PhcrTx2, a New Crab-Paralyzing Peptide Toxin from the Sea Anemone Phymanthus crucifer.

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 µM), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new ß-defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

PhcrTx2, a new crab-paralyzing peptide toxin from the sea anemone phymanthus crucifer

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 mu M), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new -defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

PhcrTx2, a new crab-paralyzing peptide toxin from the sea anemone phymanthus crucifer

Sea anemones produce proteinaceous toxins for predation and defense, including peptide toxins that act on a large variety of ion channels of pharmacological and biomedical interest. Phymanthus crucifer is commonly found in the Caribbean Sea; however, the chemical structure and biological activity of its toxins remain unknown, with the exception of PhcrTx1, an acid-sensing ion channel (ASIC) inhibitor. Therefore, in the present work, we focused on the isolation and characterization of new P. crucifer toxins by chromatographic fractionation, followed by a toxicity screening on crabs, an evaluation of ion channels, and sequence analysis. Five groups of toxic chromatographic fractions were found, and a new paralyzing toxin was purified and named PhcrTx2. The toxin inhibited glutamate-gated currents in snail neurons (maximum inhibition of 35%, IC50 4.7 mu M), and displayed little or no influence on voltage-sensitive sodium/potassium channels in snail and rat dorsal root ganglion (DRG) neurons, nor on a variety of cloned voltage-gated ion channels. The toxin sequence was fully elucidated by Edman degradation. PhcrTx2 is a new -defensin-fold peptide that shares a sequence similarity to type 3 potassium channels toxins. However, its low activity on the evaluated ion channels suggests that its molecular target remains unknown. PhcrTx2 is the first known paralyzing toxin in the family Phymanthidae.

A novel sea anemone peptide that inhibits acid-sensing ion channels.

Sea anemones produce ion channels peptide toxins of pharmacological and biomedical interest. However, peptides acting on ligand-gated ion channels, including acid-sensing ion channel (ASIC) toxins, remain poorly explored. PhcrTx1 is the first compound characterized from the sea anemone Phymanthus crucifer, and it constitutes a novel ASIC inhibitor. This peptide was purified by gel filtration, ion-exchange and reversed-phase chromatography followed by biological evaluation on ion channels of isolated rat dorsal root ganglia (DRG) neurons using patch clamp techniques. PhcrTx1 partially inhibited ASIC currents (IC50∼100 nM), and also voltage-gated K(+) currents but the effects on the peak and on the steady state currents were lower than 20% in DRG neurons, at concentrations in the micromolar range. No significant effect was observed on Na(+) voltage-gated currents in DRG neurons. The N-terminal sequencing yielded 32 amino acid residues, with a molecular mass of 3477 Da by mass spectrometry. No sequence identity to other sea anemone peptides was found. Interestingly, the bioinformatic analysis of Cys-pattern and secondary structure arrangement suggested that this peptide presents an Inhibitor Cystine Knot (ICK) scaffold, which has been found in other venomous organisms such as spider, scorpions and cone snails. Our results show that PhcrTx1 represents the first member of a new structural group of sea anemones toxins acting on ASIC and, with much lower potency, on Kv channels. Moreover, this is the first report of an ICK peptide in cnidarians, suggesting that the occurrence of this motif in venomous animals is more ancient than expected.

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.

Peptide fingerprinting of the neurotoxic fractions isolated from the secretions of sea anemones Stichodactyla helianthus and Bunodosoma granulifera. New members of the APETx-like family identified by a 454 pyrosequencing approach.

Sea anemones are known to contain a wide diversity of biologically active peptides, mostly unexplored according to recent peptidomic and transcriptomic studies. In the present work, the neurotoxic fractions from the exudates of Stichodactyla helianthus and Bunodosoma granulifera were analyzed by reversed-phase chromatography and mass spectrometry. The first peptide fingerprints of these sea anemones were assessed, revealing the largest number of peptide components (156) so far found in sea anemone species, as well as the richer peptide diversity of B. granulifera in relation to S. helianthus. The transcriptomic analysis of B. granulifera, performed by massive cDNA sequencing with 454 pyrosequencing approach allowed the discovery of five new APETx-like peptides (U-AITX-Bg1a-e - including the full sequences of their precursors for four of them), which together with type 1 sea anemone sodium channel toxins constitute a very distinguishable feature of studied sea anemone species belonging to genus Bunodosoma. The molecular modeling of these new APETx-like peptides showed a distribution of positively charged and aromatic residues in putative contact surfaces as observed in other animal toxins. On the other hand, they also showed variable electrostatic potentials, thus suggesting a docking onto their targeted channels in different spatial orientations. Moreover several crab paralyzing toxins (other than U-AITX-Bg1a-e), which induce a variety of symptoms in crabs, were isolated. Some of them presumably belong to new classes of crab-paralyzing peptide toxins, especially those with molecular masses below 2kDa, which represent the smallest peptide toxins found in sea anemones.

Antinociception produced by Thalassia testudinum extract BM-21 is mediated by the inhibition of acid sensing ionic channels by the phenolic compound thalassiolin B.

BACKGROUND: Acid-sensing ion channels (ASICs) have a significant role in the sensation of pain and constitute an important target for the search of new antinociceptive drugs. In this work we studied the antinociceptive properties of the BM-21 extract, obtained from the sea grass Thalassia testudinum, in chemical and thermal models of nociception in mice. The action of the BM-21 extract and the major phenolic component isolated from this extract, a sulphated flavone glycoside named thalassiolin B, was studied in the chemical nociception test and in the ASIC currents of the dorsal root ganglion (DRG) neurons obtained from Wistar rats. RESULTS: Behavioral antinociceptive experiments were made on male OF-1 mice. Single oral administration of BM-21 produced a significant inhibition of chemical nociception caused by acetic acid and formalin (specifically during its second phase), and increased the reaction time in the hot plate test. Thalassiolin B reduced the licking behavior during both the phasic and tonic phases in the formalin test. It was also found that BM-21 and thalassiolin B selectively inhibited the fast desensitizing (τ < 400 ms) ASIC currents in DRG neurons obtained from Wistar rats, with a nonsignificant action on ASIC currents with a slow desensitizing time-course. The action of thalassiolin B shows no pH or voltage dependence nor is it modified by steady-state ASIC desensitization or voltage. The high concentration of thalassiolin B in the extract may account for the antinociceptive action of BM-21. CONCLUSIONS: To our knowledge, this is the first report of an ASIC-current inhibitor derived of a marine-plant extract, and in a phenolic compound. The antinociceptive effects of BM-21 and thalassiolin B may be partially because of this action on the ASICs. That the active components of the extract are able to cross the blood-brain barrier gives them an additional advantage for future uses as tools to study pain mechanisms with a potential therapeutic application.

The sea anemone Bunodosoma caissarum toxin BcIII modulates the sodium current kinetics of rat dorsal root ganglia neurons and is displaced in a voltage-dependent manner.

Sea anemone toxins bind to site 3 of the sodium channels, which is partially formed by the extracellular linker connecting S3 and S4 segments of domain IV, slowing down the inactivation process. In this work we have characterized the actions of BcIII, a sea anemone polypeptide toxin isolated from Bunodosoma caissarum, on neuronal sodium currents using the patch clamp technique. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study (n=65). The main effects of BcIII were a concentration-dependent increase in the sodium current inactivation time course (IC(50)=2.8 microM) as well as an increase in the current peak amplitude. BcIII did not modify the voltage at which 50% of the channels are activated or inactivated, nor the reversal potential of sodium current. BcIII shows a voltage-dependent action. A progressive acceleration of sodium current fast inactivation with longer conditioning pulses was observed, which was steeper as more depolarizing were the prepulses. The same was observed for other two anemone toxins (CgNa, from Condylactis gigantea and ATX-II, from Anemonia viridis). These results suggest that the binding affinity of sea anemone toxins may be reduced in a voltage-dependent manner, as has been described for alpha-scorpion toxins.

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 (Na(V)) channels have become the foremost important target of animal toxins. These toxins have developed the ability to discriminate between closely related Na(V) subtypes, making them powerful tools to study Na(V) 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 Na(V) currents in rat dorsal root ganglion neurons. To illuminate the underlying Na(V) subtype-selectivity pattern, we have assayed the effects of CgNa on a broad range of mammalian isoforms (Na(V)1.2-Na(V)1.8) expressed in Xenopus oocytes. This study demonstrates that CgNa selectively slows the fast inactivation of rNa(V)1.3/ß(1), mNa(V)1.6/ß(1) and, to a lesser extent, hNa(V)1.5/ß(1), while the other mammalian isoforms remain unaffected. Importantly, CgNa was also examined on the insect sodium channel DmNa(V)1/tipE, revealing a clear phyla-selectivity in the efficacious actions of the toxin. CgNa strongly inhibits the inactivation of the insect Na(V) 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 Na(V) channel subtypes. Moreover, further structural studies could provide important information on the molecular mechanism of Na(V) channel inactivation.

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 ApC, a sea anemone toxin, on sodium currents of mammalian neurons.

We have characterized the actions of ApC, a sea anemone polypeptide toxin isolated from Anthopleura elegantissima, on neuronal sodium currents (I(Na)) using current and voltage-clamp techniques. Neurons of the dorsal root ganglia of Wistar rats (P5-9) in primary culture were used for this study. These cells express tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) I(Na). In current-clamp experiments, application of ApC increased the average duration of the action potential. Under voltage-clamp conditions, the main effect of ApC was a concentration-dependent increase in the TTX-S I(Na) inactivation time course. No significant effects were observed on the activation time course or on the current peak-amplitude. ApC also produced a hyperpolarizing shift in the voltage at which 50% of the channels are inactivated and caused a significant decrease in the voltage dependence of Na+ channel inactivation. No effects were observed on TTX-R I(Na). Our results suggest that ApC slows the conformational changes required for fast inactivation of the mammalian Na+ channels in a form similar to other site-3 toxins, although with a greater potency than ATX-II, a highly homologous anemone toxin.

A new toxin from the sea anemone Condylactis gigantea with effect on sodium channel inactivation.

A new peptide toxin exhibiting a molecular weight of 5043Da (av.) and comprising 47 amino acid residues was isolated from the sea anemone Condylactis gigantea. Purification of the peptide was achieved by a multistep chromatographic procedure monitoring its strong paralytic activity on crustacea (LD(50) approx. 1microg/kg). Complete sequence analysis of the toxic peptide revealed the isolation of a new member of type I sea anemone sodium channel toxins containing the typical pattern of the six cysteine residues. From 11kg of wet starting material, approximately 1g of the peptide toxin was isolated. The physiological action of the new toxin from C. gigantea CgNa was investigated on sodium currents of rat dorsal root ganglion neurons in culture using whole-cell patch clamp technique (n=60). Under current clamp condition (CgNa) increased action potential duration. This effect is due to slowing down of the TTX-S sodium current inactivation, without modifying the activation process. CgNa prolonged the cardiac action potential duration and enhanced contractile force albeit at 100-fold higher concentrations than the Anemonia sulcata toxin ATXII. The action on sodium channel inactivation and on cardiac excitation-contraction coupling resemble previous results with compounds obtained from this and other sea anemones [Shapiro, B.I., 1968. Purification of a toxin from tentacles of the anemone C. gigantea. Toxicon 5, 253-259; Pelhate, M., Zlotkin, E., 1982. Actions of insect toxin and other toxins derived from the venom of scorpion Androtonus australis on isolated giant axons of the cockroach Periplaneta americana. J. Exp. Biol. 97, 67-77; Salgado, V., Kem, W., 1992. Actions of three structurally distinct sea anemone toxins on crustacean and insect sodium channels. Toxicon 30, 1365-1381; Bruhn, T., Schaller, C., Schulze, C., Sanchez-Rodriquez, J., Dannmeier, C., Ravens, U., Heubach, J.F., Eckhardt, K., Schmidtmayer, J., Schmidt, H., Aneiros, A., Wachter, E., Béress, L., 2001. Isolation and characterization of 5 neurotoxic and cardiotoxic polypeptides from the sea anemone Anthopleura elegantissima. Toxicon, 39, 693-702]. Comprehensive analysis of the purified active fractions suggests that CgNa may represent the main peptide toxin of this sea anemone species.

Bioactive peptides from marine sources: pharmacological properties and isolation procedures.

Marine organisms represent a valuable source of new compounds. The biodiversity of the marine environment and the associated chemical diversity constitute a practically unlimited resource of new active substances in the field of the development of bioactive products. In this paper, the molecular diversity of different marine peptides is described as well as information about their biological properties and mechanisms of action is provided. Moreover, a short review about isolation procedures of selected bioactive marine peptides is offered. Novel peptides from sponges, ascidians, mollusks, sea anemones and seaweeds are presented in association with their pharmacological properties and obtainment methods.

The sea anemone toxins BgII and BgIII prolong the inactivation time course of the tetrodotoxin-sensitive sodium current in rat dorsal root ganglion neurons.

We have characterized the effects of BgII and BgIII, two sea anemone peptides with almost identical sequences (they only differ by a single amino acid), on neuronal sodium currents using the whole-cell patch-clamp technique. Neurons of dorsal root ganglia of Wistar rats (P5-9) in primary culture (Leibovitz's L15 medium; 37 degrees C, 95% air/5% CO2) were used for this study (n = 154). These cells express two sodium current subtypes: tetrodotoxin-sensitive (TTX-S; K(i) = 0.3 nM) and tetrodotoxin-resistant (TTX-R; K(i) = 100 microM). Neither BgII nor BgIII had significant effects on TTX-R sodium current. Both BgII and BgIII produced a concentration-dependent slowing of the TTX-S sodium current inactivation (IC50 = 4.1 +/- 1.2 and 11.9 +/- 1.4 microM, respectively), with no significant effects on activation time course or current peak amplitude. For comparison, the concentration-dependent action of Anemonia sulcata toxin II (ATX-II), a well characterized anemone toxin, on the TTX-S current was also studied. ATX-II also produced a slowing of the TTX-S sodium current inactivation, with an IC50 value of 9.6 +/- 1.2 microM indicating that BgII was 2.3 times more potent than ATX-II and 2.9 times more potent than BgIII in decreasing the inactivation time constant (tau(h)) of the sodium current in dorsal root ganglion neurons. The action of BgIII was voltage-dependent, with significant effects at voltages below -10 mV. Our results suggest that BgII and BgIII affect voltage-gated sodium channels in a similar fashion to other sea anemone toxins and alpha-scorpion toxins.