toxin II. Comparison with the known partial sequence of toxin I, which contains 46 amino acids (Wunderer, G. & Eulitz, M., in preparation) also fails to reveal homologies.     

Amino acid sequence of a sea anemone toxin from Parasicyonis actinostoloides

Amino-acid sequence of a toxin from sea anemone, Parasicyonis actinostoloides, is determined. The toxin consists of 31 amino acid residues and is cross-linked with four disulphide bridges. The sequence has some similarity to that of toxin III and no similarity to those of toxin I and toxin II both from sea anemone, Anemonia sulcata, or to that of Anthopleurin A from Anthopleura xanthogrammica.     

Immunochemistry of sea anemone toxins: structure-antigenicity relationships and toxin-receptor interactions probed by antibodies specific for one antigenic region

Two antibody subpopulations directed against Anemonia sulcata toxin I or II have been purified by immunoaffinity chromatography. These antibodies are specific for a single antigenic region and were used in a structure-antigenicity relationship study using homologous toxins and chemically modified derivatives of A. sulcata toxin II. Asp-7 and/or Asp-9 and Gln-47 of toxin II were found to be implicated in the antigenic region recognized by the two antibody subpopulations. On the contrary, Arg-14, Lys-35, -36, and -46, and alpha-NH2 of the glycine residue of A. sulcata toxin II are not involved in the corresponding antigenic region. When assayed for interaction with the sodium channel, the antigenic region of toxin II, including Asp-9 and Gln-47, appeared fully accessible to its specific antibodies, suggesting that it is not involved in the binding of the toxin to its receptor.     

Locust collagen: morphological and biochemical characterization

Natural-abundance 13C NMR spectra (at 15.04 MHz) of the polypeptide toxin II from the sea anemone Anemonia sulcata have been analysed and compared with corresponding spectra reported recently for a closely related polypeptide anthopleurin A. The spectra contain many resolved one-carbon and two-carbon resonances from carbonyl, aromatic and methyl carbons, many of which have been assigned to individual carbons in the molecule on the basis of their chemical shifts, including their pH dependence, and by comparison with the 13C NMR spectrum of anthopleurin A. Analysis of the effects of pH on the spectrum yields estimates for the pKa values of a number of functional groups in the molecule, as follows: side-chain carboxylates of the two aspartic acid residues 2 and 3.1; COOH-terminal carboxylic acid, 3.5; imidazolium moieties of the two histidine residues, 6.7 and 7.6 NH2-terminal ammonium, 8. The similarity between the pKa values of these functional groups in toxin II and those of corresponding groups in anthopleurin A, together with the close agreement between chemical shifts of conserved carbons, indicates that many local interactions are nearly identical in the two molecules, and thus supports the thesis that their overall conformations in solution are similar. However, the local interactions involving one of the aspartic acid residues are altered in toxin II. Together with other data, this leads to a proposal for the site in these two molecules which is responsible for their cardiac stimulatory activity.     

NMR studies of toxin III from the sea anemone Radianthus paumotensis and comparison of its secondary structure with related toxins

Nearly complete assignments of the proton nuclear magnetic resonance (NMR) spectrum of the polypeptide toxin III from the sea anemone Radianthus paumotensis (RP) are presented. The secondary structures of the related toxins RP II and RP III are described and are compared with each other and with another related toxin ATX Ia from Anemonia sulcata [Widmer, H., Wagner, G., Schweitz, H., Lazdunski, M., & Wüthrich, K. (1988) Eur. J. Biochem. 171, 177-192]. All of these proteins contain a highly twisted four-strand antiparallel beta-sheet core connected by loops of irregular structure. From the work done with AP-A from Anthopleura xanthogrammica [Gooley, P. R., & Norton, R. S. (1986) Biochemistry 25, 2349-2356], it is clear that this homologous toxin also has the same basic core. Some small differences are seen in the structures of these toxins, particularly in the position of the N-terminal residues that form one of the outside strands of the beta-sheet. In addition, the R. paumotensis toxins are two residues longer, extending the third strand of sheet containing the C-terminal residues. A comparison of chemical shifts for assigned residues is also presented, in general supporting the similarity of structure among these proteins.     

Photochemically induced dynamic nuclear polarisation NMR study of the aromatic residues of sea-anemone polypeptide cardiac stimulants

One-dimensional and two-dimensional photochemically induced dynamic nuclear polarisation (photo-CIDNP) nuclear magnetic resonance spectra have been recorded for the sea-anemone polypeptide cardiac stimulants anthopleurin-A and Anemonia sulcata toxins I and II. In anthopleurin-A and toxin II, all three Trp residues are accessible to the flavin dye, although Trp-23 in anthopleurin-A shows a weaker photo-CIDNP response than Trp-33 and Trp-45. Tyr-25 in anthopleurin-A also shows a strong response. In toxin I, Trp-23, Trp-33 and Tyr-45 (which replaces Trp in this molecule) are accessible to the dye. The pH dependences of the photo-CIDNP spectra of all three polypeptides have been examined. The response of Trp-33 increases significantly with pH. The two His residues of anthopleurin-A and toxin II display a response in their imidazole forms, but not their imidazolium forms. The surface accessibilities of Trp-23 and Trp-33 are discussed in relation to the interaction of these polypeptides with the Na+ channel.     

Sea anemone toxin and scorpion toxin share a common receptor site associated with the action potential sodium ionophore.

Toxin II isolated from the sea anemone Anemonia sulcata enhances activation of the action potential sodium ionophore of electrically excitable neuroblastoma cells by veratridine and batrachotoxin. This heterotropic cooperative effect is identical to that observed previously with scorpion toxin but occurs at a 110-fold higher concentration. Depolarization of the neuroblastoma cells inhibits the effect of sea anemone toxin as observed previously for scorpion toxin. Specific scorpion toxin binding is inhibited by sea anemone toxin with KD approximately equal to 90 nM. These results show that the polypeptides scorpion toxin and sea anemone toxin II share a common receptors site associated with action potential sodium ionophores.     

Developmental changes in inotropic actions of neurotoxins observed in ventricular muscle of rat heart.

Developmental changes in functions of myocardial sodium channels were examined from inotropic effects of several neurotoxins in ventricular muscle preparations obtained from prenatal (20-22 day gestation) or adult (3-4 months old) rat hearts. Tetrodotoxin caused a negative inotropic effect in low concentrations and a loss of muscle responsiveness to electrical stimulation in high concentrations in preparations obtained from either prenatal or adult rat heart. The tetrodotoxin concentration that caused a 50% decrease in developed tension was higher in prenatal rats. Anemonia sulcata toxin, Androctonus australis toxin, veratridine, and Centruroides sculpturatus toxin all produced positive inotropic effects in adult rat heart. The effects were largest with A. sulcata and A. australis toxins, intermediate with veratridine, and smallest with C. sculpturatus toxin. Prenatal heart required higher concentrations of either veratridine, or A. sulcata or A. australis toxins to produce comparable positive inotropic effects. With C. sculpturatus toxin, no significant positive inotropic effect was observed in prenatal heart muscle preparations. These results indicate that cardiac sodium channels undergo significant functional changes during development and that negative and positive inotropic effects of neurotoxins resulting from inhibition and enhancement of fast Na+ channels reflect developmental changes in the cardiac sodium channels.     

Amino acid sequence of the Anthopleura xanthogrammica heart stimulant, anthopleurin-B

Anthopleurin-B, the most potent peptide heart stimulant from the sea anemone Anthopleura xanthogrammica, was shown to exist as a single polypeptide chain consisting of 49 amino acid residues. The sequence of the peptide was shown to be: Gly-Val-Pro-Cys-Leu-Cys-Asp-Ser-Asp-Gly- Pro-Arg-Pro-Arg-Gly-Asn-Thr-Leu-Ser-Gly-Ile-Leu-Trp-Phe-Tyr-Pro-Ser- Gly-Cys-Pro-Ser-Gly-Trp-His-Asn-Cys-Lys-Ala-His-Gly-Pro-Asn-Ile-Gly- Trp-Cys-Cys-Lys-Lys. The carboxymethylcysteine derivative, tryptic and chymotryptic peptides (obtained from the derivative and separated by high performance liquid chromatography) were sequenced by manual Edman degradation. Although six carboxymethylcysteine residues were formed by reduction and alkylation of the polypeptide, no cysteine residues were detectable in the native protein, indicating that there are three cystine residues in anthopleurin-B. The amino acid sequence differs in 7 places from anthopleurin-A: at residues 3 (Pro for Ser), 12 (Arg for Ser), 13 (Pro for Val), 21 (Ile for Thr), 24 (Phe for Leu), 42 (Asn for Thr), and 49 (Lys for Gln). These differences are important since anthopleurin-B is about a 12.5-fold better heart stimulant than anthopleurin-A from A. xanthogrammica, anthopleurin-C from Anthopleura elegantissima, and toxin II from Anemonia sulcata.     

Phylogenetic changes in sensitivity to Anemonia sulcata toxin (ATX II), and impact of first interaction with the toxin (imprinting) on later response to it

The Anemonia sulcata toxin ATX II is cardiotoxic and neurotoxic, and--at a high dose level--even lethal for the mouse, neurotoxic, but non-lethal for the frog, and has no adverse influence whatever on the Planaria and Tetrahymena; it even stimulates the growth of the Tetrahymena at a low dose level. It also induces imprinting in the Tetrahymena, as judged from the altered response of the latter to ATX II on re-exposure. No similar imprinting effect was demonstrable in mice.     

The voltage-dependent Na+ channel of insect nervous system identified by receptor sites for tetrodotoxin, and scorpion and sea anemone toxins

Receptor sites for some of the most important toxins known to be specific for voltage-sensitive Na+ channel in the mammalian nervous system have been identified in a purified membrane preparation of house fly brain. Very high affinities have been found for the association of tetrodotoxin or tetrodotoxin derivatives with the insect Na+ channel (Kd = 0.03 - 0.08 nM). The gamma toxin from the Brazilian scorpion Tityus serrulatus forms a complex with the Na+ channel having a Kd of 6.1 pM. The Kd value for toxin II from the sea anemone Anemonia sulcata is 0.12 microM. These results show a high degree of conservation of the pharmacological properties of the brain Na+ channels between insects and mammals.     

A mechanistic interpretation of the action of toxin II from Anemonia sulcata on the cardiac sodium channel

Cardiac sodium channels, modified by Anemonia sulcata toxin II, have been analyzed by the patch-clamp method. The open state of the modified sodium channels proved to be prolonged highly significantly and reopening from a closed state denoted c*-state frequently occurred, interrupted by silent periods, denoted i*-state. Activation from the c*-state was apparently not affected by toxin action, whereas activation from the i*-state was markedly prolonged. Upon higher depolarizations toxin-induced sodium channels disappeared and this behaviour has been attributed to dissociation of the toxin from the channel by use of a special pulse-protocol. The onset of the toxin effect on the action potential proved to depend on stimulation, and it is concluded that the toxin binds preferentially to the open (o)-state. Taking together the results, a kinetic scheme is suggested for action of the toxin on the cardiac sodium channel.     

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.     

A proton nuclear magnetic resonance study of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata: sequential and stereospecific resonance assignment and secondary structure

The sequential resonance assignment of the 1H NMR spectrum of the antihypertensive and antiviral protein BDS-I from the sea anemone Anemonia sulcata is presented. This is carried out with two-dimensional NMR techniques to identify through-bond and through-space (less than 5 A) connectivities. Added spectral complexity arises from the fact that the sample is an approximately 1:1 mixture of two BDS-I isoproteins, (Leu-18)-BDS-I and (Phe-18)-BDS-I. Complete assignments, however, are obtained, largely due to the increased resolution and sensitivity afforded at 600 MHz. In addition, the stereospecific assignment of a large number of beta-methylene protons is achieved from an analysis of the pattern of 3J alpha beta coupling constants and the relative magnitudes of intraresidue NOEs involving the NH, C alpha H, and C beta H protons. Regular secondary structure elements are deduced from a qualitative interpretation of the nuclear Overhauser enhancement, 3JHN alpha coupling constant, and amide NH exchange data. A triple-stranded antiparallel beta-sheet is found to be related to that found in partially homologous sea anemone polypeptide toxins.     

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.     

Synthesis and biological activity of 7,8-dihydro derivatives of batrachotoxinin interacting with fast sodium channels

7,8-Dihydrobatrachotoxinin (A) (I) was synthesized from 11 alpha-hydroxyprogesterone (III) by a 37-stage procedure. Trimethylpyrrolcarboxylate, benzoate as well as 2-azido-benzoate derivatives of (I) were obtained by mixed anhydride technique, the latter two derivatives being prepared also with tritium atoms in aromatic rings (sp. radioactivity about 28 Cu/mmol). Upon interaction with rat brain synaptosomes the apparent Kd of 7,8-dihydrobatrachotoxinin A 20 alpha-[4-3H]benzoate (Iv) was about 2,5 x 10(-6) M. The (Iv) specific binding was inhibited by aconitine with K0,5 = 1,3 x 10(4) M. Anemonia sulcata toxin II (ATX II) enhanced (Iv) affinity for the receptor up to 7 x 10(-7) M, the maximum binding capacity being 2,5 pmol/mg of protein. Benzocaine and tetracaine competitively displaced specifically bound toxin with K0,5 = 3,1 x 10(-4) M and 5,7 x 10(-7) M, respectively, in the presence of 10(-5) M ATX II. 2-Azido[5-3H]benzoate derivative (Id) was shown to be an effective probe for covalent labeling of the alkaloid toxin receptor of the sodium channel.     

Assignment of aromatic resonances in the 1H nuclear magnetic resonance spectra of cardioactive polypeptides from sea anemones

High-resolution 1H NMR spectroscopy at 300 MHz has been used to investigate the aromatic residues of a series of homologous polypeptides from sea anemones: anthopleurin-A from Anthopleura xanthogrammica and toxins I and II from Anemonia sulcata. Using two-dimensional NMR techniques, specific assignments to individual protons have been made for all aromatic resonances in the spectra of these molecules. In all three polypeptides the resonances from the two conserved Trp residues, 23 and 33, are shifted significantly from their random coil values, and the indole NH resonance of Trp-23 is not observed. These shift perturbations are due in part to a mutual interaction of the two indole rings, which is also indicated by the observation of nuclear Overhauser enhancements between protons of the two rings. Several other nonpolar side chains also interact with these two Trp residues, forming a hydrophobic region, the overall structure of which is conserved throughout the series. The other aromatic residues in these polypeptides appear not to participate in this structural region.