Revisiting cangitoxin, a sea anemone peptide: purification and characterization of cangitoxins II and III from the venom of Bunodosoma cangicum.

Sodium channel toxins from sea anemones are employed as tools for dissecting the biophysical properties of inactivation in voltage-gated sodium channels. Cangitoxin (CGTX) is a peptide containing 48 amino acid residues and was formerly purified from Bunodosoma cangicum. Nevertheless, previous works reporting the isolation procedures for such peptide from B. cangicum secretions are controversial and may lead to incorrect information. In this paper, we report a simple and rapid procedure, consisting of two chromatographic steps, in order to obtain a CGTX analog directly from sea anemone venom. We also report a substitution of N16D in this peptide sample and the co-elution of an inseparable minor isoform presenting the R14H substitution. Peptides are named as CGTX-II and CGTX-III, and their effects over Nav1.1 channels in patch clamp experiments are demonstrated.

Caissarolysin I (Bcs I), a new hemolytic toxin from the Brazilian sea anemone Bunodosoma caissarum: purification and biological characterization.

Two cationic proteins, C1 and C3, were purified to homogeneity from the hemolytic fraction of the venom of Bunodosoma caissarum sea anemone. The purification processes employed gel filtration followed by ion exchange chromatography, being the purity and molecular mass confirmed by SDS-PAGE and mass spectrometry. Protein C1 represented the second major peak of the hemolytic fraction and was previously believed to be a cytolysin belonging to a new class of hemolysins. The C1 protein has a molecular mass of 15495 Da and was assayed for hemolysis, PLA2 activity and acute toxicity in crabs and mice, showing no activity in these assays. It has an amino terminal with no similarity to all known hemolysins and, therefore, should not be considered a toxin, being its function completely unknown. The protein C3 (19757 Da), that also lacks PLA2 activity, was recognized by antiserum against Eqt II and presented high hemolytic activity to human erythrocytes (ED50 of 0.270 microg/ml), being named Caissarolysin I (Bcs I). Its activity was inhibited by pre-incubation with sphingomyelin (SM) and also when in presence of erythrocytes pre-treated with the SMase P2, a phospholipase D from the brown spider Loxosceles intermedia, indicating that SM is the main target of Bcs I. Caissarolysin I is the first hemolysin purified from a sea anemone belonging to the genus Bunodosoma and belongs to the Actinoporin family of sea anemone hemolysins.

BcIV, a new paralyzing peptide obtained from the venom of the sea anemone Bunodosoma caissarum. A comparison with the Na+ channel toxin BcIII.

Sea anemones produce a wide variety of biologically active compounds, such as the proteinaceous neurotoxins and cytolysins. Herein we report a new peptide, purified to homogeneity from the neurotoxic fraction of B. caissarum venom, by using gel filtration followed by rp-HPLC, naming it as BcIV. BcIV is a 41 amino acid peptide (molecular mass of 4669 amu) possessing 6 cysteines covalently linked by three disulfide bonds. This toxin has 45 and 48% of identity when compared to APETx1 and APETx2 from Anthopleura elegantissima, respectively, and 42% of identity with Am-II and BDS-I and-II obtained from Antheopsis maculata and Anemonia sulcata, respectively. This neurotoxin presents only a weak-paralyzing action (minimal Lethal Dose close to 2000 microg/kg) in swimming crabs Callinectes danae. This appears to be a different effect to that caused by the type 1 sea anemone toxin BcIII that is lethal to the same animals at lower doses (LD50=219 microg/kg). Circular dichroism spectra of BcIII and BcIV show a high content of beta-strand secondary structure in both peptides, very similar to type 1 sodium channel toxins from various sea anemones, and to APETx1 and APETx2 from A. elegantissima, a HERG channel modulator and an ASIC3 inhibitor, respectively. Interestingly, BcIII and BcIV have similar effects on the action potential of the crab leg nerves, suggesting the same target in this tissue. As BcIII was previously reported as a Na+ channel effector and BcIV is inactive over Na+ currents of mammalian GH3 cells, we propose a species-specific action for this new molecule. A molecular model of BcIV was constructed using the structure of the APETx1 as template and putative key residues are discussed.

Decoralin, a novel linear cationic alpha-helical peptide from the venom of the solitary eumenine wasp Oreumenes decoratus.

A novel peptide, decoralin, was isolated from the venom of the solitary eumenine wasp Oreumenes decoratus. Its sequence, Ser-Leu-Leu-Ser-Leu-Ile-Arg-Lys-Leu-Ile-Thr, was determined by Edman degradation and corroborated by solid-phase synthesis. This sequence has the characteristic features of linear cationic alpha-helical peptides; rich in hydrophobic and basic amino acids with no disulfide bond, and accordingly, it can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of decoralin in the presence of TFE or SDS showed a high alpha-helical conformation content. In a biological evaluation, decoralin exhibited a significant broad-spectrum antimicrobial activity, and moderate mast cell degranulation and leishmanicidal activities, but showed virtually no hemolytic activity. A synthetic analog with C-terminal amidation showed a much more potent activity in all the biological assays.

Eumenitin, a novel antimicrobial peptide from the venom of the solitary eumenine wasp Eumenes rubronotatus.

A novel antimicrobial peptide, eumenitin, was isolated from the venom of the solitary eumenine wasp Eumenes rubronotatus. The sequence of eumenitin, Leu-Asn-Leu-Lys-Gly-Ile-Phe-Lys-Lys-Val-Ala-Ser-Leu-Leu-Thr, was mostly analyzed by mass spectrometry together with Edman degradation, and corroborated by solid-phase synthesis. This peptide has characteristic features of cationic linear alpha-helical antimicrobial peptides, and therefore, can be predicted to adopt an amphipathic alpha-helix secondary structure. In fact, the CD spectra of eumenitin in the presence of TFE or SDS showed a high content of alpha-helical conformation. Eumenitin exhibited inhibitory activity against both Gram-positive and Gram-negative bacteria, and moderately stimulated degranulation from the rat peritoneal mast cells and the RBL-2H3 cells, but showed no hemolytic activity against human erythrocytes. This antimicrobial peptide in the eumenine wasp venom may play a role in preventing potential infection by microorganisms during prey consumption by their larvae.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish.

Toxicity and toxin identification in Colomesus asellus, an Amazonian (Brazil) freshwater puffer fish. By using four different techniques--mouse bioassay, ELISA, HPLC and mass spectrometry-we evaluated the toxicity in the extracts of C. asellus, a freshwater puffer fish from the rivers of the Amazon, and identified for the first time the components responsible for its toxicity. The T20G10 monoclonal antibody raised against TTX, and employed in an indirect competitive enzyme immunoassay, showed very low affinity for the C. asellus extracts, indicating that TTX and its analogs are not the main toxic components of the extracts. This antibody was efficient in detecting presence of TTX in a total extract of Sphoeroides spengleri, which is one of the most toxic puffer fish found in the Atlantic coast. Extracts of C. asellus were toxic when administered intraperitonially into mice with an average toxicity of 38.6+/-12 mouse unit (MU)/g, while HPLC analysis indicated a lower toxin content (7.6+/-0 5MU/g). The HPLC profile showed no traces of TTX, but only the presence of PSPs (STX, GTX 2 and GTX 3). These toxins were also confirmed by electrospray ionization mass spectrometry.

Envenomation caused by Rhopalurus amazonicus Lourenço, 1986 (Scorpiones, Buthidae) in Pará State, Brazil.

Scorpions, mainly those belonging to the genus Tityus cause many deaths and injuries in Brazil, with tens of thousands of envenomations notified every year. However, injuries involving other scorpion species are scarcely registered. Among the sixteen species of the genus Rhopalurus, Thorell, 1876, described up to date, nine are found in this country, with only a confirmed case of human envenomation provoked by R. agamemnon Koch, 1839. The present case reports, for the first time, a case of scorpion sting in a human victim involving Rhopalurus amazonicus, endemic species of the west region of the Pará state, Amazon, Brazil. The symptoms of envenomation were local pain and paresthesia. This study contributes to develop the knowledge on venomous scorpions, particularly those that may cause envenomations in this region.

The proteomic profile of Stichodactyla duerdeni secretion reveals the presence of a novel O-linked glycopeptide.

Sea anemones represent one of the emerging groups of interest concerning venomous animals in toxinology and the goal of the present work was the prospection, and the structural and functional characterization of the compounds present in the secretion of the sea anemone Stichodactyla duerdeni from Brazilian coast. We used a combination of offline RPC-MALDI-TOF and online nano-RPC-ESI-LTQ-Orbitrap proteomic techniques as well as functional bioassays. The mucus was milked by electric stimulation and fractionated by gel filtration on Sephadex G-50 yielding 5 main fractions. The low molecular weight fractions were further submitted to RP-HPLC resulting in 35 new subfractions that were subsequently analyzed by offline MALDI-TOF mass spectrometry. MALDI peptide mass fingerprinting yielded up to 134 different molecular masses, ranging from m/z 901 to 10,833. Among these subfractions, a new peptide of 3431Da, named U-SHTX-Sdd1, was purified and completely sequenced by automated Edman's degradation and tandem mass spectrometry. An analysis of U-SHTX-Sdd1 revealed a modified O-HexNAc-Threonine at position 1, which, at the best of our knowledge, constitutes the first sea anemone toxin reported with such post-translational modification. Because of its sequence similarity with other sea anemone toxins, the pharmacological activity of U-SHTX-Sdd1 was assessed by electrophysiological measurements using the two electrode voltage-clamp technique on cloned voltage-gated potassium channel subtypes, expressed in Xenopus laevis oocytes. However, U-SHTX-Sdd1 did not show activity on these channels. A large-scale proteomic approach was also employed to shed lights on the sea anemone compounds, and a total 67 proteins and peptides were identified. BIOLOGICAL SIGNIFICANCE: In this manuscript, we report an extensive characterization of S. duerdeni secretion by means of peptide mass fingerprinting and high-throughput proteome analyses. Also, we report the structure of a new glycopeptide by a combination of biochemical techniques. Despite the previous studies that described proteinaceous compounds present in sea anemone secretions, the number of reported primary sequences is still low. Thus, to access the scenery of protein components from S. duerdeni mucus, including their biological functions, a robust proteomic approach was used together with bioinformatic tools. The demonstrated strategy of analysis is perfectly suitable to other sea anemone secretions and animal venoms. Moreover, new peptide structures can arise contributing to the knowledge of the diversity of these animal peptides.

Development of a rational nomenclature for naming peptide and protein toxins from sea anemones.

Sea anemone toxins are predominantly peptide and proteins that act mainly on sodium and potassium channels, as well as in a variety of target cells causing lysis. Over recent years, the number of sea anemone peptide toxins as well as cytolytic pore-forming proteins and phospholipase A(2) sequences submitted to databases has been rapidly increasing due to the developments in DNA sequencing technology and proteomic approaches. However, the lack of a systematic nomenclature has resulted in multiple names being assigned to the same toxins, toxins from unrelated species being designated by the same name, and ambiguous name designations. Therefore, in this work we propose a systematic nomenclature in which we adopted specific criteria, based on order of discovery and phylogenetic analysis, in order to avoid redundant sea anemone toxin names. Implementation of the nomenclature proposed here not only allowed us to rename the already published 191 anemone toxins without ambiguities, but it can be used to unambiguously name newly discovered toxins whether or not they are related to previously published sea anemone sequences. In the new nomenclature each toxin name contains information about the toxin's biological activity, origin and relationship to known isoforms. Ongoing increases in the speed of DNA sequencing will raise significantly the number of sea anemone toxin sequences in the literature. This will represent a constant challenge in their clear identification and logical classification, which could be solved using the proposed nomenclature.

Envenomation caused by Rhopalurus amazonicus Lourenço, 1986 (Scorpiones, Buthidae) in Pará State, Brazil

Scorpions, mainly those belonging to the genus Tityus cause many deaths and injuries in Brazil, with tens of thousands of envenomations notified every year. However, injuries involving other scorpion species are scarcely registered. Among the sixteen species of the genus Rhopalurus, Thorell, 1876, described up to date, nine are found in this country, with only a confirmed case of human envenomation provoked by R. agamemnonKoch, 1839. The present case reports, for the first time, a case of scorpion sting in a human victim involving Rhopalurus amazonicus, endemic species of the west region of the Pará state, Amazon, Brazil. The symptoms of envenomation were local pain and paresthesia. This study contributes to develop the knowledge on venomous scorpions, particularly those that may cause envenomations in this region.

Envenomation caused by Rhopalurus amazonicus Lourenço, 1986 (Scorpiones, Buthidae) in Pará State, Brazil

Scorpions, mainly those belonging to the genus Tityus cause many deaths and injuries in Brazil, with tens of thousands of envenomations notified every year. However, injuries involving other scorpion species are scarcely registered. Among the sixteen species of the genus Rhopalurus, Thorell, 1876, described up to date, nine are found in this country, with only a confirmed case of human envenomation provoked by R. agamemnonKoch, 1839. The present case reports, for the first time, a case of scorpion sting in a human victim involving Rhopalurus amazonicus, endemic species of the west region of the Pará state, Amazon, Brazil. The symptoms of envenomation were local pain and paresthesia. This study contributes to develop the knowledge on venomous scorpions, particularly those that may cause envenomations in this region.(AU)

Proteomics of the neurotoxic fraction from the sea anemone Bunodosoma cangicum venom: Novel peptides belonging to new classes of toxins.

In contrast to the many studies on the venoms of scorpions, spiders, snakes and cone snails, up to now there has been no report of the proteomic analysis of sea anemones venoms. In this work we report for the first time the peptide mass fingerprint and some novel peptides in the neurotoxic fraction (Fr III) of the sea anemone Bunodosoma cangicum venom. Fr III is neurotoxic to crabs and was purified by rp-HPLC in a C-18 column, yielding 41 fractions. By checking their molecular masses by ESI-Q-Tof and MALDI-Tof MS we found 81 components ranging from near 250 amu to approximately 6000 amu. Some of the peptidic molecules were partially sequenced through the automated Edman technique. Three of them are peptides with near 4500 amu belonging to the class of the BcIV, BDS-I, BDS-II, APETx1, APETx2 and Am-II toxins. Another three peptides represent a novel group of toxins (~3200 amu). A further three molecules (~ approximately 4900 amu) belong to the group of type 1 sodium channel neurotoxins. When assayed over the crab leg nerve compound action potentials, one of the BcIV- and APETx-like peptides exhibits an action similar to the type 1 sodium channel toxins in this preparation, suggesting the same target in this assay. On the other hand one of the novel peptides, with 3176 amu, displayed an action similar to potassium channel blockage in this experiment. In summary, the proteomic analysis and mass fingerprint of fractions from sea anemone venoms through MS are valuable tools, allowing us to rapidly predict the occurrence of different groups of toxins and facilitating the search and characterization of novel molecules without the need of full characterization of individual components by broader assays and bioassay-guided purifications. It also shows that sea anemones employ dozens of components for prey capture and defense.

Binding specificity of sea anemone toxins to Nav 1.1-1.6 sodium channels: unexpected contributions from differences in the IV/S3-S4 outer loop.

Sea anemones are an important source of various biologically active peptides, and it is known that ATX-II from Anemonia sulcata slows sodium current inactivation. Using six different sodium channel genes (from Nav1.1 to Nav1.6), we investigated the differential selectivity of the toxins AFT-II (purified from Anthopleura fuscoviridis) and Bc-III (purified from Bunodosoma caissarum) and compared their effects with those recorded in the presence of ATX-II. Interestingly, ATX-II and AFT-II differ by only one amino acid (L36A) and Bc-III has 70% similarity. The three toxins induced a low voltage-activated persistent component primarily in the Nav1.3 and Nav1.6 channels. An analysis showed that the 18 dose-response curves only partially fit the hypothesized binding of Lys-37 (sea anemone toxin Anthopleurin B) to the Asp (or Glu) residue of the extracellular IV/S3-S4 loop in cardiac (or nervous) Na+ channels, thus suggesting the substantial contribution of some nearby amino acids that are different in the various channels. As these channels are atypically expressed in mammalian tissues, the data not only suggest that the toxicity is highly dependent on the channel type but also that these toxins and their various physiological effects should be considered prototype models for the design of new and specific pharmacological tools.