RESUMEN
Bothrops jararaca is a pit viper responsible for the majority of snake envenoming accidents in Brazil. As an attempt to describe the transcriptional activity of the venom gland, ESTs of a cDNA library constructed from B. jararaca venom gland were generated and submitted to bioinformatics analysis. The results showed a clear predominance of transcripts coding for toxins instead of transcripts coding for proteins involved in cellular functions. Among toxins, the most frequent transcripts were from metalloproteinases (52.6%), followed by serine-proteinases (28.5%), C-type lectins (8.3%) and bradykinin-potentiating peptides (BPPs) (6.2%). Results were similar to that obtained from the transcriptome analysis of B. insularis, a phylogenetically close sister of B. jararaca, though some differences were observed and are pointed out, such as a higher amount of the hypotensive BPPs in B. insularis transcriptome (19.7%). Another striking difference observed is that PIII and PII-classes of metalloproteinases are similarly represented in B. jararaca in contrast to B. insularis, in which a predominance of PIII-class metalloproteinase, which present a more intense hemorrhagic action, is observed. These features may, in part, explain the higher potency of B. insularis venom. The results obtained can help in proteome studies, and the clones can be used to directly probe the genetic material from other snake species or to investigate differences in gene expression pattern in response to factors such as diet, aging and geographic localization.
Asunto(s)
Bothrops/metabolismo , Venenos de Crotálidos/biosíntesis , Perfilación de la Expresión Génica , Animales , Biblioteca de Genes , Lectinas Tipo C/genética , Metaloproteasas/genética , Filogenia , Serina Endopeptidasas/genética , Teprotido/metabolismoRESUMEN
Disintegrins are small peptides isolated from the venom of several snake families which act as integrin-antagonists or agonists, interacting with a variety of biological processes mediated by integrins. In this work we describe five new disintegrin-like domains within metalloproteinase precursor sequences, obtained from a Bothrops jararaca venom gland cDNA library. Among the new disintegrin-like domains, four were contained in PIII metalloproteinase precursors, with three of them presenting ECD-motifs and one presenting a new KCD-motif. Moreover, we found three disintegrin-like domains within PII metalloproteinase precursors. Two of them are similar to the already described disintegrins jarastatin and jararacin. The third molecule is unusual, presenting some typical PIII metalloproteinase characteristics but lacking the cysteine-rich domain being, thus, classified as a PII metalloproteinase. Only few reports presented molecules with these characteristics. Sequence analysis suggests that these molecules are intermediate steps between the more ancient PIII and the more recent PII metalloproteinases. We also investigated disintegrin N-terminus diversity in B. jararaca crude venom by purifying jarastatin and jararacin and analyzing them by mass spectrometry.
Asunto(s)
Bothrops/fisiología , Venenos de Crotálidos/genética , Desintegrinas/genética , Precursores Enzimáticos/genética , Variación Genética , Metaloproteasas/genética , Secuencia de Aminoácidos , Animales , Dominio Catalítico/genética , Venenos de Crotálidos/química , Venenos de Crotálidos/enzimología , ADN Complementario/genética , Desintegrinas/química , Desintegrinas/aislamiento & purificación , Precursores Enzimáticos/metabolismo , Metaloproteasas/metabolismo , Datos de Secuencia Molecular , Filogenia , Alineación de Secuencia , Especificidad de la Especie , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-ActividadRESUMEN
Bothrops snake venoms contain metalloproteinases that contribute to the local effects seen after envenoming. In this work, a hemorrhagic metalloproteinase (BlaH1) was purified from the venom of the snake Bothrops lanceolatus by a combination of gel filtration, affinity (metal chelating) and hydrophobic interaction chromatographies. The hemorrhagin was homogeneous by SDS-PAGE and had a molecular mass of 28 kDa that was unaltered by treatment with beta-mercaptoethanol. BlaH1 gave a single band in immunoelectrophoresis and immunoblotting using commercial bothropic antivenom. BlaH1 had hemorrhagic, caseinolytic, fibrinogenolytic, collagenolytic and elastinolytic activities, but no phospholipase A(2) activity. The hemorrhagic and caseinolytic activities were inhibited by EDTA, indicating that they were metal ion-dependent. In contrast, aprotinin, benzamidine and PMSF did not affect these activities. The caseinolytic activity of BlaH1 had a pH optimum of 8.0 and was stable in solution at up to 40 degrees C; activity was completely lost at > or =70 degrees C. The hemorrhagic activity was neutralized by commercial bothropic antivenom. These properties suggest that this new hemorrhagin belongs to class P-I snake venom metalloproteinases.
Asunto(s)
Bothrops , Venenos de Crotálidos/aislamiento & purificación , Metaloendopeptidasas/aislamiento & purificación , Animales , Aprotinina/metabolismo , Benzamidinas/metabolismo , Caseínas/metabolismo , Cromatografía de Afinidad , Cromatografía en Gel , Colagenasas/metabolismo , Venenos de Crotálidos/química , Venenos de Crotálidos/metabolismo , Ácido Edético/metabolismo , Electroforesis en Gel de Poliacrilamida , Endopeptidasas/metabolismo , Esterasas/metabolismo , Fibrinógeno/metabolismo , Immunoblotting , Masculino , Metaloendopeptidasas/química , Metaloendopeptidasas/metabolismo , Fosfolipasas A/metabolismo , Ratas , Ratas Wistar , TemperaturaRESUMEN
The ability of Bothrops lanceolatus venom to induce neutrophil migration into the peritoneal cavity of mice was investigated. Intraperitoneal injection of venom caused dose- and time-dependent neutrophil migration, which peaked with 750 ng of venom/cavity 4h after venom injection. The neutrophil migration was significantly reduced by pretreatment with dexamethasone (0.5 mg/kg, s.c.), an indirect inhibitor of phospholipase A(2) (PLA(2)), and AA861 (0.01 mg/kg, s.c.), a 5-lipoxygenase inhibitor, but in contrast, was not modified by pretreatment with indomethacin (2 mg/kg, s.c.), an inhibitor of the cyclooxygenase pathway, meloxicam (5 mg/kg, s.c.), an inhibitor of the cyclooxygenase-2 pathway, or the PAF inhibitor WEB2086 (40 mg/kg, s.c.). Dexamethasone and AA861 also inhibited the neutrophil migration by 60% when administered immediately after venom injection, and the coadministration of these two drugs caused a 75% reduction in migration. BLV-induced neutrophil migration was not due to contamination by endotoxin since polymyxin B-treated venom retained its activity. Heating the venom (97 degrees C, 2 min) reduced the PLA(2) activity by 64% and this was accompanied by a corresponding reduction (68%) in neutrophil migration. These results suggest that arachidonate-derived lipoxygenase metabolites (possibly leukotriene B(4)) are involved in the chemotaxis observed. Macrophages may be an important source of these metabolites since the migratory response to venom was potentiated in mice pretreated with thioglycollate, but reduced when the peritoneal cavity was washed with sterile saline.
Asunto(s)
Bothrops , Venenos de Crotálidos/toxicidad , Neutrófilos/efectos de los fármacos , Animales , Azepinas/farmacología , Benzoquinonas/farmacología , Inhibición de Migración Celular , Venenos de Crotálidos/administración & dosificación , Dexametasona/farmacología , Relación Dosis-Respuesta a Droga , Indometacina/farmacología , Inyecciones Intraperitoneales , Macrófagos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Fosfolipasas A/metabolismo , Triazoles/farmacologíaRESUMEN
A protein capable of inducing neuromuscular blockade in avian preparations and of depolarizing mouse diaphragm muscle was isolated from Bothrops lanceolatus venom using gel filtration and ion-exchange chromatography. The purified protein was a single chain polypeptide with an estimated molecular mass of 27.5 kDa by SDS-PAGE and had caseinolytic activity (13.3 units/mg), but no phospholipase A(2). B.lanceolatus venom (50 micro g/ml) and the caseinolytic protein (20 micro g/ml) produced contracture and progressive irreversible blockade (50% in 25+/-5 min (SEM) and 45+/-15 min, respectively), in indirectly stimulated chick biventer cervicis preparations. The contractile responses to acetylcholine (ACh; 37 and 74 micro M, n=6) were inhibited by venom and the caseinolytic protein, whereas those to potassium (13.4mM, n=6) were not. Membrane resting potential measurements in mouse hemidiaphragm preparations showed that B.lanceolatus venom and the purified protein caused depolarization which was prevented by D-tubocurarine (14.6mM). The venom produced a slight increase in the amplitude and frequency of miniature end-plate potentials, but this effect was not seen with the purified fraction. These results suggest that the purified protein acts exclusively post-synaptically.
Asunto(s)
Bothrops , Venenos de Crotálidos/farmacología , Bloqueantes Neuromusculares/farmacología , Unión Neuromuscular/efectos de los fármacos , Péptido Hidrolasas/farmacología , Animales , Caseínas/metabolismo , Pollos , Cromatografía en Gel , Cromatografía por Intercambio Iónico , Venenos de Crotálidos/química , Diafragma/efectos de los fármacos , Diafragma/fisiopatología , Electroforesis en Gel de Poliacrilamida , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Ratones , Placa Motora/efectos de los fármacos , Placa Motora/fisiopatología , Contracción Muscular/efectos de los fármacos , Contracción Muscular/fisiología , Bloqueo Neuromuscular , Bloqueantes Neuromusculares/química , Unión Neuromuscular/fisiopatología , Péptido Hidrolasas/aislamiento & purificación , Péptido Hidrolasas/metabolismoRESUMEN
La crotoxina, el mayor componente tóxico del veneno de serpiente cascabel sudamericana Crotalus durissus terrificus, es una fosolipasa A2 neurotóxica que ejerce su acción bloqueando la transmisión neuromuscular. Actúa primariamente alterando la liberación de acetilcolina de las terminales nerviosas mediante un mecanismo todavía no elucidado. Actúa también en membranas postsinápticas estabilizando el receptor de acetilcolina en una configuración inactiva semejante al estado de desensibilización. La crotoxina comprende dos subnuidades distintas: una fosoflipasa A2 básica y débilmente tóxica (componente B) y una acídica y no tóxica (componente A) que no posee actividad enzimática. La subunidad de fosfolipasa A2 se une en forma inespecífica y no saturable a membranas biológicas, mientras que en presencia delo componente A interacciona solamente con un limitado número de sitios de unión de alta afinidad presentes en membranas sinápticas pero no en eritrocitos. Experimentos de unión realizados con vesículas fosfolipídicas unilamelares de diferente composición indicaron que algunos de los fosfolípidos cargados negativamente, como los mono y difosfoinositósidos, podrían ser parte del sitio aceptor de crotoxina. La crotoxina es en realidad una mexcla de diversas isoformas de estructura peptídica similar pero no idéntica. Estas isoformas difieren levemente en su actividad enzimática y farmacológica. Estudios realizados con anticuerpos policonales preparados contra ambas subunidades anticomponente B (Fab) inhiben la actividad fosfolipasa A2 y neutralizan la potencia letal, lo que sugiere que los sitios tóxicos y catalíticos de la crotoxina están relacionados (AU)
Asunto(s)
Crotoxina , Fosfolipasas A/metabolismo , Unión Neuromuscular/fisiología , Transmisión Sináptica/efectos de los fármacos , Estructura MolecularRESUMEN
La crotoxina, el mayor componente tóxico del veneno de serpiente cascabel sudamericana Crotalus durissus terrificus, es una fosolipasa A2 neurotóxica que ejerce su acción bloqueando la transmisión neuromuscular. Actúa primariamente alterando la liberación de acetilcolina de las terminales nerviosas mediante un mecanismo todavía no elucidado. Actúa también en membranas postsinápticas estabilizando el receptor de acetilcolina en una configuración inactiva semejante al estado de desensibilización. La crotoxina comprende dos subnuidades distintas: una fosoflipasa A2 básica y débilmente tóxica (componente B) y una acídica y no tóxica (componente A) que no posee actividad enzimática. La subunidad de fosfolipasa A2 se une en forma inespecífica y no saturable a membranas biológicas, mientras que en presencia delo componente A interacciona solamente con un limitado número de sitios de unión de alta afinidad presentes en membranas sinápticas pero no en eritrocitos. Experimentos de unión realizados con vesículas fosfolipídicas unilamelares de diferente composición indicaron que algunos de los fosfolípidos cargados negativamente, como los mono y difosfoinositósidos, podrían ser parte del sitio aceptor de crotoxina. La crotoxina es en realidad una mexcla de diversas isoformas de estructura peptídica similar pero no idéntica. Estas isoformas difieren levemente en su actividad enzimática y farmacológica. Estudios realizados con anticuerpos policonales preparados contra ambas subunidades anticomponente B (Fab) inhiben la actividad fosfolipasa A2 y neutralizan la potencia letal, lo que sugiere que los sitios tóxicos y catalíticos de la crotoxina están relacionados