Urticaria induced by ingestion of anemonia sulcata

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Occupational asthma caused by actinia equina

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Animal Toxins Providing Insights into TRPV1 Activation Mechanism.

Beyond providing evolutionary advantages, venoms offer unique research tools, as they were developed to target functionally important proteins and pathways. As a key pain receptor in the nociceptive pathway, transient receptor potential vanilloid 1 (TRPV1) of the TRP superfamily has been shown to be a target for several toxins, as a way of producing pain to deter predators. Importantly, TRPV1 is involved in thermoregulation, inflammation, and acute nociception. As such, toxins provide tools to understand TRPV1 activation and modulation, a critical step in advancing pain research and the development of novel analgesics. Indeed, the phytotoxin capsaicin, which is the spicy chemical in chili peppers, was invaluable in the original cloning and characterization of TRPV1. The unique properties of each subsequently characterized toxin have continued to advance our understanding of functional, structural, and biophysical characteristics of TRPV1. By building on previous reviews, this work aims to provide a comprehensive summary of the advancements made in TRPV1 research in recent years by employing animal toxins, in particular DkTx, RhTx, BmP01, Echis coloratus toxins, APHCs and HCRG21. We examine each toxin's functional aspects, behavioral effects, and structural features, all of which have contributed to our current knowledge of TRPV1. We additionally discuss the key features of TRPV1's outer pore domain, which proves to be the target of the currently discussed toxins.

Defensin-neurotoxin dyad in a basally branching metazoan sea anemone.

Recent studies suggest that vertebrate and invertebrate defensins have evolved from two independent ancestors, and that both defensins could share origins with animal toxins. Here, we purified novel sea anemone neurotoxin (BDS)-like antimicrobial peptides (AMPs)-Crassicorin-I and its putative homolog (Crassicorin-II)-from the pharynx extract of an anthozoan sea anemone (Urticina crassicornis). Based on structural analyses and cDNA cloning, mature Crassicorin-I represents a cationic AMP likely generated from a precursor and comprising 40 amino acid residues, including six cysteines forming three intramolecular disulfide bonds. Recombinant Crassicorin-I produced in a heterologous bacterial-expression system displayed antimicrobial activity against both a gram-positive bacterium (Bacillus subtilis) and gram-negative bacteria (Escherichia coli and Salmonella enterica). The Crassicorin-I transcript was upregulated by immune challenge, suggesting its involvement in defense mechanisms against infectious pathogens in sea anemone. Sequence alignment and three-dimensional molecular modeling revealed that Crassicorin-I exhibits high degrees of structural similarity to sea anemone neurotoxins that share ß-defensin fold which is found in vertebrate defensins and invertebrate big-defensins. Consistent with its structural similarity to neurotoxins, Crassicorin-I exhibited paralytic activity toward a crustacean. These findings motivated our investigation and subsequent discovery of antimicrobial activity from other known sea anemone neurotoxins, such as APETx1 and ShK. Collectively, our work signified that Crassicorin-I is the first AMP identified from a sea anemone and provided evidence of a functional linkage between AMPs and neurotoxins in a basally branching metazoan.

Revisiting venom of the sea anemone Stichodactyla haddoni: Omics techniques reveal the complete toxin arsenal of a well-studied sea anemone genus.

More than a century of research on sea anemone venoms has shown that they contain a diversity of biologically active proteins and peptides. However, recent omics studies have revealed that much of the venom proteome remains unexplored. We used, for the first time, a combination of proteomic and transcriptomic techniques to obtain a holistic overview of the venom arsenal of the well-studied sea anemone Stichodactyla haddoni. A purely search-based approach to identify putative toxins in a transcriptome from tentacles regenerating after venom extraction identified 508 unique toxin-like transcripts grouped into 63 families. However, proteomic analysis of venom revealed that 52 of these toxin families are likely false positives. In contrast, the combination of transcriptomic and proteomic data enabled positive identification of 23 families of putative toxins, 12 of which have no homology known proteins or peptides. Our data highlight the importance of using proteomics of milked venom to correctly identify venom proteins/peptides, both known and novel, while minimizing false positive identifications from non-toxin homologues identified in transcriptomes of venom-producing tissues. This work lays the foundation for uncovering the role of individual toxins in sea anemone venom and how they contribute to the envenomation of prey, predators, and competitors. BIOLOGICAL SIGNIFICANCE: Proteomic analysis of milked venom combined with analysis of a tentacle transcriptome revealed the full extent of the venom arsenal of the sea anemone Stichodactyla haddoni. This combined approach led to the discovery of 12 entirely new families of disulfide-rich peptides and proteins in a genus of anemones that have been studied for over a century.

Damage of eukaryotic cells by the pore-forming toxin sticholysin II: Consequences of the potassium efflux.

Pore-forming toxins (PFTs) form holes in membranes causing one of the most catastrophic damages to a target cell. Target organisms have evolved a regulated response against PFTs damage including cell membrane repair. This ability of cells strongly depends on the toxin concentration and the properties of the pores. It has been hypothesized that there is an inverse correlation between the size of the pores and the time required to repair the membrane, which has been for long a non-intuitive concept and far to be completely understood. Moreover, there is a lack of information about how cells react to the injury triggered by eukaryotic PFTs. Here, we investigated some molecular events related with eukaryotic cells response against the membrane damage caused by sticholysin II (StII), a eukaryotic PFT produced by a sea anemone. We evaluated the change in the cytoplasmic potassium, identified the main MAPK pathways activated after pore-formation by StII, and compared its effect with those from two well-studied bacterial PFTs: aerolysin and listeriolysin O (LLO). Strikingly, we found that membrane recovery upon StII damage takes place in a time scale similar to LLO in spite of the fact that they form pores by far different in size. Furthermore, our data support a common role of the potassium ion, as well as MAPKs in the mechanism that cells use to cope with these toxins injury.

Pore formation: an ancient yet complex form of attack.

Bacteria, as well as higher organisms such as sea anemones or earthworms, have developed sophisticated virulence factors such as the pore-forming toxins (PFTs) to mount their attack against the host. One of the most fascinating aspects of PFTs is that they can adopt a water-soluble form at the beginning of their lifetime and become an integral transmembrane protein in the membrane of the target cells. There is a growing understanding of the sequence of events and the various conformational changes undergone by these toxins in order to bind to the host cell surface, to penetrate the cell membranes and to achieve pore formation. These points will be addressed in this review.

Model peptides mimic the structure and function of the N-terminus of the pore-forming toxin sticholysin II.

To investigate the role of the N-terminal region in the lytic mechanism of the pore-forming toxin sticholysin II (St II), we studied the conformational and functional properties of peptides encompassing the first 30 residues of the protein. Peptides containing residues 1-30 (P1-30) and 11-30 (P11-30) were synthesized and their conformational properties were examined in aqueous solution as a function of peptide concentration, pH, ionic strength, and addition of the secondary structure-inducing solvent trifluoroethanol (TFE). CD spectra showed that increasing concentration, pH, and ionic strength led to aggregation of P1-30; as a consequence, the peptide acquired beta-sheet conformation. In contrast, P11-30 exhibited practically no conformational changes under the same conditions, remaining essentially structureless. Moreover, this peptide did not undergo aggregation. These differences clearly point to the modulating effect of the first 10 hydrophobic residues on the peptides aggregation and conformational properties. In TFE both the first ten hydrophobic peptides acquired alpha-helical conformation, albeit to a different extent, P11-30 displayed lower alpha-helical content. P1-30 presented a larger fraction of residues in alpha-helical conformation in TFE than that found in St II's crystal structure for that portion of the protein. Since TFE mimics the membrane environment, such increase in helical content could also occur upon toxin binding to membranes and represent a step in the mechanism of pore formation. The peptides conformational properties correlated well with their functional behavior. Thus, P1-30 exhibited much higher hemolytic activity than P11-30. In addition, P11-30 was able to block the toxin's hemolytic activity. The size of pores formed in red blood cells by P1-30 was estimated by measuring the permeability to PEGs of different molecular mass. The pore radius (0.95 +/- 0.01 nm) was very similar to that of the pore formed by the toxin. The results demonstrate that the synthetic peptide P1-30 is a good model of St II conformation and function and emphasize the contribution of the toxin's N-terminal region, and, in particular, the hydrophobic residues 1-10 to pore formation.

Effects of recombinant Sagartia rosea cytolysin and Lapemis hardwickii phospholipase A2 on adventitial fibroblasts proliferation.

OBJECTIVE: To observe the effects of recombinant Sagartia rosea cytolysin (rSrc) and Lapemis hardwickii phospholipase A2(rPLA2) on adventitial fibroblasts proliferation. METHODS: NIH-3T3 cells were cultured and treated with rSrc and rPLA2 at different concentrations for observation of cell proliferation using non-radioactive MTS/PES assay in comparison with the control group. RESULTS: The ratio of cell proliferation was 0.840+/-0.061 in the control group, and was 0.263+/-0.044, 0.418+/-0.054, 0.605+/-0.063, 0.772+/-0.054 and 0.906+/-0.072 in rSrc groups corresponding to rSrc concentrations of 100, 10, 1 microg/ml and 100, 10 ng/ml respectively. rSrc was found to significantly inhibit fibroblast proliferation in a dose-dependent manner when the concentration used was above 1 microg/ml (P<0.05), as compared with the control group (P<0.05). The ratio of cell proliferation was 0.498+/-0.076, 0.937+/-0.112 and 0.978+/-0.145 in rPLA2 groups corresponding to rPLA2 concentrations of 100, 10, 1 microg/ml respectively, indicating that rPLA2 also significantly inhibited fibroblast proliferation at the concentration of 100 microg/ml (P<0.05). CONCLUSION: rSrc and rPLA2 can both significantly inhibit adventitial fibroblast proliferation.

Properties of St I and St II, two isotoxins isolated from Stichodactyla helianthus: a comparison.

Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins. Exposure of the toxins to high pHs (>11), a free radical source (AAPH), urea or temperature produce permanent changes in the toxin that lead to a significant loss of HA. It is significant to note that this loss of hemolytic activity occurs when other indicators, probably with the only exception of near-UV CD spectra, barely detect changes in the protein structure. This emphasizes the sensitivity of the protein function to changes in the macromolecule conformation. The most noticeable difference between both toxins is the considerably higher activity of St II, both measured in terms of erythrocyte internal K(+) exit or hemolysis; which is related to enthalpic factors. This difference is not due to an incomplete association of St I to the membrane. We consider then that the different pore forming capacity of both toxins in erythrocytes can be explained in terms of the difference in charge of the N-terminal fragment, than can considerably reduce the St I insertion rate in the membrane probably due to the negatively charged outer leaflet of the red blood cell, without a significant reduction of its capacity to bind to the cell membrane. This electrostatic effect, together with a slightly more relaxed structure in St II, could explain the higher pore forming capacity of St II in the red blood cell membrane.

Effect of pH on the conformation, interaction with membranes and hemolytic activity of sticholysin II, a pore forming cytolysin from the sea anemone Stichodactyla helianthus.

Sticholysin II (St II) is a pore forming cytolysin obtained from the sea anemone Stichodactyla helianthus. Incubation of diluted St II solutions at different pHs (ranging from 2.0 to 12) slightly changes the secondary structure of the protein. These changes are particularly manifested at high pH. Similarly, the intrinsic fluorescence of the protein indicates a progressive opening of the protein structure when the pH increases from acidic (2.0) to basic (12). These modifications are only partially reversible and do not produce any significant increase in the small capacity of the protein to bind hydrophobic dyes (ANS or Prodan). Experiments carried out with model membranes show a reduced capacity of binding to egg phosphatidyl choline:sphingomyelin (1:1) liposomes both at low (2.3) and high (11.5) pH. Preincubation of the protein in the 2. 5-9.0 pH range does not modify its hemolytic activity, measured in human red blood cells at pH 7.4. On the other hand, preincubation at pH 11.5 drastically reduces the hemolytic activity of the toxin. This strong reduction takes place without measurable modification of the toxin ability to be adsorbed to the red blood cell surface. This indicates that preincubation at high pH irreversibly reduces the capacity of the toxin to form pores without a significant decrease in its binding capacity. The present results suggest that at pH > or = 10 St II experiences irreversible conformational changes that notably reduce its biological activity. This reduced biological activity is associated with a partial defolding of the protein, which seems to contradict what is expected in terms of a molten globule formalism.