Isolation, structural and functional characterization of a new Lys49 phospholipase A2 homologue from Bothrops neuwiedi urutu with bactericidal potential.

Snake venom is a complex mixture of active compounds consisting of 80-90% proteins and peptides that exhibit a variety of biological actions that are not completely clarified or identified. Of these, phospholipase A2 is one of the molecules that has shown great biotechnological potential. The objectives of this study were to isolate, biochemically and biologically characterize a Lys49 phospholipase A2 homologue from the venom of Bothrops neuwiedi urutu. The protein was purified after two chromatographic steps, anion exchange and reverse phase. The purity and relative molecular mass were assessed by SDS-PAGE, observing a molecular weight typical of PLA2s, subsequently confirmed by mass spectrometry obtaining a mass of 13,733 Da. As for phospholipase activity, the PLA2 proved to be enzymatically inactive. The analyses by Edman degradation and sequencing of the peptide fragments allowed for the identification of 108 amino acid residues; this sequence showed high identity with other phospholipases A2 from Bothrops snake venoms, and identified this molecule as a novel PLA2 isoform from B. neuwiedi urutu venom, called BnuTX-I. In murine models, both BnuTX-I as well as the venom induced edema and myotoxic responses. The cytotoxic effect of BnuTX-I in murine macrophages was observed at concentrations above 12 µg/mL. BnuTX-I also presented antimicrobial activity against gram-positive and negative bacterial strains, having the greatest inhibitory effect on Pseudomonas aeruginosa. The results allowed for the identification of a new myotoxin isoform with PLA2 structure with promising biotechnological applications.

Venomics and antivenomics of Bothrops erythromelas from five geographic populations within the Caatinga ecoregion of northeastern Brazil.

The Caatinga lancehead, Bothrops erythromelas, is a medically relevant species, responsible for most of the snakebite accidents in most parts of its distribution range in northeastern Brazil. The spectrum and geographic variability of its venom toxins were investigated applying a venomics approach to venom pools from five geographic areas within the Caatinga ecoregion. Despite its wide habitat, populations of B. erythromelas from Ceará, Pernambuco, Juazeiro, Paraiba, and Ilha de Itaparica exhibit highly conserved venom proteomes. Mirroring their compositional conservation, the five geographic venom pools also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated in Vital Brazil (BR) and Clodomiro Picado (CR) Institutes, using different venoms in the immunization mixtures. The paraspecificity exhibited by the Brazilian SAB and the Costa Rican BCL antivenoms against venom toxins from B. erythromelas indicates large immunoreactive epitope conservation across genus Bothrops during the last ~14 million years, thus offering promise for the possibility of generating a broad-spectrum bothropic antivenom. Biological Significance Accidental snakebite envenomings represent an important public health hazard in Brazil. Ninety per cent of the yearly estimated 20-30,000 snakebite accidents are caused by species of the Bothrops genus. Bothrops erythromelas, a small, moderately stocky terrestrial venomous snake, is responsible for most of the snakebite accidents in its broad distribution range in the Caatinga, a large ecoregion in northeastern Brazil. To gain a deeper insight into the spectrum of medically important toxins present in the venom of the Caatinga lancehead, we applied a venomics approach to define the proteome and geographic variability of adult B. erythromelas venoms from five geographic regions. Although intraspecific compositional variation between venoms among specimens from different geographic regions has long been appreciated by herpetologists and toxinologists as a general feature of highly adaptable and widely distributed snake species, the five B. erythromelas populations investigated exhibit highly conserved venom proteomes. The overall toxin profile of the Caatinga lancehead's venom explains the local and systemic effects observed in envenomations by B. erythromelas. The five geographic venom pools sampled also showed qualitatively and quantitatively overlapping antivenomic profiles against antivenoms generated using different bothropic venoms in the immunization mixtures. The large immunoreactive epitope conservation across genus Bothrops offers promise for the generation of a broad-spectrum bothropic antivenom.

Obtenção e atividade biológica de uma nova disintegrina recombinante do veneno de Bothrops neuwiedi, Dis-BnMPIIx / Obtention and biological activity of a new recombinant disintegrin from Bothrops neuwiedi venom, Dis-BnMPIIx

Metalloproteinases from snake venoms are highly versatile enzymes, responsible for most of the symptoms of envenoming. Its action is related to the proteolysis of extracellular matrix components, activation or hydrolysis of components of the coagulation cascade and fibrinolysis, platelet aggregation inhibition, induction of local and systemic bleeding, pro-inflammatory activity, among others. This broad variety of biological activities is related to the structural diversity of this family of proteins. The SVMPs are divided into classes and subclasses of PI to PIII according to the organization of their domains. The SVMPs class PI are composed only by the catalytic domain, and in the class PIII, besides catalytic domain, SVMPs have disintegrin-like and cysteine-rich domains. The SVMPs class PII are composed of catalytic and disintegrin domain or only disintegrin that generally present the RGD tripeptide. For a better understanding of the structural and functional diversity of SVMPs, cDNAs from venom gland Bothrops neuwiedi encoding SVMPs were sequenced. SVMPs distinct from those described in venoms of other species were found. The BnMPIIx is a SVMP class PII showing a peculiar sequence with the fusion of a catalytic domain typical of procoagulant class P-III SVMPs with an RGDdisintegrin domain of SVMPs classP-II, with the inclusion of cysteines at positions undescribed previously. The aim of this work was to obtain of BnMPIIx and its disintegrin domain in recombinant form using vectors directing to the E. coli periplasm (pET20) or vectors that produce the recombinant protein in fusion with chaperones (pET32 and pSMT3). For this, the cDNAs encoding BnMPIIx and its disintegrin domain were amplified by PCR, digested with endonucleases BamHI and HindIII and cloned into pET20 vector. The disintegrin domain was also cloned into pET32 and pSMT3 vectors that express proteins with chaperones thioredoxin (Trx) and SUMO, respectively. The sequencing of the inserts did not show any mutations introduced during the cloning procedures. These vectors were transformed into the E. coli BL21 (DE3) or C43 (DE3) strains, and expression was evaluated at two temperatures (30°C and 37°C). To test platelet aggregation, human blood was collected in a 3,8% sodium citrate (1:9) from healthy donors. The mixture was centrifuged and the platelet-rich plasma (PRP) was submitted to an ADP-induced platelet aggregation assay. The analysis by SDS-PAGE showed that both BnMPIIx as its disintegrin domain were obtained by pET20 partially soluble in both temperatures (30°C and 37°C), resulting in inclusion bodies. Using the vectors pET32 and pSMT3 the disintegrin domain was expressed in a soluble form and was called Dis-Trx or Dis-SUMO respectively. However, the fusion proteins did not show biological activity. After cleavage of Trx it was not possible to isolate the disintegrin to validate the biological activity of Trx-Dis, but after cleavage of SUMO, the isolated disintegrin completely inhibited platelet aggregation in a concentration of approximately 2 µM. After several attempts using different expression vectors, we obtained a new recombinant disintegrin from Bothrops neuwiedi venom in pSMT3 vector, with preserved biological activity.This molecule may contribute to the study of hemostasis and cancer.

As metaloproteinases de venenos de serpentes (SVMPs) são enzimas altamente versáteis, responsáveis por grande parte dos sintomas do envenenamento. Sua ação está relacionada com a proteólise dos componentes da matriz extracelular, ativação ou hidrólise de componentes da cascata de coagulação e fibrinólise, inibição da agregação plaquetária, indução de hemorragia local e sistêmica, atividade pró-inflamatória, entre outras. Essa ampla gama de atividades biológicas está relacionada com a diversidade estrutural dessa família de proteínas. As SVMPs são divididas em classes e subclasses de PI a PIII de acordo com a organização dos seus domínios. As SVMPs de classe PI são compostas apenas pelo domínio catalítico, já as PIII possuem domínio catalítico, tipo disintegrina e rico em cisteína. As SVMPs PII, possuí domínio catalítico e disintegrina ou apenas de disintegrina livre que geralmente apresenta o tripeptídeo RGD. Para melhor entender a diversidade estrutural e funcional das SVMPs, foram sequenciados cDNAs da glândula de veneno Bothrops neuwiedi que codificam SVMPs. Foram encontradas novas SVMPs, distintas das descritas em venenos de outras espécies. A BnMPIIx, uma SVMP de classe PII,apresentou uma sequência bastante peculiar com a fusão de um domínio catalítico similar ao de SVMPs pró-coagulantes da classe P-III com um domínio RGD-disintegrina, de SVMPs classe P-II, contendo cisteínas em posições não descritas anteriormente. O objetivo deste trabalho foi obter a BnMPIIx e seu domínio disintegrina na forma recombinante através de vetores de E. coli que direcionam para o periplasma (pET20) ou vetores que proporcionam a fusão com chaperonas (pET32 e pSMT3). Para isto, o cDNA da BnMPIIx e de seu dominínio disintegrina foram amplificados por PCR, digeridos com as endonucleases BamHI e HindIII e clonados em vetor pET20. O domínio disintegrina também foi clonado nos vetores pET32 e pSMT3 que expressam as proteínas com as chaperonas Tioredoxina (Trx) e SUMO, respectivamente. O sequenciamento de todos os insertos não apresentou mutações. Estes vetores foram transformados nas linhagens de E. coli BL21 (DE3) ou C43 (DE3) e a expressão avaliada em duas temperaturas (30ºC e 37ºC). Para o ensaio de inibição da agregação plaquetária, foi coletado sangue humano de doadores voluntários saudáveis em 3,8% de citrato de sódio (1:9). O sangue foi centrifugado e o plasma rico em plaquetas (PRP) submetido à agregação plaquetária induzida por ADP. A análise por SDS-PAGE mostrou que tanto a BnMPIIx quanto seu domínio disintegrina foram obtidos parcialmente solúveis em ambas as temperaturas (30ºC e 37ºC), gerando corpos de inclusão em vetor pET20. Já a disintegrina foi obtida com sucesso nos vetores pET32 e pSMT3 e foram denominadas Dis-Trx e Dis-SUMO respectivamente, no entanto não apresentaram atividade biológica. Após a clivagem da Trx, não foi possível isolar a disintegrina para validar a atividade biológica da Dis-Trx, já após a clivagem da SUMO a disintegrina inibiu completamente a agregação plaquetária em concentrações da ordem de 2 µM. Após diversas tentativas de clonagem usando diferentes vetores, nós obtivemos uma nova disintegrina recombinante de Bothrops neuwiedi em vetor pSMT3 com atividade biológica preservada, podendo esta molécula contribuir para os estudos de hemostasia e câncer.

Neutralización heteróloga e interacción de amanitinas con venenos de Bothropsy Crotalus y de abeja / Heterologous neutralization and interaction of toxic amanitins with Bothrops and Crotalus venoms, and honey bee venom / NeutralizaþÒo heteróloga e interaþÒo das amanitinas tóxicas com veneno de Bothrops e Crotalus e veneno de abelha

En el presente trabajo se examinó la interacción de las amanitinas de Amanita phalloides (Basidiomycetes) con los venenos de las serpientes Bothrops neuwiedi diporus ("yarará pequeña"), B. alternatus ("yarará grande"), Crotalus durissus terrificus ("serpiente de cascabel") y de la abeja mielera Apis mellifera. Se aplicaron las técnicas de Ouchterlony, inmunotransferencia, electroforesis rocket y electroforesis en gel de poliacrilamida a los anti-venenos y anti-toxinas obtenidos por inmunización en caballos y/o en conejos. Los anti¡sueros de serpientes y las amanitinas reaccionaron en forma cruzada, así como el veneno de abeja y las amanitinas. Cuando los venenos de Bothrops neuwiedii diporus y Crotalus durissus terrificus se preincubaron con las amanitinas y se analizaron por electroforesis en gel de poliacrilamida-dodecilsulfato de sodio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algunas bandas de proteínas desaparecieron y otras se redujeron notablemente. Estos resultados revelan por primera vez la interacción y la degradación de las proteínas de los venenos de serpientes por las amanitinas. Por otra parte, la modificación del tiempo de coagulación de la sangre humana, debida a los venenos, se corrigió con los ciclopéptidos de Amanita. Estos resultados también se informan por primera vez en este trabajo. La presencia de polipéptidos tóxicos en los venenos de serpientes y abejas, así como en A. phalloides y la reactividad cruzada demostradas en este trabajo, sugieren la existencia de epítopos comunes a todos ellos. Teniendo en cuenta estas reacciones, el uso de anti-venenos heterólogos parece ser de utilidad en el tratamiento del envenenamiento.(AU)

In the present work, the interaction of the amanitins of Amanita phalloides (Basidiomycetes) with the venoms of Bothrops neuwiedi diporus ("small yarará snake"), B. alternatus ("big yarará"), Crotalus durissus terrificus ("rattlesnake"), and honey bee Apis mellifera was examined. Ouchterlony, immunotransfer, rocket-electrophoresis, and polyacrylamide gel electrophoresis techniques were applied to anti-venoms and anti-toxins obtained by immunization in horses and/or in rabbits. Snake antisera and amanitins cross-reacted as well as bee venom and amanitins. When venoms of Bothrops neuwiedii diporus and Crotalus durissus terrificus were preincubated with amanitins and analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some protein bands disappeared and others were significantly reduced. These results reveal for the first time the interaction and degradation of proteins in snake venoms by amanitins. Moreover, the modification of the human blood clotting time due to snake venoms was corrected by the Amanita cyclopeptides. These results are also reported for the first time in this work. The occurrence of toxic polypeptides in the snake and bee venoms as well as in A. phalloides, and the cross-reactivity demostrated herein, suggest the occurrence of epitopes common to all of them. Taking into account these reactions,the use of heterologous anti-venoms seems to be of value in envenomation treatment.(AU)

No presente trabalho foi examinada a interaþÒo das amanitinas de Amanita phalloides (Basidiomy¡cetes) com os venenos das serpentes Bothrops neuwiedi diporus ("jararaca-cruzeira"), B. alternatus ("urutu"), Crotalus durissus terrificus ("serpente cascavel") e da abelha-europeia Apis mellifera. Foram aplicadas as técnicas de Ouchterlony, imunotransferÛncia, eletroforese rocket e eletroforese em gel de poliacrilamida aos anti-venenos e anti-toxinas obtidos por imunizaþÒo em cavalos e/ou em coelhos. Os anti-soros de serpentes e as amanitinas reagiram em forma cruzada, bem como o veneno de abelha e as amanitinas. Quando os venenos de Bothrops neuwiedii diporus e Crotalus durissus terrificus foram incubados previamente com as amanitinas e foram analisados por eletroforese em gel de poliacrilamida-dodecilsulfato de sódio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algumas faixas de proteínas desapareceram e outras se reduziram notavelmente. Estes resultados revelam por primeira vez a interaþÒo e a degradaþÒo das proteínas dos venenos de serpentes pelas amanitinas. Por outra parte, a modificaþÒo do tempo de coagulaþÒo do sangue humano, devido aos venenos, se corrigiu com os ciclopeptídeos de Amanita. Estes resultados também se informam por primeira vez neste trabalho. A presenþa de polipeptídeos tóxicos nos venenos de serpentes e abelhas, bem como em A. phalloides e a reatividade cruzada demonstradas neste trabalho, sugerem a existÛncia de epítopos comuns a todos eles. Levando em consideraþÒo estas reaþ§es, o uso de anti-venenos heterólogos parece ser de utilidade no tratamento do envenenamento.(AU)

Neutralización heteróloga e interacción de amanitinas con venenos de Bothropsy Crotalus y de abeja / Heterologous neutralization and interaction of toxic amanitins with Bothrops and Crotalus venoms, and honey bee venom / Neutralização heteróloga e interação das amanitinas tóxicas com veneno de Bothrops e Crotalus e veneno de abelha

En el presente trabajo se examinó la interacción de las amanitinas de Amanita phalloides (Basidiomycetes) con los venenos de las serpientes Bothrops neuwiedi diporus ("yarará pequeña"), B. alternatus ("yarará grande"), Crotalus durissus terrificus ("serpiente de cascabel") y de la abeja mielera Apis mellifera. Se aplicaron las técnicas de Ouchterlony, inmunotransferencia, electroforesis rocket y electroforesis en gel de poliacrilamida a los anti-venenos y anti-toxinas obtenidos por inmunización en caballos y/o en conejos. Los anti­sueros de serpientes y las amanitinas reaccionaron en forma cruzada, así como el veneno de abeja y las amanitinas. Cuando los venenos de Bothrops neuwiedii diporus y Crotalus durissus terrificus se preincubaron con las amanitinas y se analizaron por electroforesis en gel de poliacrilamida-dodecilsulfato de sodio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algunas bandas de proteínas desaparecieron y otras se redujeron notablemente. Estos resultados revelan por primera vez la interacción y la degradación de las proteínas de los venenos de serpientes por las amanitinas. Por otra parte, la modificación del tiempo de coagulación de la sangre humana, debida a los venenos, se corrigió con los ciclopéptidos de Amanita. Estos resultados también se informan por primera vez en este trabajo. La presencia de polipéptidos tóxicos en los venenos de serpientes y abejas, así como en A. phalloides y la reactividad cruzada demostradas en este trabajo, sugieren la existencia de epítopos comunes a todos ellos. Teniendo en cuenta estas reacciones, el uso de anti-venenos heterólogos parece ser de utilidad en el tratamiento del envenenamiento.

In the present work, the interaction of the amanitins of Amanita phalloides (Basidiomycetes) with the venoms of Bothrops neuwiedi diporus ("small yarará snake"), B. alternatus ("big yarará"), Crotalus durissus terrificus ("rattlesnake"), and honey bee Apis mellifera was examined. Ouchterlony, immunotransfer, rocket-electrophoresis, and polyacrylamide gel electrophoresis techniques were applied to anti-venoms and anti-toxins obtained by immunization in horses and/or in rabbits. Snake antisera and amanitins cross-reacted as well as bee venom and amanitins. When venoms of Bothrops neuwiedii diporus and Crotalus durissus terrificus were preincubated with amanitins and analysed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), some protein bands disappeared and others were significantly reduced. These results reveal for the first time the interaction and degradation of proteins in snake venoms by amanitins. Moreover, the modification of the human blood clotting time due to snake venoms was corrected by the Amanita cyclopeptides. These results are also reported for the first time in this work. The occurrence of toxic polypeptides in the snake and bee venoms as well as in A. phalloides, and the cross-reactivity demostrated herein, suggest the occurrence of epitopes common to all of them. Taking into account these reactions,the use of heterologous anti-venoms seems to be of value in envenomation treatment.

No presente trabalho foi examinada a interação das amanitinas de Amanita phalloides (Basidiomy­cetes) com os venenos das serpentes Bothrops neuwiedi diporus ("jararaca-cruzeira"), B. alternatus ("urutu"), Crotalus durissus terrificus ("serpente cascavel") e da abelha-europeia Apis mellifera. Foram aplicadas as técnicas de Ouchterlony, imunotransferência, eletroforese rocket e eletroforese em gel de poliacrilamida aos anti-venenos e anti-toxinas obtidos por imunização em cavalos e/ou em coelhos. Os anti-soros de serpentes e as amanitinas reagiram em forma cruzada, bem como o veneno de abelha e as amanitinas. Quando os venenos de Bothrops neuwiedii diporus e Crotalus durissus terrificus foram incubados previamente com as amanitinas e foram analisados por eletroforese em gel de poliacrilamida-dodecilsulfato de sódio (dodecylsulfate-polyacrylamide gel electrophoresis: SDS-PAGE), algumas faixas de proteínas desapareceram e outras se reduziram notavelmente. Estes resultados revelam por primeira vez a interação e a degradação das proteínas dos venenos de serpentes pelas amanitinas. Por outra parte, a modificação do tempo de coagulação do sangue humano, devido aos venenos, se corrigiu com os ciclopeptídeos de Amanita. Estes resultados também se informam por primeira vez neste trabalho. A presença de polipeptídeos tóxicos nos venenos de serpentes e abelhas, bem como em A. phalloides e a reatividade cruzada demonstradas neste trabalho, sugerem a existência de epítopos comuns a todos eles. Levando em consideração estas reações, o uso de anti-venenos heterólogos parece ser de utilidade no tratamento do envenenamento.

Action of neuwiedase, a metalloproteinase isolated from bothrops neuwiedi venom, on skeletal muscle: an ultrastructural and immunocytochemistry study

The damaging effects of neuwiedase, a non-hemorrhagic snake venom metalloproteinase from P-I class, on gastrocnemius muscle are studied herein. Following neuwiedase injection, ultrastructural alterations were detected early showing disarrangement of skeletal muscle fibers (characterized by discontinuity of Z lines), mitochondrial swelling, and disruption of plasma membrane and basal lamina. Degradation of skeletal muscle and the appearance of an amorphous substance, primarily composed of cellular debris, were noted after 24 hours. The presence of neuwiedase at the injection site (detected by immunocytochemistry) revealed highly specific labeling of myofibril components of damaged myocytes. In addition, proteolysis of muscle proteins assayed through myofibrils extracted from gastrocnemius muscle indicated that neuwiedase provoked degradation of myofibrils, especially myosin. These results suggest that skeletal muscle damage, induced by neuwiedase, is probably due to its proteolytic action on myofibrils, which are responsible for the maintenance of the cellular architecture.

Myonecrosis induced in rat by a myotoxin isolated from the venom of Bothrops neuwiedifrom Alfenas, MG

Bothrops venoms are composed by several protein fractions. Among these fractions there are myotoxins which induce an important muscle lesion. The purification of this component involves some steps, although providing a pure material, is time consuming. In the present study, we describe a quick method for myotoxin fraction isolation from the venom of Bolhrops nsuwiedi using one-step high performance liquid chromatography (HPLC). The complete procedure took 30 min. The pur6+y of the two myotoxic fractions isolated was assessed by SDS-PAGE. Upon i.m. injection into rat tibialis anterior, both toxins induced early morphological changes, indicating that the plasma membrane was the first cellular structure affected. Afterwards, the lesion was typically myonecrotic and inflammatory infiltrate was present.

Os venenos de Bolhrops são compostos por várias frações protéicas. Dentre elas, as miotoxinas induzem lesões musculares importantes. A purificação deste componente envolve várias etapas e, embora forneça frações puras, é muito trabalhosa. Neste trabalho foi descrito um método rápido para o isolamento da fração miotóxica do veneno de Bothrops neuwiedi (jararaca pintada), utilizando-se uma etapa única de purificação por Cromatografia Liquida de Alta Eficiência (CLAE). O procedimento completo demorou apenas 30 min. Foram isoladas duas frações com atividade miotóxica, cuja pureza foi verificada por eletroforese em gel de poliacrilamida-SDS. A injeção intramuscular no músculo tibial anterior de ratos induziu alterações morfológicas precoces, indicando que a membrana plasmática foi a primeira estrutura celular afetada pela fração miotóxica. A lesão muscular obtida foi tipicamente mionecrótica e foi observado infiltrado inflamatório.