RESUMEN
The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 µg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 µg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.
Asunto(s)
Venenos de los Peces , Perciformes , Animales , Eritrocitos , Venenos de los Peces/farmacología , Peces , Ratones , Conejos , PielRESUMEN
The scorpionfish Scorpaena plumieri is one of the most venomous fish species in the Brazilian coast. Amongst many biological activities, the S. plumieri fish venom (SpV) promotes hemagglutination. Although this activity appears to be associated to the presence of C-type lectins in the venom, it has not yet been chemically or functionally characterized. In the present work we sought to advance the characterization of the hemagglutinating activity associated to this venom. By fractionating SpV through saline precipitation followed by size exclusion chromatography we obtained two purified fractions - HF1 and HF3 - with Ca2+-dependent agglutinating activity against rabbit erythrocytes, which remained stable upon storage at 4 and -80oC. HF1 and HF3 were bacteriostatic against Gram-positive bacteria (Staphylococcus aureus), displaying minimum inhibitory concentration (MIC) of 50 and 200 μg/mL, respectively. In addition, a resazurin-based viability assay revealed that both fractions, at doses up to 370 μg/mL, were cytotoxic against tumor and non-tumor cell lines. Finally, a tendency towards edema formation could be detected when the fractions - particularly HF1 - were injected into mice footpads. We believe our data contribute to a better understanding of the biological properties of the so often neglected fish venoms.
RESUMEN
Cardiovascular effects induced by snake venoms, in spite of having a crucial role in the outcome of the envenomation, have been less studied than other toxic activities displayed by these venoms. In this study we evaluated acute cardiovascular responses to Bothrops leucurus venom - Bl-V - both in vivo, in anesthetized rats, and in vitro, in isolated rat mesenteric resistance arteries. Bl-V (10-100 µg protein/kg) caused dose-dependent hypotension, followed by gradual recovery (2-20 min) to basal levels, and induced dose-dependent (1-20 µg/mL) vasodilation in pre-contracted arteries, what was more pronounced when the endothelium remained intact. These effects were partially counteracted by pre-treatment with indomethacin (cyclooxygenase inhibitor). Prior incubation of Bl-V with commercial pentavalent Bothrops antivenom also attenuated the cardiovascular effects induced by the venom, in spite of it not being among the venoms used for the development of the bothropic antivenom. Through an approach based on two chromatographic steps and mass spectrometry (MALDI-ToF and MALDI-ISD), a component with acute cardiovascular effects was isolated and identified as the basic phospholipase blD-PLA2, previously purified from the venom of B. leucurus. Taken together, our results show that, at low doses, the venom of B. leucurus induces transient, acute hypotension in anesthetized rats following systemic vasodilation in a dose-dependent way. In addition, we provide clear evidence of the involvement of the enzymatic activity of blD-PLA2 in this cardiovascular response, acting via the production of vasodilating prostanoids.
Asunto(s)
Bothrops , Venenos de Crotálidos/toxicidad , Fosfolipasas A2/metabolismo , Animales , Hipotensión/inducido químicamente , Ratas , Venenos de SerpienteRESUMEN
The biological activities observed upon envenomation by Scorpaena plumieri could be linked to both the venom and the skin mucus. Through a proteomic/functional approach we analyzed protein composition and biological activities of the venom and skin mucus. We identified 885 proteins: 722 in the Venomous Apparatus extracts (Sp-VAe) and 391 in the Skin Mucus extract (Sp-SMe), with 494 found exclusively in Sp-VAe, being named S. plumieri Venom Proteins (Sp-VP), while 228 were found in both extracts. The majority of the many proteins identified were not directly related to the biological activities reported here. Nevertheless, some were classified as toxins/potentially interesting molecules: lectins, proteases and protease inhibitors were detected in both extracts, while the pore-forming toxin and hyaluronidase were associated with Sp-VP. Proteolytic and anti-microbial activities were linked to both extracts, while the main toxic activities - cardiovascular, inflammatory, hemolytic and nociceptive - were elicited only by Sp-VAe. Our study provided a clear picture on the composition of the skin mucus and the venom. We also show that the classic effects observed upon envenomation are produced by molecules from the venomous gland. Our results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins. SIGNIFICANCE: In this study a large number of proteins - including classical and non-classical toxins - were identified in the venomous apparatus and the skin mucus extracts of the Scorpaena plumieri fish through shotgun proteomic approach. It was shown that the toxic effects observed upon envenomation are elicited by molecules originated from the venomous gland. These results add to the growing catalogue of scorpaeniform fish venoms and their skin mucus proteins - so scarcely explored when compared to the venoms and bioactive components of terrestrial animals. Data are available via ProteomeXchange with identifier PXD009983.
Asunto(s)
Proteínas de Peces/análisis , Proteínas de Peces/fisiología , Venenos de los Peces/análisis , Moco/química , Perciformes/metabolismo , Proteómica/métodos , Piel/química , Animales , Proteínas de Peces/metabolismo , Venenos de los Peces/metabolismo , Venenos de los Peces/fisiología , Masculino , Ratones , Moco/metabolismo , Ratas , Ratas Wistar , Piel/metabolismo , Extractos de Tejidos/análisis , Extractos de Tejidos/metabolismoRESUMEN
Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventer spider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventer venom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.
Asunto(s)
Proteómica , Venenos de Araña/metabolismo , Arañas/metabolismo , Transcriptoma , Secuencia de Aminoácidos , Animales , Biomarcadores de Tumor/química , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Péptidos/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Arañas/genética , Toxinas Biológicas/genética , Toxinas Biológicas/metabolismo , Proteína Tumoral Controlada Traslacionalmente 1RESUMEN
Phoneutria nigriventer is one of the largest existing true spiders and one of the few considered medically relevant. Its venom contains several neurotoxic peptides that act on different ion channels and chemical receptors of vertebrates and invertebrates. Some of these venom toxins have been shown as promising models for pharmaceutical or biotechnological use. However, the large diversity and the predominance of low molecular weight toxins in this venom have hampered the identification and deep investigation of the less abundant toxins and the proteins with high molecular weight. Here, we combined conventional and next-generation cDNA sequencing with Multidimensional Protein Identification Technology (MudPIT), to obtain an in-depth panorama of the composition of P. nigriventerspider venom. The results from these three approaches showed that cysteine-rich peptide toxins are the most abundant components in this venom and most of them contain the Inhibitor Cysteine Knot (ICK) structural motif. Ninety-eight sequences corresponding to cysteine-rich peptide toxins were identified by the three methodologies and many of them were considered as putative novel toxins, due to the low similarity to previously described toxins. Furthermore, using next-generation sequencing we identified families of several other classes of toxins, including CAPs (Cysteine Rich Secretory Protein-CRiSP, antigen 5 and Pathogenesis-Related 1-PR-1), serine proteinases, TCTPs (translationally controlled tumor proteins), proteinase inhibitors, metalloproteinases and hyaluronidases, which have been poorly described for this venom. This study provides an overview of the molecular diversity of P. nigriventervenom, revealing several novel components and providing a better basis to understand its toxicity and pharmacological activities.
RESUMEN
AIMS: Extensive evidence supports a role for voltage-gated calcium channels (VGCC) and TRPV1 receptors in pain transmission and modulation. We investigated the profile of analgesic interaction between Phα1ß toxin (a VGCC blocker) and SB366791 (selective TRPV1 antagonist) in a model of acute pain induced by capsaicin. Changes in body temperature induced by combination regimens were also evaluated. MAIN METHODS: Isobolographic approach with a fixed dose-ratio of combined drugs was used to determine whether antinociceptive interaction of Phα1ß and SB366791 are subadditive, additive or synergic. Body temperature was obtained by thermal infrared imaging. KEY FINDINGS: Phα1ß and SB366791 interact in a synergistic manner to cause antinociception. We found an interaction index (α) of 0.07 for Phα1ß and SB366791 when these drugs were injected together intraplantarly, which indicates that in vivo interaction between these drugs is greater than additive interaction. Synergism also occurred when intraplantar SB366791 was administered simultaneously with intrathecal Phα1ß (interaction index α=0.06) suggesting a 15 fold rise in potency on the analgesic effect of these drugs when they are added together. It was observed no significant alterations in body temperature of animals treated with this combination regimen. SIGNIFICANCE: Our data reveal that Phα1ß toxin potentiates in 15 fold the antinociceptive action of the TRPV1 blocker SB366791. Therefore, lower doses of these drugs are required to achieve antinociceptive effects when these agents are given in combination.
Asunto(s)
Dolor Agudo/tratamiento farmacológico , Analgésicos/farmacología , Anilidas/farmacología , Cinamatos/farmacología , Venenos de Araña/farmacología , Canales Catiónicos TRPV/antagonistas & inhibidores , Analgésicos/administración & dosificación , Anilidas/administración & dosificación , Animales , Temperatura Corporal , Bloqueadores de los Canales de Calcio/administración & dosificación , Bloqueadores de los Canales de Calcio/farmacología , Capsaicina , Cinamatos/administración & dosificación , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Ratones , Venenos de Araña/administración & dosificaciónRESUMEN
The venom of the Brazilian armed spider Phoneutria nigriventer is a rich source of biologically active peptides that have potential as analgesic drugs. In this study, we investigated the analgesic and adverse effects of peptide 3-5 (Tx3-5), purified from P. nigriventer venom, in several mouse models of pain. Tx3-5 was administered by intrathecal injection to mice selected as models of postoperative (plantar incision), neuropathic (partial sciatic nerve ligation) and cancer-related pain (inoculation with melanoma cells) in animals that were either sensitive or tolerant to morphine. Intrathecal administration of Tx3-5 (3-300 fmol/site) in mice could either prevent or reverse postoperative nociception, with a 50 % inhibitory dose (ID50) of 16.6 (3.2-87.2) fmol/site and a maximum inhibition of 87 ± 10 % at a dose of 30 fmol/site. Its effect was prevented by the selective activator of L-type calcium channel Bay-K8644 (10 µg/site). Tx3-5 (30 fmol/site) also produced a partial antinociceptive effect in a neuropathic pain model (inhibition of 67 ± 10 %). Additionally, treatment with Tx3-5 (30 fmol/site) nearly abolished cancer-related nociception with similar efficacy in both morphine-sensitive and morphine-tolerant mice (96 ± 7 and 100 % inhibition, respectively). Notably, Tx3-5 did not produce visible adverse effects at doses that produced antinociception and presented a TD50 of 1125 (893-1418) fmol/site. Finally, Tx3-5 did not alter the normal mechanical or thermal sensitivity of the animals or cause immunogenicity. Our results suggest that Tx3-5 is a strong drug candidate for the treatment of painful conditions.
Asunto(s)
Analgésicos/uso terapéutico , Dolor en Cáncer/tratamiento farmacológico , Neuralgia/tratamiento farmacológico , Neuropéptidos/uso terapéutico , Neurotoxinas/uso terapéutico , Venenos de Araña/uso terapéutico , Analgésicos/efectos adversos , Analgésicos/farmacología , Animales , Agonistas de los Canales de Calcio/farmacología , Masculino , Ratones , Ratones Endogámicos C57BL , Neuropéptidos/efectos adversos , Neuropéptidos/farmacología , Neurotoxinas/efectos adversos , Neurotoxinas/farmacología , Nocicepción/efectos de los fármacos , Venenos de Araña/efectos adversos , Venenos de Araña/farmacologíaRESUMEN
UNLABELLED: Tarantula spiders, Theraphosidae family, are spread throughout most tropical regions of the world. Despite their size and reputation, there are few reports of accidents. However, like other spiders, their venom is considered a remarkable source of toxins, which have been selected through millions of years of evolution. The present work provides a proteomic overview of the fascinating complexity of the venomous extract of the Grammostola iheringi tarantula, obtained by electrical stimulation of the chelicerae. For analysis a bottom-up proteomic approach Multidimensional Protein Identification Technology (MudPIT) was used. Based on bioinformatics analyses, PepExplorer, a similarity-driven search tool that identifies proteins based on phylogenetically close organisms, a total of 395 proteins were identified in this venomous extract. Most of the identifications (~70%) were classified as predicted (21%), hypothetical (6%) and putative (37%), while a small group (6%) had no predicted function. Identified molecules matched with neurotoxins that act on ions channels; proteases, such as serine proteases, metalloproteinases, cysteine proteinases, aspartic proteinases, carboxypeptidases and cysteine-rich secretory enzymes (CRISP) and some molecules with unknown target. Additionally, non-classical venom proteins were also identified. Up to now, this study represents, to date, the first broad characterization of the composition of G. iheringi venomous extract. Our data provides a tantalizing insight into the diversity of proteins in this venom and their biotechnological potential. SIGNIFICANCE: Animal venoms contain a diversity of molecules able to bind to specific cell targets. Due to their biochemical and physiological properties, these molecules are interesting for medical and biotechnological purposes. In this study, a large number of components of the venomous extract of the spider Grammostola iheringi were identified by the MudPIT technique. It was demonstrated that this approach is a sensitive and adequate method to achieve a broad spectrum of information about animal venoms. Using this bottom-up proteomic method, classical and non-classical venom proteins were identified which stimulate new interest in the systematic research of their protein components.
Asunto(s)
Proteínas de Artrópodos/metabolismo , Proteómica , Venenos de Araña/metabolismo , Arañas/metabolismo , AnimalesRESUMEN
We report the comparative proteomic characterization of the venoms of Bothrops atrox, B. barnetti and B. pictus. The venoms were subjected to RP-HPLC and the resulting fractions analyzed by SDS-PAGE. The proteins were cut from the gels, digested with trypsin and identified via peptide mass fingerprint and manual sequencing of selected peptides by MALDI-TOF/TOF mass spectrometry. Around 20-25 proteins were identified belonging to only 6-7 protein families. Metalloproteinases of the classes P-I and P-III were the most abundant proteins in all venoms (58-74% based on peak area A214 nm), followed by phospholipases-A(2) (6.4-14%), disintegrins (3.2-9%) and serine proteinases (7-11%), and some of these proteins occurred in several isoforms. In contrast cysteine-rich secretory proteins and L-amino acid oxidases appeared only as single isoforms and were found only in B. atrox and B. barnetti. C-type lectins were also detected in all venoms but at low levels (~ 5%). Furthermore, the venoms contain variable numbers of peptides (<3 kDa) and non-protein compounds which were not considered in this work. The protein composition of the investigated Bothrops species is in agreement with their pharmacological and pathological effects.
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Bothrops/metabolismo , Venenos de Crotálidos/metabolismo , Proteoma/metabolismo , Animales , Perú , Proteómica/métodos , Especificidad de la EspecieRESUMEN
The systematic investigation of the peptidic composition of the skin secretion of Phasmahyla jandaia, a phyllomedusine anuran endemic to the southern region of the Espinhaço range in Brazil, is herein reported. By means of de novo interpretation of tandem mass spectrometric data, Edman N-terminal sequencing and similarity searches, 57 peptides - including phylloseptins, dermaseptins stricto sensu, dermatoxins, hyposins, tryptophyllins, caerulein-related, bradykinin-related, bradykinin potentiating, tyrosine-rich, and opioid peptides - were sequenced. Moreover, five peptide families without significant similarity to other known molecules were verified. Differently from most Phyllomedusinae genera, the molecular diversity in the skin of representatives of Phasmahyla remained unprospected until now. Therefore, besides disclosing novel natural variants of number of bioactive peptides, the present study contributes to the understanding of the evolution of biochemical characters of the phyllomedusines.
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Anuros/fisiología , Mapeo Peptídico/métodos , Péptidos/química , Proteómica/métodos , Piel/metabolismo , Secuencia de Aminoácidos , Animales , Brasil , Cromatografía/métodos , Masculino , Datos de Secuencia Molecular , Análisis de Secuencia de Proteína , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
We report the isolation and structure-function relationship of a 23kDa metalloproteinase named atroxlysin-I from the venom of the Peruvian Bothrops atrox (Jergón). Atroxlysin is a P-I metalloproteinase and contains 204 residues. Its proteolytic activity towards dimethylcasein is enhanced by Ca2+ but inhibited by EDTA, dithiothreitol, excessive Zn2+ and alpha2-macroglobulin. Unlike other structurally homologous P-I metalloproteinases, atroxlysin-I causes hemorrhages. To examine its hemorrhagic activity mechanistically, we studied its function in vitro and in vivo. It cleaved the Ala14-Leu15 and Tyr16-Leu17 bonds in oxidized insulin B-chain and specifically hydrolyzed the alpha-chains of fibrin(ogen) in a dose- and time-dependent manner. Atroxlysin-I cleaved plasma fibronectin and other extracellular matrix proteins (collagens I and IV) and the triple-helical fragment CB3 of collagen IV, but did not degrade laminin-111. Complementarily, the laminin and collagen binding integrins alpha7beta1 and alpha1beta1 were cleaved by atroxlysin. Even without catalytic activity atroxlysin-I inhibited collagen- and ADP-triggered platelet aggregation.
Asunto(s)
Plaquetas/efectos de los fármacos , Vasos Sanguíneos/citología , Bothrops , Matriz Extracelular/efectos de los fármacos , Metaloproteasas/toxicidad , Venenos de Serpiente/enzimología , Secuencia de Aminoácidos , Animales , Plaquetas/metabolismo , Vasos Sanguíneos/efectos de los fármacos , Vasos Sanguíneos/metabolismo , Matriz Extracelular/metabolismo , Fibrina/metabolismo , Fibrinógeno/metabolismo , Fibronectinas/metabolismo , Hemorragia/inducido químicamente , Hemostasis/efectos de los fármacos , Humanos , Integrinas/metabolismo , Macroglobulinas/metabolismo , Metaloproteasas/química , Metaloproteasas/metabolismo , Datos de Secuencia Molecular , Especificidad por SustratoRESUMEN
Brazilian freshwater stingrays, Potamotrygon gr. orbigyni, are relatively common in the middle-western regions of Brazil, where they are considered an important public health threat. In order to identify some of their naturally occurring toxin peptides available in very low amounts, we combine analytical protocols such as reversed-phase high-performance liquid chromatography (RP-HPLC), followed by a biological microcirculatory screening and mass spectrometry analysis. Using this approach, one bioactive peptide was identified and characterized, and two analogues were synthesized. The natural peptide named Porflan has the primary structure ESIVRPPPVEAKVEETPE (MW 2006.09 Da) and has no similarity with any bioactive peptide or protein found in public data banks. Bioassay protocols characterized peptides as presenting potent activity in a microcirculatory environment. The primary sequences and bioassay results, including interactions with the membrane phospholipids, suggest that these toxins are a new class of fish toxins, directly involved in the inflammatory processes of a stingray sting.
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Venenos de los Peces/metabolismo , Péptidos/química , Péptidos/metabolismo , Rajidae/metabolismo , Secuencia de Aminoácidos , Animales , Cromatografía Líquida de Alta Presión , Venenos de los Peces/química , Espectrometría de Masas , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Espectrometría de Masas en TándemRESUMEN
MALDI-TOF-TOF and de novo sequencing were employed to assess the Tityus serrulatus venom peptide diversity. Previous works has shown the cornucopia of molecular masses, ranging from 800 to 3000Da, present in the venom from this and other scorpions species. This work reports the identification/sequencing of several of these peptides. The majority of the peptides found were fragments of larger venom toxins. For instance, 28 peptides could be identified as fragments from Pape proteins, 10 peptides corresponded to N-terminal fragments of the TsK beta (scorpine-like) toxin and fragments of potassium channel toxins (other than the k-beta) were sequenced as well. N-terminal fragments from the T. serrulatus hypotensins-I and II and a novel hypotensin-like peptide could also be found. This work also reports the sequencing of novel peptides without sequence similarities to other known molecules.
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Venenos de Escorpión/química , Secuencia de Aminoácidos , Animales , Fraccionamiento Químico , Cromatografía en Gel , Datos de Secuencia Molecular , Péptidos/química , Alineación de Secuencia , Análisis de Secuencia de Proteína , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masas en TándemRESUMEN
A proteinase, named BmooMPalpha-I, from the venom of Bothrops moojeni, was purified by DEAE-Sephacel, Sephadex G-75 and heparin-agarose column chromatography. The enzyme was purified to homogeneity as judged by its migration profile in SDS-PAGE stained with coomassie blue, and showed a molecular mass of about 24.5 kDa. Its complete cDNA was obtained by RT-PCR and the 615 bp codified for a mature protein of 205 amino acid residues. The multiple alignment of its deduced amino acid sequence and those of other snake venom metalloproteinases showed a high structural similarly, mainly among class P-IB proteases. The enzyme cleaves the Aalpha-chain of fibrinogen first, followed by the Bbeta-chain, and shows no effects on the gamma-chain. On fibrin, the enzyme hydrolyzed only the beta-chain, leaving the gamma-dimer apparently untouched. It was devoid of phospholipase A(2), hemorrhagic and thrombin-like activities. Like many venom enzymes, it is stable at pH values between 4 and 10 and stable at 70 degrees C for 15 min. The inhibitory effects of EDTA on the fibrinogenolytic activity suggest that BmooMPalpha-I is a metalloproteinase and inhibition by beta-mercaptoethanol revealed the important role of the disulfide bonds in the stabilization of the native structure. Aprotinin and benzamidine, specific serine proteinase inhibitors, had no effect on BmooMPalpha-I activity. Since the BmooMPalpha-I enzyme was found to cause defibrinogenation when administered i.p. on mice, it is expected that it may be of medical interest as a therapeutic agent in the treatment and prevention of arterial thrombosis.
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Bothrops/metabolismo , Venenos de Crotálidos/enzimología , Fibrinógeno/antagonistas & inhibidores , Metaloproteasas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/química , Datos de Secuencia MolecularRESUMEN
Centipedes are venomous arthropods responsible for a significant number of non-lethal human envenomations. Despite this, information about the composition and function of their venom contents is scarce. In this study, we have used a 'structure to function' proteomic approach combining two-dimensional chromatography (2D-LC), electrospray ionization quadrupole/time-of-flight mass spectrometry (ESI-Q-TOF/MS), N-terminal sequencing and similarity searching to better understand the complexities of the venoms from two Brazilian centipede species: Scolopendra viridicornis nigra and Scolopendra angulata. Comparisons between the LC profiles and the mass compositions of the venoms of the two species are provided. The observed molecular masses ranged from 3019.62 to 20996.94Da in S. viridicornis nigra (total: 62 molecular masses) and from 1304.73 to 22639.15Da in S. angulata (total: 65 molecular masses). Also, the N-termini of representatives of 10 protein/peptide families were successfully sequenced where nine of them showed no significant similarity to other protein sequences deposited in the Swiss-Prot database. A screening for insecto-toxic activities in fractions from S. viridicornis venom has also been performed. Six out of the 12 tested fractions were responsible for clear toxic effects in house flies. This work demonstrates that centipede venoms might be a neglected but important source of new bioactive compounds.
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Venenos de Artrópodos/química , Artrópodos , Secuencia de Aminoácidos , Animales , Venenos de Artrópodos/toxicidad , Cromatografía Liquida , Dípteros/efectos de los fármacos , Femenino , Masculino , Datos de Secuencia Molecular , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Pruebas de ToxicidadRESUMEN
Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.