Amino acid sequence of neurotoxin II from the sea anemone Radianthus macrodactylus

Amino acid sequence of neurotoxin II isolated from the sea anemone Radianthus macrodactylus was determined by analysis of peptides obtained after its digestion with trypsin and staphylococcal proteinase. It is shown that the polypeptide chain of the toxin consists of 48 amino acid residues, including six cysteines.     

Tenebrosin-A, a new cardiostimulant protein from the Australian sea anemone Actinia tenebrosa

A new cardiac stimulatory protein, tenebrosin-A, has been isolated from the Australian sea anemone Actinia tenebrosa by gel filtration and cation-exchange chromatography, followed by cation-exchange HPLC. Its purity is established by analytical reversed-phase HPLC and N-terminal sequence analysis. According to SDS-PAGE, its apparent Mr is 20,000 daltons. Amino acid analysis indicates that it contains 186 residues, and is devoid of cysteine or cystine. Tenebrosin-A exerts a strong positive inotropic effect on isolated guinea pig atria at a concentration of 1.4 nM, with little chronotropic activity.     

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.     

The 18S ribosomal RNA sequence of the sea anemone Anemonia sulcata and its evolutionary position among other eukaryotes

Evolutionary trees based on partial small ribosomal subunit RNA sequences of 22 metazoa species have been published [(1988) Science 239, 748-753]. In these trees, cnidarians (Radiata) seemed to have evolved independently from the Bilateria, which is in contradiction with the general evolutionary view. In order to further investigate this problem, the complete srRNA sequence of the sea anemone Anemonia sulcata was determined and evolutionary trees were constructed using a matrix optimization method. In the tree thus obtained the sea anemone and Bilateria together form a monophyletic cluster, with the sea anemone forming the first line of the metazoan group.     

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.     

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.     

海葵神经毒素基因的克隆和序列分析

根据已发现的海葵神经毒素蛋白的两端保守氨基酸序列 ,设计简并引物 .用RT PCR方法 ,从侧花海葵 (Anthopleurasp .)触手总RNA中分离出多个神经毒素新基因 .它们分别编码 3个长度都是 4 7个氨基酸的毒素蛋白 ,它们的氨基酸序列与海葵神经毒素C(Ap C)最为相似 .新基因的发现为进一步研究其生物活性及应用打下了基础 .     

Molecular evolution of protein: Internal homology in the amino acid sequence of calmodulin

Calmodulin is one of the calcium-binding proteins and is distributed widely in eukaryotes. The amino acid sequences were studied of calmodulin taken from bovine brain, scallop (Patinopecten), sea anemone (Metridium senile) and tetrahymena (Tetrahymena pyriformis). One notable feature of the primary structure of calmodulin is its internal homology. It can be subdivided into four domains with similar amino acid sequences. This homology implies that the primary structure of calmodulin has been elongated twice by intragenic duplication. Using this intragenic duplication model, the amino acid sequences of calmodulin from those four species were analyzed in detail. This kind of approach has proved very useful for investigation of the origin and evolution of this protein.     

Amino acid sequence of a neurotoxin from the anemone Radianthus macrodactylus

Amino acid sequence of neurotoxin I isolated from the anemone Radianthus macrodactylus, and consisted of 48 amino acid residues, including six cysteines, was determined by analysis of products of its trypsin and chymotrypsin digestion.     

Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.     

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.     

Do the Anemonefish Amphiprion ocellaris (Pisces: Pomacentridae) Imprint Themselves to Their Host Sea Anemone Heteractis magnifica (Anthozoa: Actinidae)?

Juvenile anemonefishes detect their host sea anemone by olfactory stimuli; in order to investigate whether this behaviour is innate or acquired, the anemonefish species Amphiprion ocellaris was bred in two different ways: 1. With no host sea anemone present at all (–A); and 2. With the specific host sea anemone Heteractis magnifica present in the hatching aquarium, so that these eggs were laid and hatched close to the sea anemone, as in nature (+A). The two different types of juvenile A. ocellaris were presented to the odours of the host sea anemone H. magnifica in two sets of short-term experiments with the host (a) visually hidden in a net cage, and (b) visible but physically separated from the anemonefishes. In both cases, a water flow was established between fishes and host. The +A-fishes found their host by olfactory and not by visual stimuli. In both series, the –A-fishes showed a significantly lower affinity behaviour towards the odour compounds from the host sea anemone than the +A-fishes did. A third type of experiment was a direct confrontation between fishes and host; here, the –A-fishes were indifferent towards the host sea anemone for almost 48 h, while the +A-fishes acclimated to the host sea anemone within the first 5 min of the direct confrontation. The results of this study suggest that Amphiprion ocellaris imprints itself olfactorily to its species-specific host sea anemone Heteractis magnifica, and, furthermore, may be genetically disposed towards olfactory recognition of the host sea anemone.     

Amino Acid Sequence of the α- and β-Globin Chains of the Hiroo Sea Snake (Laticauda laticaudata)

We determined the hemoglobin complete amino acid sequences of the Hiroo sea snake (Laticaudia laticuada) from the intact globin chain, enzymatically digested fragments, and chemical cleavage fragments to analyze molecular evolution for classification of the sea snake. The Hiroo sea snake has two hemoglobin components, Hb-I and Hb-II, which contain different α- and β-chains, respectively. This is the first report of the complete primary structure of a snake hemoglobin. The sequences were compared with those of other reptilian hemoglobins. Amino acid replacements at positions critical for structure and physiological role of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors at β-globins seemed to be fullfilled.     

Primary structures of actinoporins from sea anemone Oulactis orientalis

Partial amino acid sequences of the actinoporins Or-A (136 aa) and Or-G (144 aa) isolated from the Sea of Japan sea anemone Oulactis orientalis were determined by sequencing the clones obtained by the amplification of genomic DNA and cDNA with specific primers to the N-terminal regions of the 0. orientalis actinoporin sequences and to the C-terminal region, which is known to be highly conservative in all the known actinoporin sequences. The complete structures of Or-A (165 aa) and Or-G (173 aa) were established by sequencing the cDNA clones obtained by the fast amplification of 3'-ends of cDNA. A comparative analysis of the amino acid sequences of the Oulactis actinoporins with those of actinoporins from tropical species revealed considerable differences in the structures of their N-terminal fragments and their membrane-binding sites. We believe that these differences could explain lower hemolytic activities of Or-A and Or-G than that of actinoporins from the tropical species.     

A common motif in proparts of Cnidarian toxins and nematocyst collagens and its putative role

In Cnidarians, cnidoblast cells contain organelles called cnidocysts, which are believed to be the product of an extremely complex regulated secretory pathway. When matured, these stinging organelles are capable of storing and delivering toxins. We hypothesized that translated nematocyst proteins might comprise specific sequences serving as signals in sorting to the organelle. A sodium channel neurotoxin from the sea anemone Actinia equina was cloned and the toxin precursor sequence was compared to those of nematocyst collagens, pore-forming toxins and ion channel neurotoxins. It was found that all the analyzed sequences possess a highly conserved stretch of nine amino acid residues ending with Lys-Arg N-terminally of the mature region.     

Amino acid sequence of the alpha- and beta-globin chains of the Erabu sea snake (Laticaudia semifasciata)

We determined the complete amino acid sequences of the Erabu sea snake (Laticaudia semifasciata) hemoglobin by analyzing the intact globin chains, enzymatically digested fragments, and chemical cleavage fragments to clarify the molecular evolution and phylogenetic classification of the sea snake. The Erabu sea snake has two types of hemoglobin components, Hb-I and Hb-II, which contain different alpha- and beta-chains. This is the second report of the complete primary structure for hemoglobin of snakes. The sequences were compared with those of other reptilian hemoglobins. Amino acids at positions critical for the structure and physiological functions of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors of beta-globins seemed to be fulfilled.     

Functional discrimination of sea anemone neurotoxins using 3D-plotting

One of the most important goals in structural biology is the identification of functional relationships among the structure of proteins and peptides. The purpose of this study was to (1) generate a model based on theoretical and computational considerations among amino acid sequences within select neurotoxin peptides, and (2) compare the relationship these values have to the various toxins tested. We employed isolated neurotoxins from sea anemones with established specific potential to act on voltage-dependent sodium and potassium channel activity as our model. Values were assigned to each amino acid in the peptide sequence of the neurotoxins tested using the Number of Lareo and Acevedo algorithm (NULA). Once the NULA number was obtained, it was then plotted using three dimensional space coordinates. The results of this study allow us to report, for the first time, that there is a different numerical and functional relationship between the sequences of amino acids from sea anemone neurotoxins, and the resulting numerical relationship for each peptide, or NULA number, has a unique location in three-dimensional space.     

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.