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.     

Experimental habituation of aggression in the sea anemone Actinia equina

Behavioural plasticity in Actinia equina (L.) was examined in experimental contests using a range of pedal disc colour phenotypes, which characterize 3 known, ecologically distinct morphs. With repeated pairing of individuals in auto-phenotypic encounters, habituation was easily induced in the 2 mid-shore morphs, but was not obvious in the less aggressive, low-shore form. Subsequent pairing with a different partner revealed that anemones remained aggressive towards a novel opponent. Following novel contact, repairing of the dark red pedal phenotype with the original partner provided some evidence of retention of habituation to a previous opponent, and thus of a specific inducible memory.     

The cytotoxic and cytolytic activity of equinatoxin II from the sea anemone Actinia equina

The cytotoxic and cytolytic effects of equinatoxin II (EqT II) from the sea anemone Actinia equina L. were studied on exponentially growing and synchronized V-79-379 A cell line in culture. The cell viability test and the determination of the cytolytic effect by cell counting confirmed both cytotoxic and cytolytic activity of EqT II. Additionally, cytocidal and cytostatic effects depending on the toxin concentration were observed. The presence of fetal calf serum in the cell culture medium reduced both cytocidal and cytostatic effects by two magnitudes and prevented cytolysis. Combining EqT II and serum resulted in an insoluble complex which was cytostatic even when isolated and resuspended in the culture medium, while the supernatant retained both cytocidal and cytostatic activity. No significant difference in sensitivity between synchronized and exponentially growing cells could be detected after EqT II treatment.     

Crystal structure of the soluble form of equinatoxin II, a pore-forming toxin from the sea anemone Actinia equina

BACKGROUND: Membrane pore-forming toxins have a remarkable property: they adopt a stable soluble form structure, which, when in contact with a membrane, undergoes a series of transformations, leading to an active, membrane-bound form. In contrast to bacterial toxins, no structure of a pore-forming toxin from an eukaryotic organism has been determined so far, an indication that structural studies of equinatoxin II (EqtII) may unravel a novel mechanism. RESULTS: The crystal structure of the soluble form of EqtII from the sea anemone Actinia equina has been determined at 1.9 A resolution. EqtII is shown to be a single-domain protein based on a 12 strand beta sandwich fold with a hydrophobic core and a pair of alpha helices, each of which is associated with the face of a beta sheet. CONCLUSIONS: The structure of the 30 N-terminal residues is the largest segment that can adopt a different structure without disrupting the fold of the beta sandwich core. This segment includes a three-turn alpha helix that lies on the surface of a beta sheet and ends in a stretch of three positively charged residues, Lys-30, Arg-31, and Lys-32. On the basis of gathered data, it is suggested that this segment forms the membrane pore, whereas the beta sandwich structure remains unaltered and attaches to a membrane as do other structurally related extrinsic membrane proteins or their domains. The use of a structural data site-directed mutagenesis study should reveal the residues involved in membrane pore formation.     

Conformational stability of serine proteinase inhibitor from the sea anemone Heteractis crispa

The influence of different environmental values of the pH and temperature on the spatial organization of serine proteinase inhibitor from the sea anemone Heteractis crispa (=Radianthus macrodactylus) on the level of tertiary and secondary structure was studied by CD spectroscopy. The molecule InhVJ was shown to possess a high conformational thermo- and pH-stability. We determined the point of conformational thermotransition of polypeptide (70 degrees C) after which the molecule gets denaturational stable state with conservation of 80% proteinase inhibitory activity. The significant partial reversible changes of molecule spatial organization were established to occur at the level of tertiary structure in the process of acid-base titration in the range of pH 11.0-13.0. This can be explained by of ionization of tyrosine residues. The molecule InhVJ is conformationally stable at the low pH values (2.0). The quenching of tyrosine residues by acrylamide showed that two of these residues are accessible to the quencher in full, while the third part is available.     

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.     

Sensory transducers of E. coli are composed of discrete structural and functional domains

The tar and tsr genes of E. coli encode functionally analogous transducer proteins that mediate two distinct classes of chemotactic response. The tap gene lies adjacent to tar, and is thought to encode another transducer protein. We present here the complete nucleotide sequence of the tar-tap region of the E. coli genome, together with a comparative analysis of the sequences of the Tar, Tap, and Tsr proteins. The proteins appear to have a simple transmembrane structure consisting of an extracytoplasmic amino-terminal domain, a membrane-spanning domain, and an intracellular carboxy-terminal domain. The carboxy-terminal domains of three proteins possess highly homologous sequences and contain sites of methylation involved in sensory adaptation, while the amino-terminal sequences are only distantly related to one another, consistent with their serving as chemoreceptor domains that have diverged functionally.     

A serine protease inhibitor from the anemone Radianthus macrodactylus: isolation and physicochemical characteristics

A serine protease inhibitor with a molecular mass of 6106 +/- 2Da (designated as InhVJ) was isolated from the tropical anemone Radianthus macrodactylus by a combination of liquid chromatography methods. The molecule of InhVJ consists of 57 amino acid residues, has three disulfide bonds, and contains no Met or Trp residues. The N-terminal amino acid sequence of the inhibitor (19 aa residues) was established. It was shown that this fragment has a high degree of homology with the N-terminal amino acid sequences of serine protease inhibitors from other anemone species, reptiles, and mammals. The spatial organization of the inhibitor at the levels of tertiary and secondary structures was studied by the methods of UV and CD spectroscopy. The specific and molar absorption coefficients of InhVJ were determined. The percentage of canonical secondary structure elements in the polypeptide was calculated. The inhibitor has a highly ordered tertiary structure and belongs to mixed alpha/beta or alpha + beta polypeptides. It was established that InhVJ is highly specific toward trypsin (Ki 2.49 x 10(-9) M) and alpha-chymotrypsin (Ki 2.17 x 10(-8) M) and does not inhibit other proteases, such as thrombin, kallikrein, and papain. The inhibitor InhVJ was assigned to the family of the Kunitz inhibitor according to its physicochemical properties.     

A new inhibitor of elastase from the sea anemone (Anemonia sulcata)

A proteinase inhibitor for elastases was isolated from extracts of the sea anemone Anemonia sulcata and purified to apparent homogeneity. The procedure comprises ethanolic extraction of the deep-frozen animals followed by gel filtration on Sephadex G-50 and by ion exchange chromatography on DEAE-Sephadex A-25 and SP-Sephadex C-25 and by hydroxylapatite chromatography. The slightly acidic inhibitor (isoelectric point 5.9) is a small protein consisting of 48 amino-acid residues without tryptophan and phenylalanine. The single chain molecule contains two methionines and no free sulfhydryl group but six cysteines presumably forming disulfide bonds. Reaction with cyanogen bromide abolishes the inhibitory properties. The inhibitor exhibits a rather narrow specificity for elastases. It strongly inhibits porcine pancreatic elastase in a permanent fashion with an equilibrium dissociation constant Ki of about 10(-10)M and somewhat weaker the elastase from human leucocytes with a Ki of about 10(-7)M. No obvious inhibition is observed of other serine proteinase such as bovine trypsin, bovine chymotrypsin, subtilisin from Bacillus subtilis and cathepsin G from human leucocytes when tested with synthetic substrates.     

Functional domains of the human epididymal protease inhibitor, eppin

Eppin has two potential protease inhibitory domains: a whey acid protein or four disulfide core domain and a Kunitz domain. The protein is also reported to have antibacterial activity against Gram-negative bacteria. Eppin and its whey acid protein and Kunitz domains were expressed in Escherichia coli and their ability to inhibit proteases and kill bacteria compared. The Kunitz domain inhibits elastase (EC 3.4.21.37) to a similar extent as intact eppin, whereas the whey acid protein domain has no such activity. None of these fragments inhibits trypsin (EC 3.4.21.4) or chymotrypsin (EC 3.4.21.1) at the concentrations tested. In a colony forming unit assay, both domains have some antibacterial activity against E. coli, but this was not to the same degree as intact eppin or the two domains together. When bacterial respiratory electron transport was measured using a 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide assay, eppin and its domains caused an increase in the rate of respiration. This suggests that the mechanism of cell killing may be partly through the permeablization of the bacterial inner membrane, resulting in uncoupling of respiratory electron transport and consequent collapse of the proton motive force. Thus, we conclude that although both of eppin's domains are involved in the protein's antibacterial activity, only the Kunitz domain is required for selective protease inhibition.     

Assignment of the three disulfide bonds in ShK toxin: A potent potassium channel inhibitor from the sea anemone Stichodactyla helianthus

Summary ShK toxin, a 35-residue peptide isolated from the Caribbean sea anemone Stichodactyla helianthus, is a potent inhibitor of the Kv 1.3 potassium channel in lymphocytes. The natural toxin contains three disulfide bonds. The disulfide pairings of the synthetic ShK toxin were elucidated as a prerequisite for studies on its structure-function relationships. The toxin was fragmented at pH 6.5 using either thermolysin or a mixture of trypsin and chymotrypsin followed by thermolysin. The fragments were isolated by RP-HPLC and were identified by sequence analysis and MALDI-TOF mass spectrometry. The three disulfides were unambiguously identified in either proteolytic digest: Cys³ to Cys³⁵, Cys¹² to Cys²⁸ and Cys¹⁷ to Cys³². The Cys³-Cys³⁵ disulfide, linking the amino- and carboxyl-termini, defines the characteristic cyclic structure of the molecule. A similar disulfide pairing motif is found in the snake venom-derived potassium channel blocker dendrotoxin and the mammalian antibiotic peptide defensins.     

Study of the interaction of trypsin inhibitor from the sea anemone Radianthus macrodactylus with proteases

The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: K_D = 7.38 × 10^?8 M and 9.93 × 10^?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively.     

Conformational stability of the serine protease inhibitor InhVJ from the sea anemone <Emphasis Type=Italic>Heteractis crispa</Emphasis>

The effect of temperature and pH of medium on the spatial organization of a molecule of the serine protease inhibitor InhVJ from the sea anemone Heteractis crispa (=Radianthus macrodactylus) at the level of the tertiary and secondary structures has been studied by CD spectroscopy. It has been shown that the conformation of an InhVJ molecule is highly stable to changes in temperature and pH. The point of the thermal conformational transition of the polypeptide (70°C) has been determined, after which the molecule turns into a denatured stable state with the retention of 80% of the inhibitory activity. It was found that significant, partially reversible changes in the spatial organization of the molecule occur on the level of the tertiary structure in the pH range 11.0–13.0, which may be explained by the ionization of tyrosine residues. At a low pH value (2.0), the InhVJ molecule is conformationally stable. The results of quenching of tyrosine residues by acrylamide showed that two residues are completely accessible for the quencher, whereas the third residue is partially accessible.     

Analgesic compound from sea anemone Heteractis crispa is the first polypeptide inhibitor of vanilloid receptor 1 (TRPV1)

Venomous animals from distinct phyla such as spiders, scorpions, snakes, cone snails, or sea anemones produce small toxic proteins interacting with a variety of cell targets. Their bites often cause pain. One of the ways of pain generation is the activation of TRPV1 channels. Screening of 30 different venoms from spiders and sea anemones for modulation of TRPV1 activity revealed inhibitors in tropical sea anemone Heteractis crispa venom. Several separation steps resulted in isolation of an inhibiting compound. This is a 56-residue-long polypeptide named APHC1 that has a Bos taurus trypsin inhibitor (BPTI)/Kunitz-type fold, mostly represented by serine protease inhibitors and ion channel blockers. APHC1 acted as a partial antagonist of capsaicin-induced currents (32 +/- 9% inhibition) with half-maximal effective concentration (EC(50)) 54 +/- 4 nm. In vivo, a 0.1 mg/kg dose of APHC1 significantly prolonged tail-flick latency and reduced capsaicin-induced acute pain. Therefore, our results can make an important contribution to the research into molecular mechanisms of TRPV1 modulation and help to solve the problem of overactivity of this receptor during a number of pathological processes in the organism.     

Gametogenic cycles and reproduction in the beadlet sea anemone Actinia equina (Cnidaria: Anthozoa)

Sexual Actinia equina have been maintained in the laboratory. Monthly biopsies show that these animals have annual gametogenic cycles. Regular samples from several natural populations suggest that animals in the wild show similar gametogenic periodicity. In the laboratory, males release sperm, but females brood young, at least for a few weeks. Members of these broods are released sporadically, usually after a water change. Males and non-sexual adults kept in isolation for over a year release a few juveniles during and at the end of that time. These young are likely to be asexually reproduced. There appears to be geographical variation of anemone size and in the frequency of sexual individuals in populations round the British Coast, so that modes of reproduction may vary.     

The role of lysine, histidine and carboxyl residues in biological activity of equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L.

Equinatoxin II, a pore forming polypeptide from the sea anemone Actinia equina L. was subjected to chemical modifications with group specific reagents. Lysine residues were modified with pyridoxal-5'-phosphate, histidine residues with diethyl pyrocarbonate and carboxyl groups with the use of a water soluble carbodiimide. Modification of charged residues had no significant influence on the toxin interaction with serum lipoproteins. Lysine 5'-phosphopyridoxylated and histidine carbethoxylated derivatives of the toxin retained lethal and hemolytic activities, but the pH profile of hemolytic activity of 5'-phospho-pyridoxylequinatoxin II was markedly altered. Modification of the toxin carboxyl groups impaired both hemolytic and lethal activities, the latter, however, to the greater extent.     

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.