RESUMEN
The phenolic extracts of jabuticaba skin flour (JSF) were characterized by HPLC, and evaluated for their modulating action upon phospholipases A2 and proteases of snake venom, aiming at their possible use in the treatment of the various diseases associated with the action of venom toxins. Two types of extracts were prepared from JSF: aqueous and methanolic. These extracts, evaluated at different ratios, (venom: extract, m/m), significantly inhibited the phospholipase activity induced by the venom of Bothrops moojeni and Crotalus durissus terrificus, except for Bothrops atrox venom. The greatest hemolysis inhibitory action was observed for the methanolic extract, when incubated with venoms of B. moojeni and C. durissus terrificus, with inhibitions between 21 and 100%. Thrombolysis induced by venoms of B. moojeni and C. durissus terrificus was inhibited by both extracts, ranging from 32 to 83% and 51 to 83% for the aqueous and methanolic extracts, respectively. Both extracts extended coagulation time, induced by the venoms of B. moojeni and Lachesis muta muta. Inhibitory actions are related to phenolic compounds, such as gallic, syringic and p-coumaric acids, besides catechin, epigallocatechin gallate, epicatechin; resveratrol and quercetin, present in the extracts of jabuticaba skin flour, confirming their potential for nutraceutical use.
Asunto(s)
Myrtaceae/química , Inhibidores de Fosfolipasa A2/farmacología , Extractos Vegetales/farmacología , Inhibidores de Proteasas/farmacología , Venenos de Víboras/antagonistas & inhibidores , Animales , Células Cultivadas , Cromatografía Líquida de Alta Presión , Humanos , Inhibidores de Fosfolipasa A2/aislamiento & purificación , Inhibidores de Proteasas/aislamiento & purificación , Venenos de Víboras/enzimologíaRESUMEN
Snake venom serine proteinases are toxins that perturb hemostasis acting on proteins from the blood coagulation cascade, the fibrinolytic or the kallikrein-kinin system. Despite the relevance of these enzymes in envenomations by viper bites, the characterization of the antibody response to these toxins at the molecular level has not been previously addressed. In this work surface-located B cell recognized linear epitopes from a Lachesis stenophrys venom serine proteinase (UniProt accession number Q072L7) were predicted using an artificial neuronal network at the ABCpred server, the corresponding peptides were synthesized and their immunoreactivity was analyzed against a panel of experimental and therapeutic antivenoms. A molecular model of the L. stenophrys enzyme was built using as a template the structure of the D. acutus Dav-PA serine proteinase (Q9I8X1), which displays the highest degree of sequence similarity to the L. stenophrys enzyme among proteins of known 3D structure, and the surface-located epitopes were identified in the protein model using iCn3D. A total of 13 peptides corresponding to the surface exposed predicted epitopes from L. stenophrys serine proteinase were synthesized and, their reactivity with a rabbit antiserum against the recombinant enzyme and a panel of antivenoms was evaluated by a capture ELISA. Some of the epitopes recognized by monospecific and polyspecific antivenoms comprise sequences overlapping motifs conserved in viper venom serine proteinases. The identification and characterization of relevant epitopes recognized by B cells in snake venom toxins may provide valuable information for the preparation of immunogens that help in the production of improved therapeutic antivenoms.
Asunto(s)
Linfocitos B/inmunología , Epítopos/inmunología , Serina Proteasas/inmunología , Venenos de Víboras/inmunología , Viperidae , Secuencia de Aminoácidos , Animales , Formación de Anticuerpos , Antivenenos/inmunología , Conejos , Serina Proteasas/química , Venenos de Víboras/enzimologíaRESUMEN
Pain-producing animal venoms contain evolutionarily honed toxins that can be exploited to study and manipulate somatosensory and nociceptive signaling pathways. From a functional screen, we have identified a secreted phospholipase A2 (sPLA2)-like protein, BomoTx, from the Brazilian lancehead pit viper (Bothrops moojeni). BomoTx is closely related to a group of Lys49 myotoxins that have been shown to promote ATP release from myotubes through an unknown mechanism. Here we show that BomoTx excites a cohort of sensory neurons via ATP release and consequent activation of P2X2 and/or P2X3 purinergic receptors. We provide pharmacological and electrophysiological evidence to support pannexin hemichannels as downstream mediators of toxin-evoked ATP release. At the behavioral level, BomoTx elicits nonneurogenic inflammatory pain, thermal hyperalgesia, and mechanical allodynia, of which the latter is completely dependent on purinergic signaling. Thus, we reveal a role of regulated endogenous nucleotide release in nociception and provide a detailed mechanism of a pain-inducing Lys49 myotoxin from Bothrops species, which are responsible for the majority of snake-related deaths and injuries in Latin America.
Asunto(s)
Adenosina Trifosfato/metabolismo , Bothrops/fisiología , Fosfolipasas A2 Grupo II/toxicidad , Dolor/metabolismo , Proteínas de Reptiles/toxicidad , Células Receptoras Sensoriales/efectos de los fármacos , Mordeduras de Serpientes/metabolismo , Toxinas Biológicas/toxicidad , Venenos de Víboras/enzimología , Animales , Bothrops/genética , Brasil , Femenino , Fosfolipasas A2 Grupo II/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Dolor/etiología , Dolor/genética , Dolor/parasitología , Ratas , Receptores Purinérgicos/metabolismo , Proteínas de Reptiles/genética , Células Receptoras Sensoriales/metabolismo , Transducción de Señal , Mordeduras de Serpientes/genética , Mordeduras de Serpientes/parasitología , Venenos de Víboras/toxicidadRESUMEN
The Lansberg's hognose pitviper, Porthidium lansbergii lansbergii, inhabits northern Colombia. A recent proteomic characterization of its venom (J. Proteomics [2015] 114, 287-299) revealed the presence of phospholipases A2 (PLA2) accounting for 16.2% of its proteins. The two most abundant PLA2s were biochemically and functionally characterized. Pllans-I is a basic, dimeric enzyme with a monomer mass of 14,136 Da, while Pllans-II is an acidic, monomeric enzyme of 13,901 Da. Both have Asp49 in their partial amino acid sequences and, accordingly, are catalytically active upon natural or synthetic substrates. Nevertheless, these two enzymes differ markedly in their bioactivities. Pllans-I induces myonecrosis, edema, and is lethal by intracerebro-ventricular injection in mice, as well as cytolytic and anticoagulant in vitro. In contrast, Pllans-II is devoid of these effects, except for the induction of a moderate edema. In spite of lacking myotoxicity, Pllans-II enhances the muscle damaging action of Pllans-I in vivo. Altogether, results further illustrate the divergent functional profiles of basic and acidic PLA2s in viperid venoms, and suggest that Pllans-I plays a myotoxic role in envenomings by P. l. lansbergii, whereas Pllans-II, apparently devoid of toxicity, enhances muscle damage caused by Pllans-I.
Asunto(s)
Fosfolipasas A2/metabolismo , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Animales , Línea Celular , Cromatografía Líquida de Alta Presión , Cromatografía de Fase Inversa , Electroforesis en Gel de Poliacrilamida , Femenino , Concentración de Iones de Hidrógeno , Masculino , Ratones , Fosfolipasas A2/química , Fosfolipasas A2/toxicidad , Homología de Secuencia de Aminoácido , Venenos de Víboras/toxicidad , ViperidaeRESUMEN
The action of LAAO, an L-amino acid oxidase isolated from Calloselasma rhodosthoma snake venom, on isolated human neutrophil function was investigated. Cr-LAAO showed no toxicity on neutrophils. Cr-LAAO in its native form induced the neutrophil chemotaxis, suggesting that its primary structure is essential for stimulation the cell. p38 MAPK and PI3K have a role as signaling pathways of CR-LAAO induced chemotaxis. This toxin also induced the production of hydrogen peroxide and stimulated phagocytosis in neutrophils. Furthermore, Cr-LAAO was able to stimulate neutrophils to release IL-6, IL-8, MPO, LTB4 and PGE2. Together, the data showed that the Cr-LAAO triggers relevant proinflammatory events.
Asunto(s)
Quimiotaxis de Leucocito/efectos de los fármacos , L-Aminoácido Oxidasa/toxicidad , Venenos de Víboras/enzimología , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Animales , Citocinas/metabolismo , Dinoprostona/metabolismo , Humanos , Leucotrieno B4/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/enzimología , Neutrófilos/metabolismo , Peroxidasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , ViperidaeRESUMEN
The historical development of discoveries and conceptual frames for understanding the hemorrhagic activity induced by viperid snake venoms and by hemorrhagic metalloproteinases (SVMPs) present in these venoms is reviewed. Histological and ultrastructural tools allowed the identification of the capillary network as the main site of action of SVMPs. After years of debate, biochemical developments demonstrated that all hemorrhagic toxins in viperid venoms are zinc-dependent metalloproteinases. Hemorrhagic SVMPs act by initially hydrolyzing key substrates at the basement membrane (BM) of capillaries. This degradation results in the weakening of the mechanical stability of the capillary wall, which becomes distended owing of the action of the hemodynamic biophysical forces operating in the circulation. As a consequence, the capillary wall is disrupted and extravasation occurs. SVMPs do not induce rapid toxicity to endothelial cells, and the pathological effects described in these cells in vivo result from the mechanical action of these hemodynamic forces. Experimental evidence suggests that degradation of type IV collagen, and perhaps also perlecan, is the key event in the onset of microvessel damage. It is necessary to study this phenomenon from a holistic, systemic perspective in which the action of other venom components is also taken into consideration.
Asunto(s)
Hemorragia/inducido químicamente , Metaloendopeptidasas/toxicidad , Proteínas de Reptiles/toxicidad , Venenos de Víboras/enzimología , Animales , Membrana Basal/efectos de los fármacos , Células Endoteliales/efectos de los fármacos , Células Endoteliales/patología , Hemorragia/patología , Microvasos/efectos de los fármacos , Microvasos/patologíaRESUMEN
BACKGROUND: An alarming number of fatal accidents involving snakes are annually reported in Africa and most of the victims suffer from permanent local tissue damage and chronic disabilities. Envenomation by snakes belonging to the genus Bitis, Viperidae family, are common in Sub-Saharan Africa. The accidents are severe and the victims often have a poor prognosis due to the lack of effective specific therapies. In this study we have biochemically characterized venoms from three different species of Bitis, i.e., Bitis arietans, Bitis gabonica rhinoceros and Bitis nasicornis, involved in the majority of the human accidents in Africa, and analyzed the in vitro neutralizing ability of two experimental antivenoms. METHODOLOGY/PRINCIPAL FINDINGS: The data indicate that all venoms presented phospholipase, hyaluronidase and fibrinogenolytic activities and cleaved efficiently the FRET substrate Abz-RPPGFSPFRQ-EDDnp and angiotensin I, generating angiotensin 1-7. Gelatinolytic activity was only observed in the venoms of B. arietans and B. nasicornis. The treatment of the venoms with protease inhibitors indicated that Bitis venoms possess metallo and serinoproteases enzymes, which may be involved in the different biological activities here evaluated. Experimental antivenoms produced against B. arietans venom or Bitis g. rhinoceros plus B. nasicornis venoms cross-reacted with the venoms from the three species and blocked, in different degrees, all the enzymatic activities in which they were tested. CONCLUSION: These results suggest that the venoms of the three Bitis species, involved in accidents with humans in the Sub-Saharan Africa, contain a mixture of various enzymes that may act in the generation and development of some of the clinical manifestations of the envenomations. We also demonstrated that horse antivenoms produced against B. arietans or B. g. rhinoceros plus B. nasicornis venoms can blocked some of the toxic activities of these venoms.
Asunto(s)
Antivenenos/metabolismo , Inhibidores de Proteasas/metabolismo , Venenos de Víboras/enzimología , África del Sur del Sahara , Animales , Antivenenos/uso terapéutico , Reacciones Cruzadas , Caballos , Humanos , Venenos de Víboras/antagonistas & inhibidores , Venenos de Víboras/metabolismo , Viperidae/metabolismoRESUMEN
Snake-venom proteins form multi-component defence systems by the recruitment and rapid evolution of nonvenomous proteins and hence serve as model systems to understand the structural modifications that result in toxicity. L-Amino-acid oxidases (LAAOs) are encountered in a number of snake venoms and have been implicated in the inhibition of platelet aggregation, cytotoxicity, haemolysis, apoptosis and haemorrhage. An L-amino-acid oxidase from Lachesis muta venom has been purified and crystallized. The crystals belonged to space group P21, with unit-cell parameters a=66.05, b=79.41, c=100.52â Å, ß=96.55°. The asymmetric unit contained two molecules and the structure has been determined and partially refined at 3.0â Å resolution.
Asunto(s)
L-Aminoácido Oxidasa/química , Venenos de Víboras/química , Venenos de Víboras/enzimología , Viperidae , Animales , Cristalización , L-Aminoácido Oxidasa/aislamiento & purificación , Difracción de Rayos XRESUMEN
The study of venom components is an important step toward understanding the mechanism of action of such venoms and is indispensable for the development of new therapies. This work aimed to investigate the venom of Lachesis muta rhombeata and evaluate enzymes related to its toxicity. Phospholipase A2 (PLA(2)), L-amino acid oxidase (LAAO), and proteinase activities were measured, and the molecular weights were estimated. We found the venom to contain one PLA(2) (17 kDa), one LAAO (132 kDa), and three serine proteinases (40, 31, and 20 kDa). Although only serine proteinases were observed in the zymogram, metalloproteinases were found to contribute more to the total proteolytic activity than did serine proteinases. The work confirmed the presence of highly active enzymes; and, moreover, we proposed a novel method for confirming the presence of LAAOs by zymography. We also suggested a simple step to increase the sensitivity of proteinase assays.
Asunto(s)
L-Aminoácido Oxidasa/química , Péptido Hidrolasas/química , Fosfolipasas A2/química , Venenos de Víboras/enzimología , Viperidae , Animales , Caseínas/química , Embrión de Pollo , Eritrocitos/efectos de los fármacos , Hemólisis/efectos de los fármacos , Leucina/química , Ratones , Peso Molecular , Oxidación-Reducción , Fosfolipasas A2/farmacología , Inhibidores de Proteasas/química , Proteolisis , Albúmina Sérica Bovina/química , Venenos de Víboras/química , Venenos de Víboras/farmacologíaRESUMEN
Snake Venom Metalloproteinases (SVMPs) are the most abundant components present in Viperidae venom. They are important in the induction of systemic alterations and local tissue damage after envenomation. In the present study, a metalloproteinase named BpMPI was isolated from Bothropoides pauloensis snake venom and its biochemical and enzymatic characteristics were determined. BpMPI was purified in two chromatography steps on ion exchange CM-Sepharose Fast flow and Sephacryl S-300. This protease was homogeneous on SDS-PAGE and showed a single chain polypeptide of 20kDa under non reducing conditions. The partial amino acid sequence of the enzyme showed high similarity with other SVMPs enzymes from snake venoms. BpMPI showed proteolytic activity upon azocasein and bovine fibrinogen and was inhibited by EDTA, 1,10 phenanthroline and ß-mercaptoethanol. Moreover, this enzyme showed stability at neutral and alkaline pH and it was inactivated at high temperatures. BpMPI was able to hydrolyze glandular and tissue kallikrein substrates, but was unable to act upon factor Xa and plasmin substrates. The enzyme did not induce local hemorrhage in the dorsal region of mice even at high doses. Taken together, our data showed that BpMP-I is in fact a fibrinogenolytic metalloproteinase and a non hemorrhagic enzyme.
Asunto(s)
Fibrinógeno/química , Metaloproteasas/aislamiento & purificación , Proteínas de Reptiles/aislamiento & purificación , Venenos de Víboras/enzimología , Viperidae , Secuencia de Aminoácidos , Animales , Pruebas de Coagulación Sanguínea , Caseínas/química , Secuencia Conservada , Hemorragia/inducido químicamente , Masculino , Metaloproteasas/química , Metaloproteasas/toxicidad , Ratones , Datos de Secuencia Molecular , Proteolisis , Proteínas de Reptiles/química , Proteínas de Reptiles/toxicidad , Alineación de Secuencia , Análisis de Secuencia de ProteínaRESUMEN
The snake genus Lachesis provokes 2 to 3% of snakebites in Colombia every year. Two Lachesis species, L. acrochorda and L. muta, share habitats with snakes from another genus, namely Bothrops asper and B. atrox. Lachesis venom causes systemic and local effects such as swelling, hemorrhaging, myonecrosis, hemostatic disorders and nephrotoxic symptoms similar to those induced by Bothrops, Portidium and Bothriechis bites. Bothrops antivenoms neutralize a variety of Lachesis venom toxins. However, these products are unable to avoid coagulation problems provoked by Lachesis snakebites. Thus, it is important to ascertain whether the envenomation was caused by a Bothrops or Lachesis snake. The present study found enzyme linked immunosorbent assay (ELISA) efficient for detecting Lachesis acrochorda venom in a concentration range of 3.9 to 1000 ng/mL, which did not show a cross-reaction with Bothrops, Portidium, Botriechis and Crotalus venoms. Furthermore, one fraction of L. acrochorda venom that did not show crossreactivity with B. asper venom was isolated using the same ELISA antibodies; some of its proteins were identified including one Gal-specific lectin and one metalloproteinase. This test may be useful to physicians, since it could be applicable for tracking the kinetic distribution of antigens in patients or experimentally envenomed animals.(AU)
Asunto(s)
Animales , Venenos de Víboras/enzimología , /métodos , Ensayo de Inmunoadsorción Enzimática/veterinaria , Reacciones CruzadasRESUMEN
The LYS49-PLA2s myotoxins have attracted attention as models for the induction of myonecrosis by a catalytically independent mechanism of action. Structural studies and biological activities have demonstrated that the myotoxic activity of LYS49-PLA2 is independent of the catalytic activity site. The myotoxic effect is conventionally thought to be to due to the C-terminal region 111-121, which plays an effective role in membrane damage. In the present study, Bn IV LYS49-PLA2 was isolated from Bothrops neuwiedi snake venom in complex with myristic acid (CH3(CH2)12COOH) and its overall structure was refined at 2.2 Å resolution. The Bn IV crystals belong to monoclinic space group P21 and contain a dimer in the asymmetric unit. The unit cell parameters are a = 38.8, b = 70.4, c = 44.0 Å. The biological assembly is a "conventional dimer" and the results confirm that dimer formation is not relevant to the myotoxic activity. Electron density map analysis of the Bn IV structure shows clearly the presence of myristic acid in catalytic site. The relevant structural features for myotoxic activity are located in the C-terminal region and the Bn IV C-terminal residues NKKYRY are a probable heparin binding domain. These findings indicate that the mechanism of interaction between Bn IV and muscle cell membranes is through some kind of cell signal transduction mediated by heparin complexes.
Asunto(s)
Bothrops , Lisina , Ácido Mirístico/metabolismo , Fosfolipasas A2/química , Fosfolipasas A2/metabolismo , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cristalografía por Rayos X , Heparina/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Multimerización de Proteína , Estructura Cuaternaria de Proteína , Análisis de Secuencia de ADNRESUMEN
A serine protease from Bothrops alternatus snake venom was isolated using DEAE-Sephacel, Sephadex G-75 and Benzamidine-Sepharose column chromatography. The purified enzyme, named Bhalternin, ran as a single protein band on analytical polyacrylamide gel electrophoresis (SDS-PAGE) and showed molecular weights of 31,500 and 27,000 under reducing and non-reducing conditions, respectively. Its complete cDNA was obtained by RT-PCR and the 708bp codified for a mature protein of 236 amino acid residues. The multiple alignment of its deduced amino acid sequence showed a structural similarly with other serine proteases from snake venoms. Bhalternin was proteolytically active against bovine fibrinogen and albumin as substrates. When Bhalternin and bovine fibrinogen were incubated at 37 degrees C, at a ratio of 1:100 (w/w), the enzyme cleaved preferentially the Aalpha-chain, apparently not degrading the Bbeta and gamma-chains. Stability tests showed that the intervals of optimum temperature and pH for the fibrinogenolytic activity were 30-40 degrees C and 7.0-8.0, respectively. Also, the inhibitory effects of benzamidine on the fibrinogenolytic activity of Bhalternin indicate that it is a serine protease. This enzyme caused morphological alterations in heart, liver, lung and muscle of mice and it was found to cause blood clotting in vitro and defibrinogenation when intraperitoneally administered to mice, suggesting it to be a thrombin-like enzyme. Therefore, Bhaltenin may be of interest as a therapeutic agent in the treatment and prevention of thrombotic disorders.
Asunto(s)
Bothrops , Venenos de Crotálidos/enzimología , Trombina/química , Venenos de Víboras/enzimología , Albúminas/química , Secuencia de Aminoácidos , Animales , Coagulación Sanguínea/efectos de los fármacos , Caseínas/química , Venenos de Crotálidos/farmacología , ADN Complementario/biosíntesis , ADN Complementario/genética , Electroforesis en Gel de Poliacrilamida , Glándulas Exocrinas/química , Fibrinógeno/química , Fibrinolíticos/farmacología , Biblioteca de Genes , Masculino , Ratones , Datos de Secuencia Molecular , Peso Molecular , Músculo Esquelético/patología , Necrosis/inducido químicamente , Necrosis/patología , Péptido Hidrolasas/química , Proteínas/química , Serina Proteasas/farmacología , Venenos de Víboras/farmacologíaRESUMEN
Phospholipases A(2) (PLA(2)s) with a lysine substituting for the highly conserved aspartate 49, Lys49 PLA(2) homologues, are important myotoxic components in venoms from snakes of Viperidae family. These proteins induce conspicuous myonecrosis by a catalytically-independent mechanism. Traditionally, the Lys49 PLA(2) homologues are classified as non-neurotoxic myotoxins given their inability to cause lethality or paralytic effects when injected in vivo, even at relatively high doses. However, a series of in vitro studies has shown that several Lys49 PLA(2) homologues from Bothrops snake venoms induce neuromuscular blocking activity on nerve-muscle preparations in vitro. The interpretation of these findings has created some confusion in the literature, raising the question whether the Lys49 PLA(2) homologues present some neurotoxic activity. The present article reviews the in vitro neuromuscular effects of Lys49 PLA(2) homologues and discusses their possible mechanisms of action. It was concluded that the neuromuscular blockade induced by Lys49 PLA(2) homologues in isolated preparations is mainly a consequence of the general membrane-destabilizing effect of these toxins.
Asunto(s)
Unión Neuromuscular/efectos de los fármacos , Fosfolipasas A2/toxicidad , Proteínas de Reptiles/toxicidad , Venenos de Víboras/enzimología , Animales , Humanos , Membranas/efectos de los fármacos , Músculos/efectos de los fármacos , Neuronas/efectos de los fármacos , Especificidad de la Especie , ViperidaeRESUMEN
Hemostatically active snake venom metalloproteinases (SVMPs) perturb the blood coagulation cascade at specific points and due to their potential application as thrombolytic agents, the fibrin(ogen)olytic non-hemorrhagic SVMPs have been employed as biochemical tools in coagulation research and diagnosis. Structural studies complemented by the design of metalloproteinase inhibitors have been instrumental in understanding their stereo specificity and action mechanism. We present here, details of the crystal structure of BmooMPalpha-I, a 22.6 kDa non-hemorrhagic P-I class SVMP isolated from Bothrops moojeni venom, determined at 1.76 A resolution. In this structure, the catalytic zinc ion displays an unusual octahedral coordination formed by the three canonical histidines (His(142), His(146) and His(152)) and additionally, by three solvent molecules. Comparative sequence and structural studies indicate that the motif comprising amino acid segments 153-164 and 167-176 adjacent to the methionine-turn is a salient feature that differentiates both non and hemorrhagic P-I class SVMPs and could directly be involved in the development of the hemorrhagic activity.
Asunto(s)
Bothrops/fisiología , Metaloproteasas/química , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Animales , Cristalización , Electroforesis en Gel de Poliacrilamida , Hemorragia/inducido químicamente , Metaloproteasas/antagonistas & inhibidores , Metaloproteasas/farmacología , Modelos Moleculares , Datos de Secuencia Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Unión Proteica , Conformación Proteica , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Especificidad por Sustrato , Venenos de Víboras/farmacología , Difracción de Rayos X , Zinc/químicaRESUMEN
Various toxins isolated from Bothrops snake venoms induce inflammatory reactions and have been claimed to contribute to the severity of local symptoms present in this envenomation. Notwithstanding, the relative participation of serine proteases, metalloproteases and phospholipases A(2) in the inflammatory reaction produced by crude Bothrops venoms is poorly understood. Herein, crude Bothrops jararaca venom was treated with phenylmethanesulfonyl fluoride (PMSF), 1,10-phenanthroline (oPhe), or p-bromophenacyl-bromide (p-BPB) to inhibit those classes of enzymes, respectively, and inflammatory parameters were evaluated and compared to those induced by the control crude venom. The intensity of edema and hyperalgesia/allodynia was remarkably reduced in animals administered with oPhe-treated venom. Leukocyte-endothelium interactions (LEI), such as adhesion and migration of leukocytes, were also modified at 2h and 24h. Edema and LEI parameters induced by p-BPB-treated venom were similar to those observed with the control venom, but hyperalgesia/allodynia was significantly lower. Inflammatory parameters induced by PMSF-treated venom were similar to those induced by the crude venom, except for a mild reduction in edema intensity. Our results indicate that metalloproteases have a pivotal role in the inflammatory reactions induced by B. jararaca venom, and phospholipases A(2) and serine proteases have a minor role.
Asunto(s)
Bothrops , Inflamación/inducido químicamente , Metaloproteasas/toxicidad , Fosfolipasas A2/toxicidad , Serina Proteasas/toxicidad , Venenos de Víboras/enzimología , Venenos de Víboras/toxicidad , Animales , Movimiento Celular/efectos de los fármacos , Edema/inducido químicamente , Edema/patología , Células Endoteliales/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/psicología , Inflamación/patología , Masculino , Ratones , Neutrófilos/efectos de los fármacos , Dimensión del Dolor/efectos de los fármacos , Inhibidores de Fosfolipasa A2 , Estimulación Física , Inhibidores de Proteasas/química , Inhibidores de Proteasas/farmacología , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/farmacologíaRESUMEN
A particular subgroup of toxins with phospholipase A(2) (PLA(2)) structure, but devoid of this enzymatic activity, is commonly found in the venoms of snakes of the family Viperidae, and known as the PLA(2) homologues. Among these, the most frequent type presents a lysine residue at position 49 (Lys49), in substitution of the otherwise conserved aspartate (Asp49) of catalytically-active PLA(2)s. A brief and updated overview of these toxic PLA(2) homologues is presented, emphasizing their various biological activities, both in vivo and in vitro. The relevance of these bioactivities in relation to their possible adaptive roles for the snakes is discussed. Finally, experiments designed to assess the validity of such hypothetical roles are suggested, to stimulate future studies in this field.
Asunto(s)
Fosfolipasas A2/metabolismo , Venenos de Víboras/enzimología , Viperidae , Animales , Fosfolipasas A2/químicaRESUMEN
Phospholipases A(2) homologues are found in the venom of Crotalinae snakes, being their main action related to myonecrosis induction. Although many studies on these toxins had already been performed, their mechanism of action remains unclear. Here, important aspects about these toxins are reviewed, including their correct biological assembly and how essential is the natural substitution D49K for their catalytic inactivity.
Asunto(s)
Fosfolipasas A2/química , Venenos de Víboras/enzimología , Viperidae , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Fosfolipasas A2/metabolismo , Fosfolipasas A2/toxicidad , Alineación de SecuenciaRESUMEN
HF3 and bothropasin are P-III hemorrhagic snake venom metalloproteinases (SVMPs) of Bothrops jararaca. The DC protein is composed of the disintegrin-like/cysteine-rich domains derived from the autolysis of P-III SVMPs. Here we describe simplified procedures for the isolation of HF3, bothropasin, the DC protein, and BJ-PI, a novel P-I SVMP. The isolated proteins were identified by mass spectrometry. BJ-PI is a potent caseinolytic enzyme devoid of hemorrhagic activity. HF3, bothropasin and BJ-PI show distinct fibrinogenolytic activities.
Asunto(s)
Bothrops/fisiología , Venenos de Crotálidos/aislamiento & purificación , Metaloendopeptidasas/aislamiento & purificación , Metaloproteasas/aislamiento & purificación , Venenos de Víboras/enzimología , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Venenos de Crotálidos/toxicidad , Electroforesis en Gel de Poliacrilamida , Hemorragia/inducido químicamente , Hemorragia/patología , Metaloendopeptidasas/toxicidad , Metaloproteasas/toxicidad , Ratones , Datos de Secuencia Molecular , Tripsina/químicaRESUMEN
Venoms from the bee Apis mellifera, the caterpillar Lonomia achelous, the spiders Lycosa sp. and Phoneutria nigriventer, the scorpions Tityus bahiensis and Tityus serrulatus, and the snakes Bothrops alternatus, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni, Bothrops neuwiedi, Crotalus durissus terrificus, and Lachesis muta were assayed (800mug/mL) for activity against Staphylococcus aureus. Venoms from B. jararaca and B. jararacussu showed the highest S. aureus growth inhibition and also against other Gram-positive and Gram-negative bacteria. To characterize the microbicidal component(s) produced by B. jararaca, venom was fractionated through gel exclusion chromatography. The high molecular weight, anti-S. aureus P1 fraction was further resolved by anion exchange chromatography through Mono Q columns using a 0-0.5M NaCl gradient. Bactericidal Mono Q fractions P5 and P6 showed significant LAAO activity using l-leucine as substrate. These fractions were pooled and subjected to Heparin affinity chromatography, which rendered a single LAAO activity peak. The anti-S. aureus activity was abolished by catalase, suggesting that the effect is dependent on H(2)O(2) production. SDS-PAGE of isolated LAAO indicated the presence of three isoforms since deglycosylation with a recombinant N-glycanase rendered a single 38.2 kDa component. B. jararaca LAAO specific activity was 142.7 U/mg, based on the oxidation of l-leucine. The correlation between in vivo neutralization of lethal toxicity (ED(50)) and levels of horse therapeutic antibodies anti-LAAO measured by ELISA was investigated to predict the potency of Brazilian antibothropic antivenoms. Six horses were hyperimmunized with Bothrops venoms (50% from B. jararaca and 12.5% each from B. alternatus, B. jararacussu, B. neuwiedii and B. moojeni). To set up an indirect ELISA, B. jararaca LAAO and crude venom were used as antigens. Correlation coefficients (r) between ED(50) and ELISA antibody titers against B. jararaca venom and LAAO were 0.846 (p<0.001) and 0.747 (p<0.001), respectively. The hemolytic and leishmanicidal (anti-Leishmania amazonensis) activity of LAAO was also determined.