Lászlo Béress

Purification of three polypeptides with neuroand cardiotoxic activity from the sea anemone Anemonia sulcata

Abstract Three toxic polypeptides, Toxins I, II and III have been isolated from the sea anemone Anemonia sulcata Pennant (Actinaria). Toxin isolation was accomplished by alcoholic extraction of the homogenized sea anemones, batchwise adsorption on cation exchangers, gel filtration on Sephadex (G 50, G 25, G 10) and ion exchange chromatography on SP-Sephadex and QAE-Sephadex. It was demonstrated that the same toxins (Toxin I and Toxin II) are also present in the separated tentacles. Toxin I contains 45 amino acids, mol. wt 4702, Toxin II contains 44 amino acids, mol. wt 4197, Toxin III contains 24 amino acids, mol. wt 2678. The toxins were tested on the shore crab Carcinus maenas by intramuscular injection. For both Toxins I and II the lethal dose is less than 2 μg per kg Carcinus , whereas for Toxin III it is less than 50 μg per kg. Toxins I and II also have a strong paralysing effect on mammals and fishes.

Anticoagulant fucoidan fractions from Fucus vesiculosus induce platelet activation in vitro

Anticoagulant fucoidan fractions of different molecular weight and sulfate content were prepared and investigated for their effects on platelet function in vitro. The fucoidan fractions were incubated with human platelet rich plasma (PRP) at concentrations of 5, 10 and 50 micrograms/ml. Platelet activation was subsequently studied by a standard aggregation assay and flow cytometric determination of the activation dependent platelet-surface markers CD62p (P-selectin, GMP-140) and CD63 (GP53). All fucoidan fractions induced irreversible platelet aggregation in a dose-dependent manner. Comparing fractions of identical molecular weight (100 kDa) the low sulfate content fucoidan FF5 (S = 7.6%) exerted a significantly greater effect than the highly sulfated fucoidan FF7 (S = 10.2%) over the whole concentration range (n = 5, P < 0.05). Among fractions of identical sulfate content fucoidan-induced platelet aggregation was also found to depend on the molecular weight of the fucoidan. At concentrations of 10 and 50 micrograms/ml the high molecular weight fraction FF7/1 (150 kDa) showed a significantly greater effect than the 50 kDa fraction FF7/3 (24.8 +/- 6.7 vs. 7.0 +/- 3.5 and 54.6 +/- 13.5 vs. 15.0 +/- 9.0%, respectively; mean +/- SD, n = 5, P < 0.05). The molecular weight dependence of the fucoidan effect was also reflected by the flow cytometric data. Coincubation of FF7/1 and FF7/3 (10 micrograms/ml) with PRP increased the number of CD62p and CD63 positive platelets by 9.0 +/- 3.3 vs. 2 +/- 1.9 and 7.1 +/- 2.4 vs. 3.2 +/- 2.6% over control values, respectively (n = 5, P < 0.05). In conclusion, our results show that the low molecular weight fucoidan FF7/3 combines potent anticoagulant and fibrinolytic properties with only minor platelet activating effects and is therefore a suitable substance for further pharmacological studies.

Isolation and characterisation of five neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima

Five toxins (APE 1 to APE 5) of the sea anemone species Anthopleura elegantissima (Brandt) have been isolated from a toxic by-product fraction of its concentrated crude watery-methanolic extract, prepared previously for the isolation of a neuropeptide (the head-activator) by Schaller and Bodenmüller (Proc. Natl. Acad. Sci. USA 78 (1981) 7000) from 200kg sea anemones. Toxin purification was performed by desalting of the starting material by dialysis (MWCO 3500) against distilled water, anion exchange chromatography on QAE-Sephadex A25 at pH 8, twice gel filtration on Sephadex G50 m, repeated chromatography on QAE-Sephadex at pH 10 and chromatography on the cation exchanger Fractogel EMD SO(3)(-)-650 M.Final purification of the toxins was achieved by HPLC on MN SP 250/10 Nucleosil 500-5 C(18) PPN and MN SP 250/21 Nucleosil 300-7 C(18). Each toxin was composed of at least two isotoxins of which APE 1-1, APE 1-2, APE 2-1, APE 2-2 and APE 5-3 were isolated in preparative scale. With exception of APE 5-3 the sequences of the isotoxins have been elucidated. They resemble the 47 residue type-I long polypeptide toxins native to Anemonia sulcata (Pennant). All isotoxins paralyse the shore crab (Carcinus maenas) by tetanic contractions after i.m. application. The toxins modify current passing through the fast Na(+) channel in neuroblastoma cells, leading to delayed and incomplete inactivation. APE 1-1, APE 2-1 and APE 5-3 produce a positive inotropic effect in mammalian heart muscle, although they differ in potency. The order of potency is APE 2-1>APE 1-1>APE 5-3 (i.e. threshold concentrations are 1, 10 and 300nM, respectively). In addition, they enhance the spontaneous beating frequency in isolated right atria (guinea pig). The most potent cardiotoxic isotoxin is APE 2-1, its sequence is identical with that of AP-C, a toxin isolated and characterised previously by Norton et al. (Drugs and Foods from the Sea, 1978, University of Oklahoma Press, p. 37-50).LD50 APE 2-1:1 micro g/kg b.w. C. maenas (i.m.). LD50 APE 1-1:10 microg/kg b.w. C. maenas (i. m.). LD50 APE 5-3:50 microg/kg b.w. C. maenas (i.m.).

A method for the isolation of the caribbean palytoxin (C-PTX) from the coelenterate (zooanthid) Palythoa caribaeorum

Chromatographically pure toxin was isolated from the zooanthid Palythoa caribaeorum. Toxin isolation was achieved by extraction with 50% ethanol from the homogenized specimens, gel filtration on Sephadex G-50 and ion exchange chromatography on QAE- and SP-Sephadex. Final purification was obtained by gel filtration on Biogel P-6. The LD50 tested on the shore crab Carcinus maenas was 62.5 ng/kg.

The influence of the rate of electrical stimulation on the effects of the Anemonia sulcata toxin ATX II in guinea pig papillary muscle

In guinea pig papillary muscle, the rate of electrical stimulation (0.1-2 Hz) strongly influenced the effects of the Anemonia sulcata toxin ATX II on action potential duration (APD) and contractile force. In the concentration range studied (10-8-10-7 M), ATX II always produced a larger prolongation in APD at low rates of stimulation. At 0.1 Hz there was a temporal dissociation between the onset of the APD-prolonging and the positive inotropic effect. However, under equilibrium conditions there was a positive relationship between the APD expressed as a fraction of the time during which the membrane was depolarized, and the contractile force irrespective of the change in experimental conditions being variation of stimulation frequency or the addition of ATX II. The results suggest that the positive inotropic effects of both ATX II and increased stimulation frequency could be induced by a similar mechanism, e.g. an increase in sodium of the heart muscle.

Structural properties of toxin II of sea anemone (Anemone sulcata) determined by laser Raman spectroscopy.

The sea anemone, Anemonia sulcata, paralyses its prey by injecting potent neurotoxins into it [ 1] . Recently, Beress et al. [2] isolated from Anemonia sulcata three neurotoxic polypeptides (toxins I, II and III) and characterized each according to its gross amino-acid composition, molecular weight, N-terminus residue and molar absorptivity. Toxin II was the most easily purified [2] and its amino-acid composition and sequence have been under further investigation [3]. Toxin II is of particular interest since recent studies suggest that its effect is neurotoxic on some organisms (e.g., crayfish) but cardiotoxic on others (e.g., guinea pig) [4-71. Because toxicological specificity may be related to the protein conformation, we have undertaken an examination of the laser Raman spectrum of toxin II to identify specific features of its conformational structure. The Raman spectrum consists of a series of lines or frequencies, due to scattering by vibrations of specific molecular subgroups [8]. For proteins and polypeptides, Raman frequencies of both the peptidyl backbone and side-chain groups are revealed, and

Combining multidimensional liquid chromatography and MALDI-TOF-MS for the fingerprint analysis of secreted peptides from the unexplored sea anemone species Phymanthus crucifer.

Sea anemones are sources of biologically active proteins and peptides. However, up to date few peptidomic studies of these organisms are known; therefore most species and their peptide diversity remain unexplored. Contrasting to previous venom peptidomic works on sea anemones and other venomous animals, in the present study we combined pH gradient ion-exchange chromatography with gel filtration and reversed-phase chromatography, allowing the separation of the 1-10 kDa polypeptides from the secretion of the unexplored sea anemone Phymanthus crucifer (Cnidaria/Phymanthidae). This multidimensional chromatographic approach followed by MALDI-TOF-MS detection generated a peptide fingerprint comprising 504 different molecular mass values from acidic and basic peptides, being the largest number estimated for a sea anemone exudate. The peptide population within the 2.0-3.5 kDa mass range showed the highest frequency whereas the main biomarkers comprised acidic and basic peptides with molecular masses within 2.5-6.9 kDa, in contrast to the homogeneous group of 4-5 kDa biomarkers found in sea anemones such as B. granulifera and B. cangicum (Cnidaria/Actiniidae). Our study shows that sea anemone peptide fingerprinting can be greatly improved by including pH gradient ion-exchange chromatography into the multidimensional separation approach, complemented by MALDI-TOF-MS detection. This strategy allowed us to find the most abundant and unprecedented diversity of secreted components from a sea anemone exudate, indicating that the search for novel biologically active peptides from these organisms has much greater potential than previously predicted.

Phyla- and Subtype-Selectivity of CgNa, a Na+ Channel Toxin from the Venom of the Giant Caribbean Sea Anemone Condylactis Gigantea

Because of their prominent role in electro-excitability, voltage-gated sodium (NaV) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related NaV subtypes, making them powerful tools to study NaV channel function and structure. CgNa is a 47-amino acid residue type I toxin isolated from the venom of the Giant Caribbean Sea Anemone Condylactis gigantea. Previous studies showed that this toxin slows the fast inactivation of tetrodotoxin-sensitive NaV currents in rat dorsal root ganglion neurons. To illuminate the underlying NaV subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (NaV1.2–NaV1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNaV1.3/β1, mNaV1.6/β1 and, to a lesser extent, hNaV1.5/β1, while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNaV1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect NaV channel, resulting in a dramatic increase in peak current amplitude and complete removal of fast and steady-state inactivation. Together with the previously determined solution structure, the subtype-selective effects revealed in this study make of CgNa an interesting pharmacological probe to investigate the functional role of specific NaV channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of NaV channel inactivation.

Sea Anemone Toxins, Acting on Na+ Channels and K+ Channels: Isolation and Characterization

Sea anemones are nettling animals; they belong to the phylum Cnidaria, class Anthozoa, family Actiniidae. From an evolutionary point of view they represent the simplest level of organization within the metazoa. All sea anemones possess stinging organelles called nematocysts which play an important role in paralysing prey and also in defence. They contain potent toxins with an immediate paralysing action on crustaceans and fishes after injection.

Preparation and properties of fluorescence labeled neuro- and cardiotoxin II from the sea anemone (Anemonia sulcata)

Labeling of the sea anemone toxin II with fluoresceinisothiocyanate is described. The native toxin and low molecular weight fluorescent compounds were completely separated from the derivative. Compared to fluorescein, the fluorescence of the toxin-derivative has a red shift and a decreased quantum yield. Like the native toxin, the fluorescent derivative affects the voltage-dependent sodium channel in nerve membranes. Under voltage-clamp conditions, the effect of the modified toxin on the inactivation of sodium channels was 45% of that of the unlabeled toxin.