RESUMO
Subtilisin-like enzymes are recognized as key players in many infectious agents. In this context, its inhibitors are very valuable molecular lead compounds for structure based drug discovery and design. Marine invertebrates offer a great source of bioactive molecules, including protease inhibitors. In this work, we describe a new subtilisin inhibitor, from the sea anemone Condylactis gigantea (CogiTx1). CogiTx1 was purified using a combination of cation exchange chromatography, size exclusion chromatography and RP-HPLC chromatography. CogiTx1 it is a protein with 46 amino acid residues, with 4970.44 Da and three disulfide bridges. Is also able to inhibit subtilisin-like enzymes and pancreatic elastase. According to the amino acid sequence, it belongs to the defensin 4 family of proteins. The sequencing showed that CogiTx1 has an amidated C-terminal end, which was confirmed by the presence of the typical -XGR signal for amidation in the protein sequence deduced from the cDNA. This modification was described at protein level for the first time in this family of proteins. CogiTx1 is the first subtilisin inhibitor from the defensin 4 family and accordingly it has a folding consisting primarily in beta-strands in agreement with the analysis by CD and 3D modelling. Therefore, future in-depth functional studies may allow a more detailed characterization and will shed light on structure-function properties.
Assuntos
Anêmonas-do-Mar , Animais , Anêmonas-do-Mar/química , Anêmonas-do-Mar/metabolismo , Subtilisinas/metabolismo , Sequência de Aminoácidos , Inibidores de Proteases/metabolismo , Defensinas/genética , Defensinas/farmacologiaRESUMO
Actinoporins have emerged as archetypal α-pore-forming toxins (PFTs) that promote the formation of pores in membranes upon oligomerization and insertion of an α-helix pore-forming domain in the bilayer. These proteins have been used as active components of immunotoxins, therefore, understanding their lytic mechanism is crucial for developing this and other applications. However, the mechanism of how the biophysical properties of the membrane modulate the properties of pores generated by actinoporins remains unclear. Here we studied the effect of membrane fluidity on the permeabilizing activity of sticholysin I (St I), a toxin that belongs to the actinoporins family of α-PFTs. To modulate membrane fluidity we used vesicles made of an equimolar mixture of phosphatidylcholine (PC) and egg sphingomyelin (eggSM), in which PC contained fatty acids of different acyl chain lengths and degrees of unsaturation. Our detailed single-vesicle analysis revealed that when membrane fluidity is high, most of the vesicles are partially permeabilized in a graded manner. In contrast, more rigid membranes can be either completely permeabilized or not, indicating an all-or-none mechanism. Altogether, our results reveal that St I pores can be heterogeneous in size and stability, and that these properties depend on the fluid state of the lipid bilayer. We propose that membrane fluidity at different regions of cellular membranes is a key factor to modulate the activity of the actinoporins, which has implications for the design of different therapeutic strategies based on their lytic action.
Assuntos
Venenos de Cnidários , Anêmonas-do-Mar , Animais , Fluidez de Membrana , Compostos Orgânicos/química , Bicamadas Lipídicas , Membrana Celular/metabolismo , Fosfatidilcolinas , Venenos de Cnidários/química , Anêmonas-do-Mar/químicaRESUMO
Sea anemones produce venoms characterized by a complex mixture of low molecular weight compounds, proteins and peptides acting on voltage-gated ion channels. Mammal sperm cells, like neurons, are characterized by their ion channels. Calcium channels seem to be implicated in pivotal roles such as motility and capacitation. In this study, we evaluated the effect of a low molecular weight fraction from the venom of the sea anemone Lebrunia neglecta on boar sperm cells and in HVA calcium channels from rat chromaffin cells. Spermatozoa viability seemed unaffected by the fraction whereas motility and sperm capacitation were notoriously impaired. The sea anemone fraction inhibited the HVA calcium current with partial recovery and no changes in chromaffin cells' current kinetics and current-voltage relationship. These findings might be relevant to the pharmacological characterization of cnidarian venoms and toxins on voltage-gated calcium channels.
Assuntos
Venenos de Cnidários , Hidrozoários , Anêmonas-do-Mar , Animais , Canais de Cálcio/metabolismo , Venenos de Cnidários/química , Masculino , Ratos , Anêmonas-do-Mar/química , Espermatozoides , SuínosRESUMO
Actinoporins (APs) are soluble pore-forming proteins secreted by sea anemones that experience conformational changes originating in pores in the membranes that can lead to cell death. The processes involved in the binding and pore-formation of members of this protein family have been deeply examined in recent years; however, the intracellular responses to APs are only beginning to be understood. Unlike pore formers of bacterial origin, whose intracellular impact has been studied in more detail, currently, we only have knowledge of a few poorly integrated elements of the APs' intracellular action. In this review, we present and discuss an updated landscape of the studies aimed at understanding the intracellular pathways triggered in response to APs attack with particular reference to sticholysin II, the most active isoform produced by the Caribbean Sea anemone Stichodactyla helianthus. To achieve this, we first describe the major alterations these cytolysins elicit on simpler cells, such as non-nucleated mammalian erythrocytes, and then onto more complex eukaryotic cells, including tumor cells. This understanding has provided the basis for the development of novel applications of sticholysins such as the construction of immunotoxins directed against undesirable cells, such as tumor cells, and the design of a cancer vaccine platform. These are among the most interesting potential uses for the members of this toxin family that have been carried out in our laboratory.
Assuntos
Morte Celular/efeitos dos fármacos , Venenos de Cnidários/metabolismo , Venenos de Cnidários/toxicidade , Imunotoxinas/química , Imunotoxinas/metabolismo , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anêmonas-do-Mar/química , AnimaisRESUMO
Sea anemones of the genus Bunodosoma possess along their body column, longitudinally arranged brown-colored vesicles. We have shown that in B. cangicum, these warty structures contain a mixture of potent toxins. This work highlights the neuro-inhibitory effects exhibited by two decapod crustacean species exposed to the extracts from these vesicles. For this, we use the unrefined toxin in doses, exposure times, and different exposure pathways. The findings show that at least one neuro-inhibitory compound is present and remains active regardless of the exposure method or dose tested. This toxin affects neuro-motor pathways but not neuro-sensory pathways. Shrimp exposed to toxin could continue to perceive and track food pellets but could not secure and consume their ration. Of six anatomical reflexes tested under the toxin's influence, voluntary movements of the mouthparts were impacted most commonly. Interestingly, all subject animals recovered from the toxin exposure within 2 h. Finally, we propose Reflexive Action Analysis (RAMP) as a tool to evaluate the potency of other neurotoxic or neuro-inhibitory compounds in crustacea. This work is the first to show the neuro-inhibitory activity of extracts from these sea anemone columnar vesicle structures and the first to evaluate these effects using RAMP reflex analysis.
Assuntos
Comportamento Animal/efeitos dos fármacos , Braquiúros/efeitos dos fármacos , Venenos de Cnidários/toxicidade , Palaemonidae/efeitos dos fármacos , Anêmonas-do-Mar/química , Animais , Braquiúros/fisiologia , Água Doce/química , Monitorização Neurofisiológica/métodos , Palaemonidae/fisiologia , Anêmonas-do-Mar/metabolismo , Alimentos MarinhosRESUMO
BACKGROUND: Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina. OBJECTIVE: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. METHODS: In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed. RESULTS: We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography. CONCLUSION: Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.
Assuntos
Corantes/química , Proteínas Luminescentes/química , Pigmentos Biológicos/química , Proteínas/química , Anêmonas-do-Mar/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cor , Corantes/metabolismo , Escherichia coli/metabolismo , Expressão Gênica , Concentração de Íons de Hidrogênio , Luz , Proteínas Luminescentes/metabolismo , Modelos Moleculares , Oxirredução , Pigmentos Biológicos/metabolismo , Conformação Proteica , Desnaturação Proteica , Proteínas/metabolismo , Espectrofotometria , TemperaturaRESUMO
Voltage-gated potassium (KV) channels regulate diverse physiological processes and are an important target for developing novel therapeutic approaches. Sea anemone (Cnidaria, Anthozoa) venoms comprise a highly complex mixture of peptide toxins with diverse and selective pharmacology on KV channels. From the nematocysts of the sea anemone Actinia bermudensis, a peptide that we named AbeTx1 was purified and functionally characterized on 12 different subtypes of KV channels (KV1.1â»KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; KV11.1; and, Shaker IR), and three voltage-gated sodium channel isoforms (NaV1.2, NaV1.4, and BgNaV). AbeTx1 was selective for Shaker-related K⺠channels and is capable of inhibiting K⺠currents, not only by blocking the K⺠current of KV1.2 subtype, but by altering the energetics of activation of KV1.1 and KV1.6. Moreover, experiments using six synthetic alanine point-mutated analogs further showed that a ring of basic amino acids acts as a multipoint interaction for the binding of the toxin to the channel. The AbeTx1 primary sequence is composed of 17 amino acids with a high proportion of lysines and arginines, including two disulfide bridges (Cys1â»Cys4 and Cys2â»Cys3), and it is devoid of aromatic or aliphatic amino acids. Secondary structure analysis reveals that AbeTx1 has a highly flexible, random-coil-like conformation, but with a tendency of structuring in the beta sheet. Its overall structure is similar to open-ended cyclic peptides found on the scorpion κ-KTx toxins family, cone snail venoms, and antimicrobial peptides.
Assuntos
Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Anêmonas-do-Mar/química , Anêmonas-do-Mar/metabolismo , Toxinas Biológicas/química , Toxinas Biológicas/farmacologia , Sequência de Aminoácidos , Aminoácidos/metabolismo , Animais , Venenos de Cnidários/química , Venenos de Cnidários/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Potássio/metabolismo , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/farmacologia , Alinhamento de SequênciaRESUMO
Sea anemones contain a variety of interesting biologically active compounds, including some potent toxins. PLA2 from Bunodosoma caissarum, a sea anemone endemic in the Brazilian southern coast, has shown renal alterations on isolated kidney. The aim of this study was to evaluate the renal and vascular effects of B. caissarum crude extract (BcE) on isolated perfused kidney and arteriolar mesenteric bed, as well the involvement of prostaglandins and endothelin. BcE did not show any effect on arteriolar mesenteric bed, but increased perfusion pressure, renal vascular resistance, urinary flow, glomerular filtration rate and decreased the percentage of sodium tubular transport on isolated perfused kidney. Indomethacin blocked the renal effects induced by BcE and tezosentan only partially blocked these effects. These results demonstrate the effects of BcE on kidney in situ, suggesting the involvement of prostaglandins and endothelin.
Assuntos
Venenos de Cnidários/toxicidade , Rim/efeitos dos fármacos , Anêmonas-do-Mar/química , Animais , Endotelinas , Taxa de Filtração Glomerular/efeitos dos fármacos , Indometacina/farmacologia , Masculino , Artérias Mesentéricas/efeitos dos fármacos , Prostaglandinas , Piridinas/farmacologia , Ratos Wistar , Tetrazóis/farmacologia , Resistência Vascular/efeitos dos fármacosRESUMO
Sticholysin II is a pore-forming toxin produced by the sea anemone Stichodactyla helianthus that belongs to the actinoporin protein family. The high affinity of actinoporins for sphingomyelin (SM)-containing membranes has been well documented. However, the molecular determinants that define this affinity have not been fully clarified. Here, we have examined the binding and permeabilizing activity of StII to different single and mixed lipidic systems by combining lipid monolayers, liposomes, and permeabilizing assays. This study characterizes the contribution of ceramide-derived compounds for StII-membrane interaction. Molecular dynamics simulations revealed a differential binding mode of StII with the polar head group of SM and PC. The electrostatic interaction energies were the major energetic contributors to the better affinity of StII for SM compared to PC, while the van der Waals interaction energies were the major driving forces of the better affinity of StII for SM respect to Cer. Furthermore, the presence of sugar residues in glycosphingolipids modulated binding and pore-formation by actinoporins probably by hindering StII to reach relevant structural motifs in membrane for binding or inducing a non-competent adsorption to membrane. Our results demonstrate that StII-membrane interaction, leading to pore formation, may critically respond to changes in lipid head group properties, and the access to SM interfacial structural motif.
Assuntos
Venenos de Cnidários/metabolismo , Simulação de Dinâmica Molecular , Anêmonas-do-Mar/química , Esfingomielinas/metabolismo , Termodinâmica , Animais , Venenos de Cnidários/química , Interações Hidrofóbicas e Hidrofílicas , Bicamadas Lipídicas/química , Lipossomos/química , Esfingomielinas/químicaRESUMO
Total mercury concentrations in the mussel Perna perna and the sea anemone Bunodosoma caissarum were determined to assess Hg contamination in Guanabara Bay, Rio de Janeiro, Brazil, and an adjacent sea area. Concentrations in the tissues of these species were compared. Average total mercury concentrations ranged from 3.54 to 21.01 µg kg-1 (wet wt.) in P. perna and from 4.51 to 23.19 µg kg-1 (wet wt.) for B. caissarum. Concentrations varied according to the sampling stations. Distribution of concentrations for both species was similar along the sampling stations, and a significant correlation was observed. Results suggest that B. caissarum could be a suitable biomonitor species for mercury contamination in the study area and could be used as a complementary species for monitoring studies. Further research is, however, needed to assess how environmental conditions and other variables affect Hg concentrations in B. caissarum.
Assuntos
Mercúrio/análise , Perna (Organismo)/química , Anêmonas-do-Mar/química , Poluentes Químicos da Água/análise , Animais , Brasil , Monitoramento AmbientalRESUMO
The Zoanthids are an order of cnidarians whose venoms and toxins have been poorly studied. Palythoa caribaeorum is a zoanthid commonly found around the Mexican coastline. In this study, we tested the activity of P. caribaeorum venom on voltage-gated sodium channel (NaV1.7), voltage-gated calcium channel (CaV2.2), the A-type transient outward (IA) and delayed rectifier (IDR) currents of KV channels of the superior cervical ganglion (SCG) neurons of the rat. These results showed that the venom reversibly delays the inactivation process of voltage-gated sodium channels and inhibits voltage-gated calcium and potassium channels in this mammalian model. The compounds responsible for these effects seem to be low molecular weight peptides. Together, these results provide evidence for the potential use of zoanthids as a novel source of cnidarian toxins active on voltage-gated ion channels.
Assuntos
Venenos de Cnidários/farmacologia , Canais Iônicos/metabolismo , Neurônios/efeitos dos fármacos , Neurotoxinas/farmacologia , Anêmonas-do-Mar/química , Gânglio Cervical Superior/citologia , Animais , Canais de Cálcio Tipo N/metabolismo , Canais de Cálcio Tipo N/fisiologia , Células Cultivadas , Venenos de Cnidários/química , Venenos de Cnidários/isolamento & purificação , Fenômenos Eletrofisiológicos , Canais Iônicos/fisiologia , Masculino , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/fisiologia , Neurônios/metabolismo , Neurotoxinas/química , Neurotoxinas/isolamento & purificação , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Canais de Potássio de Abertura Dependente da Tensão da Membrana/fisiologia , Ratos Wistar , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Sticholysin II (St II) is a haemolytic toxin isolated from the sea anemone Stichodactyla helianthus. The high haemolytic activity of this toxin is strongly dependent on the red cell status and the macromolecule conformation. In the present communication we evaluate the effect of human serum albumin on St II haemolytic activity and its capacity to form pores in the bilayer of synthetic liposomes. St II retains its pore forming capacity in the presence of large concentrations (up to 500 µM) of human serum albumin. This effect is observed both in its capacity to produce red blood cells haemolysis and to generate functional pores in liposomes. In particular, the capacity of the toxin to lyse red blood cells increases in the presence of human serum albumin (HSA). Regarding the rate of the pore forming process, it is moderately decreased in liposomes and in red blood cells, in spite of an almost total coverage of the interface by albumin. All the data obtained in red cells and model membranes show that St II remains lytically active even in the presence of high HSA concentrations. This stubbornness can explain why the toxin is able to exert its haemolytic activity on membranes immersed in complex plasma matrixes such as those present in living organisms.
Assuntos
Venenos de Cnidários/metabolismo , Hemólise/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/metabolismo , Anêmonas-do-Mar/química , Albumina Sérica/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Venenos de Cnidários/isolamento & purificação , Eritrócitos/efeitos dos fármacos , Eritrócitos/patologia , Humanos , Lipossomos/metabolismo , Proteínas Citotóxicas Formadoras de Poros/isolamento & purificaçãoRESUMO
Sticholysins (Sts) I and II (StI/II) are pore-forming toxins (PFTs) produced by the Caribbean Sea anemone Stichodactyla helianthus belonging to the actinoporin family, a unique class of eukaryotic PFTs exclusively found in sea anemones. The role of lipid phase co-existence in the mechanism of the action of membranolytic proteins and peptides is not clearly understood. As for actinoporins, it has been proposed that phase separation promotes pore forming activity. However little is known about the effect of sticholysins on the phase separation of lipids in membranes. To gain insight into the mechanism of action of sticholysins, we evaluated the effect of these proteins on lipid segregation using differential scanning calorimetry (DSC) and atomic force microscopy (AFM). New evidence was obtained reflecting that these proteins reduce line tension in the membrane by promoting lipid mixing. In terms of the relevance for the mechanism of action of actinoporins, we hypothesize that expanding lipid disordered phases into lipid ordered phases decreases the lipid packing at the borders of the lipid raft, turning it into a more suitable environment for N-terminal insertion and pore formation.
Assuntos
Venenos de Cnidários/farmacologia , Lipídeos/química , Microdomínios da Membrana/metabolismo , Anêmonas-do-Mar/metabolismo , Animais , Varredura Diferencial de Calorimetria , Microscopia de Força Atômica , Compostos Orgânicos/farmacologia , Anêmonas-do-Mar/químicaRESUMO
It has been reported that serine peptidase activities of Trypanosoma cruzi play crucial roles in parasite dissemination and host cell invasion and therefore their inhibition could affect the progress of Chagas disease. The present study investigates the interference of the Stichodactyla helianthus Kunitz-type serine protease inhibitor (ShPI-I), a 55-amino acid peptide, in T. cruzi serine peptidase activities, parasite viability, and parasite morphology. The effect of this peptide was also studied in Leishmania amazonensis promastigotes and it was proved to be a powerful inhibitor of serine proteases activities and the parasite viability. The ultrastructural alterations caused by ShPI-I included vesiculation of the flagellar pocket membrane and the appearance of a cytoplasmic vesicle that resembles an autophagic vacuole. ShPI-I, which showed itself to be an important T. cruzi serine peptidase inhibitor, reduced the parasite viability, in a dose and time dependent manner. The maximum effect of peptide on T. cruzi viability was observed when ShPI-I at 1×10(-5)M was incubated for 24 and 48h which killed completely both metacyclic trypomastigote and epimastigote forms. At 1×10(-6)M ShPI-I, in the same periods of time, reduced parasite viability about 91-95% respectively. Ultrastructural analysis demonstrated the formation of concentric membranar structures especially in the cytosol, involving organelles and small vesicles. Profiles of endoplasmic reticulum were also detected, surrounding cytosolic vesicles that resembled autophagic vacuoles. These results suggest that serine peptidases are important in T. cruzi physiology since the inhibition of their activity killed parasites in vitro as well as inducing important morphological alterations. Protease inhibitors thus appear to have a potential role as anti-trypanosomatidal agents.
Assuntos
Antiprotozoários/farmacologia , Produtos Biológicos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Anêmonas-do-Mar/química , Serpinas/farmacologia , Trypanosoma cruzi/efeitos dos fármacos , Animais , Antiprotozoários/isolamento & purificação , Organismos Aquáticos/química , Produtos Biológicos/isolamento & purificação , Doença de Chagas/parasitologia , Relação Dose-Resposta a Droga , Humanos , Leishmania/citologia , Leishmania/efeitos dos fármacos , Leishmania/fisiologia , Microscopia Eletrônica , Organelas/ultraestrutura , Serpinas/isolamento & purificação , Trypanosoma cruzi/citologia , Trypanosoma cruzi/fisiologiaRESUMO
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.
Assuntos
Glicopeptídeos , Ativação do Canal Iônico/efeitos dos fármacos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Proteômica , Anêmonas-do-Mar , Animais , Glicopeptídeos/química , Glicopeptídeos/genética , Glicopeptídeos/metabolismo , Glicopeptídeos/farmacologia , Ativação do Canal Iônico/genética , Toxinas Marinhas/química , Toxinas Marinhas/genética , Toxinas Marinhas/metabolismo , Toxinas Marinhas/farmacologia , Oócitos , Canais de Potássio de Abertura Dependente da Tensão da Membrana/biossíntese , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Anêmonas-do-Mar/química , Anêmonas-do-Mar/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Xenopus laevisRESUMO
Sea anemone (Cnidaria, Anthozoa) venom is an important source of bioactive compounds used as tools to study the pharmacology and structure-function of voltage-gated K+ channels (KV). These neurotoxins can be divided into four different types, according to their structure and mode of action. In this work, for the first time, two toxins were purified from the venom of Bunodosoma caissarum population from Saint Peter and Saint Paul Archipelago, Brazil. Sequence alignment and phylogenetic analysis reveals that BcsTx1 and BcsTx2 are the newest members of the sea anemone type 1 potassium channel toxins. Their functional characterization was performed by means of a wide electrophysiological screening on 12 different subtypes of KV channels (KV1.1-KV1.6; KV2.1; KV3.1; KV4.2; KV4.3; hERG and Shaker IR). BcsTx1 shows a high affinity for rKv1.2 over rKv1.6, hKv1.3, Shaker IR and rKv1.1, while Bcstx2 potently blocked rKv1.6 over hKv1.3, rKv1.1, Shaker IR and rKv1.2. Furthermore, we also report for the first time a venom composition and biological activity comparison between two geographically distant populations of sea anemones.
Assuntos
Venenos de Cnidários/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Anêmonas-do-Mar/química , Animais , Brasil , Venenos de Cnidários/química , Fenômenos Eletrofisiológicos , Humanos , Filogenia , Bloqueadores dos Canais de Potássio/química , Bloqueadores dos Canais de Potássio/isolamento & purificação , Ratos , Alinhamento de SequênciaRESUMO
Sea anemones possess a number of peptide toxins that target ion channels which provide powerful tools to study the molecular basis of diverse signaling pathways. It is also acknowledged that currents through Erg1 K(+) channels in cardiac myocytes are important for electrical stability of the heart and alterations in its activity has been linked to the onset of a potentially life-threatening heart condition named long QT syndrome type 2. Here, we report that a crude extract from sea anemone Condylactis gigantea significantly increases the QT interval and has arrhythmogenic effects in the rat heart. Furthermore, a bioassay-guided purification procedure allowed the isolation of a chromatographic fraction containing a major component with a molecular mass of 4478 Da from the crude extract, which causes a significant inhibition of whole-cell patch-clamp currents through recombinant Erg1 channels, responsible of the rapid delayed rectifying current crucial for electrical activity in the heart. Further studies could provide relevant information on the molecular mechanism of C. gigantea peptide toxins which represent promising tools in studying the physiology of diverse ion channels.
Assuntos
Venenos de Cnidários/farmacologia , Canais de Potássio Éter-A-Go-Go/efeitos dos fármacos , Síndrome do QT Longo/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Anêmonas-do-Mar/química , Extratos de Tecidos/farmacologia , Animais , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Células HEK293/efeitos dos fármacos , Células HEK293/metabolismo , Coração/efeitos dos fármacos , Humanos , Síndrome do QT Longo/fisiopatologia , Masculino , Miócitos Cardíacos/fisiologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Proteínas Recombinantes/metabolismoRESUMO
Cnidarians comprise an old and diverse animal phylum, and possess a wide variety of biologically active substances. Sea anemones contain a diversity of interesting biologically active compounds including some potent toxins. In the present work, the sea anemones Stichodactyla mertensii and Stichodactyla gigantea, collected from the Mandapam coast, are characterized biomedically and pharmacologically. The crude protein was obtained by using methanol and aqueous extracts. The respective protein contents of S. mertensii and S. gigantea were found to be 2.10 µg/mL and 1.87 µg/mL. The methanol and aqueous extracts of S. mertensii and S. gigantea yielded six and nine bands by SDS-PAGE on 12 percent gel. In the hemolytic assay, both extracts exhibited hemolytic effect on chicken, goat, cow and human erythrocytes ('A', 'B' and 'O'). The neurotoxic effects of these crude extracts were determined in vivo using the sea shore crab Ocypode macrocera and mortality was observed. The mouse bioassay for lethality was performed on male albino mice. The crude extract of S. mertensii showed higher lethality (58 seconds at 1 mL-dose) than that of S. gigantea (2 minutes and 10 seconds at 0.75 mL-dose). The analgesic activity test was also carried out on albino mice by Eddy's hot plate and tail-flick methods. The extracts showed moderate analgesic effect by both hot-plate and tail-flick methods. These characteristics emphasize the need for the isolation and molecular characterization of new active toxins in S. mertensii and S. gigantea.(AU)
Assuntos
Animais , Masculino , Ratos , Anêmonas-do-Mar/química , Antivenenos , Venenos de Cnidários/toxicidade , Neurotoxinas/química , Bioensaio/métodos , Proteínas Hemolisinas/isolamento & purificação , Proteínas Hemolisinas/efeitos dos fármacos , Analgésicos/farmacologiaRESUMO
A new acylamino acid, bunodosine 391 (BDS 391), was isolated from the venom of the sea anemone Bunodosoma cangicum. The structure was elucidated by spectroscopic analyses (2D NMR, ESIMS/MS) and verified by its synthesis. Intraplantar injection of BDS 391 into the hind paw of a rat induced a potent analgesic effect. This effect was not altered by naloxone (an opioid receptor antagonist), but was completely reversed by methysergide (a serotonin receptor antagonist), indicating that the effect is mediated by activation of serotonin receptors.
Assuntos
Analgésicos/isolamento & purificação , Analgésicos/farmacologia , Anêmonas-do-Mar/química , Analgésicos/química , Animais , Venenos de Cnidários/síntese química , Venenos de Cnidários/química , Venenos de Cnidários/isolamento & purificação , Venenos de Cnidários/farmacologia , Edema/induzido quimicamente , Edema/tratamento farmacológico , Membro Posterior/efeitos dos fármacos , Masculino , Estrutura Molecular , Naloxona/farmacologia , Antagonistas de Entorpecentes/farmacologia , Ressonância Magnética Nuclear Biomolecular , Ratos , Ratos Wistar , Receptores de Serotonina/efeitos dos fármacos , Receptores de Serotonina/metabolismoRESUMO
Stichodactyla heliantus II (St II) is a haemolytic toxin whose activity depends of the characteristics of red blood cells (RBC). Among the factors that may tune the response of the RBC to the toxin activity stand the oxidative status of the cell. This study investigates how pre-oxidation of RBC modifies St II activity employing two oxidants, peroxynitrite and hypochlorous acid. Results show that peroxynitrite-treated RBC are more resistant to St II activity. On the other hand, hypochlorous acid-treated RBC become more susceptible to St II. This contrasting behaviour of both oxidants is related to the modifications elicited in RBC by both oxidant agents. Peroxynitrite does not modify RBC osmotic fragility but reduces anion transport through band 3 protein. This effect, together with an increase in K+ efflux, can explain the increased resistance to the toxin activity. On the other hand, results obtained with hypochlorous acid can be explained in terms of a disruption of the membrane organization without the compensating effect of a reduction in band 3-mediated anion transport. The present results, obtained employing the effect of a model haemolytic toxin on RBC, emphasize the specificity of the RBC response to different endogenous oxidative agents.