Venenos de serpentes do gênero Bothrops: impacto da glicosilação na complexidade dos proteomas e função de toxinas / Bothrops snake venoms: impact of glycosylation on the complexity of proteomes and toxins function

A variabilidade estrutural é uma característica das proteínas de venenos de serpentes, e a glicosilação é uma das principais modificações pós-traducionais que contribui para a diversificação de seus proteomas. Recentes estudos de nosso grupo demonstraram que venenos do gênero Bothrops são marcadamente definidos pelo seu conteúdo de glicoproteínas, e que a maioria das estruturas de N-glicanos dos tipos híbrido e complexo identificados em oito venenos deste gênero contêm unidades de ácido siálico. Em paralelo, em glicoproteínas do veneno de B. cotiara foi identificada a presença de uma estrutura de N-acetilglicosamina bissecada. Assim, com o objetivo de investigar a variação do conteúdo de glicoproteínas, assim como os mecanismos envolvidos na geração dos diferentes venenos de Bothrops, neste estudo foram analisados comparativamente os glicoproteomas de nove venenos do gênero Bothrops (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi). As abordagens glicoproteômicas envolveram cromatografia de afinidade e ensaio de pull-down utilizando, respectivamente, as lectinas SNA (aglutinina de Sambucus nigra) e MAL I (lectina de Maackia amurensis), que mostram afinidade por unidades de ácido siálico nas posições, respectivamente, α2,6 e α2,3; e cromatografia de afinidade com a lectina PHA-E (eritroaglutinina de Phaseolus vulgaris), que reconhece N-acetilglicosamina bissecada. Ainda, eletroforese de proteínas, blot de lectina, e identificação de proteínas por espectrometria de massas foram empregadas para caracterizar os glicoproteomas. As lectinas geraram frações dos venenos enriquecidas de diferentes componentes, onde as principais classes de glicoproteínas identificadas foram metaloprotease, serinoprotease, e L-amino ácido oxidase, além de outras enzimas pouco abundantes nos venenos. Os diferentes conteúdos de proteínas reconhecidas por essas lectinas, com especificidades distintas, ressaltaram novos aspectos da variabilidade dos subproteomas de glicoproteínas desses venenos, dependendo da espécie. Ainda, considerando que metaloproteases e serinoproteases são componentes abundantes nesses venenos e fundamentais no quadro de envenenamento botrópico, e que estas enzimas contêm diversos sítios de glicosilação, o papel das unidades de ácido siálico na atividade proteolítica das mesmas foi avaliado. Assim, a remoção enzimática de ácido siálico (i) alterou o padrão de gelatinólise em zimografia da maioria dos venenos, (ii) diminuiu a atividade proteolítica de alguns venenos sobre o fibrinogênio e a atividade coagulante do plasma humano de todos os venenos, e (iii) alterou o perfil de hidrólise de proteínas plasmáticas pelo veneno de B. jararaca, indicando que este carboidrato pode desempenhar um papel na interação das proteases com seus substratos proteicos. Em contraste, o perfil da atividade amidolítica dos venenos não se alterou após a remoção de ácido siálico e incubação com o substrato Bz-Arg-pNA, indicando que ácido siálico não é essencial em N-glicanos de serinoproteases atuando sobre substratos não proteicos. Em conjunto, esses resultados expandem o conhecimento sobre a variabilidade de proteomas de venenos do gênero Bothrops e apontam a importância das cadeias de carboidratos contendo ácido siálico nas atividades enzimáticas das proteases desses venenos

Structural variability is a feature of snake venom proteins, and glycosylation is one of the main post-translational modifications that contributes to the diversification of venom proteomes. Recent studies by our group have shown that Bothrops venoms are markedly defined by their glycoprotein content, and that most hybrid and complex N-glycan structures identified in eight venoms of this genus contain sialic acid units. In parallel, the presence of a bisected N-acetylglucosamine structure was identified in B. cotiara venom glycoproteins. Thus, with the aim of investigating the variation in the content of glycoproteins, as well as the mechanisms involved in the generation of different Bothrops venoms, in this study the glycoproteomes of nine Bothrops venoms (B. atrox, B. cotiara, B. erythromelas, B. fonsecai, B. insularis, B. jararaca, B. jararacussu, B. moojeni e B. neuwiedi) were comparatively analyzed. The glycoproteomic approaches involved affinity chromatography and pulldown using, respectively, the lectins SNA (Sambucus nigra agglutinin) and MAL I (Maackia amurensis lectin), which show affinity for sialic acid units at positions, respectively, α2,6 and α2,3, and affinity chromatography with PHA-E (Phaseolus vulgaris erythroagglutinin), which recognizes bisected N-acetylglucosamine. In addition, protein electrophoresis, lectin blot, and protein identification by mass spectrometry were employed for glycoproteome characterization. The lectins generated venom fractions enriched with different components, where the main classes of glycoproteins identified were metalloprotease, serine protease, and L-amino acid oxidase, in addition to other low abundant enzymes. The different contents of proteins recognized by these lectins of distinct specificities highlighted new aspects of the variability of the glycoprotein subproteomes of these venoms, depending on the species. Furthermore, considering that metalloproteases and serine proteases are abundant components of these venoms and essential in Bothrops envenomation, and that these enzymes contain several glycosylation sites, the role of sialic acid units in their proteolytic activities was evaluated. Thus, enzymatic removal of sialic acid (i) altered the pattern of gelatinolysis in zymography of most venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the hydrolysis profile of plasma proteins by B. jararaca venom, indicating that this carbohydrate may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of amidolytic activity of the venoms did not change after removal of sialic acid and incubation with the substrate Bz-Arg-pNA, indicating that sialic acid is not essential in N-glycans of serine proteases acting on small substrates. Together, these results expand the knowledge about the variability of proteomes of Bothrops venoms and point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases

Extractivism of palm tree fruits: A risky activity because of snakebites in the state of Acre, Western Brazilian Amazon

Abstract INTRODUCTION: We present here the risk from snakebites because of palm extractivism in western Amazonia. METHODS: The data were extracted from a cross-sectional sample study, from January 2016 to April 2018, at the Juruá Regional Hospital of Cruzeiro do Sul (Acre). RESULTS: There were 14 Bothrops incidents involving males, most of them occurring during the harvesting of "açaí" (Euterpe precatoria). CONCLUSIONS: During the harvesting of "açaí," there is the risk of the presence of B. atrox on the ground near the palm tree, and of B. bilineatus at the top of the palm tree.

An overview of Bothrops erythromelas venom

Abstract This review discusses studies on the venom of Bothrops erythromelas published over the past 36 years. During this period, many contributions have been made to understand the venomous snake, its venom, and its experimental and clinical effects better. The following chronological overview is based on 29 articles that were published between 1979 and 2015, with emphasis on diverse areas. The complexity of this task demands an integration of multidisciplinary research tools to study toxinology. This science is in need of renewed conceptual and experimental platforms aimed at obtaining a profound understanding of the highly complex pathophysiology of snakebite envenoming and toxins isolated from snakes.

Preliminary assessment of Hedychium coronarium essential oil on fibrinogenolytic and coagulant activity induced by Bothrops and Lachesis snake venoms

The search for new inhibitors of snake venom toxins is essential to complement or even replace traditional antivenom therapy, especially in relation to compounds that neutralize the local effects of envenomations. Besides their possible use as alternative to traditional antivenom therapy, some plant species possess bioactive secondary metabolites including essential oils, which can be extracted from weeds that are considered substantial problems for agriculture, such as Hedychium coronarium. The essential oils of leaves and rhizomes from H. coronarium were extracted by hydrodistillation, and their potential inhibitory effects on the coagulant and fibrinogenolytic activities induced by the venoms of Lachesis muta, Bothrops atrox and Bothrops moojeni were analyzed. Citrated human plasma was used to evaluate the clotting time whereas changes in fibrinogen molecules were visualized by electrophoresis in polyacrylamide gel. The experimental design used for testing coagulation inhibition was randomized in a 3 × 2 factorial arrangement (concentration × essential oils), with three replications. The essential oils were compared since they were extracted from different organs of the same botanical species, H. coronarium. The results suggest that the oils interact with venom proteases and plasma constituents, since all oils evaluated, when previously incubated with venoms, were able to inhibit the clotting effect, with less inhibition when oils and plasma were preincubated prior to the addition of venoms. Thus, after extensive characterization of their pharmacological and toxicological effects, the essential oils can be used as an alternative to complement serum therapy, especially considering that these plant metabolites generally do not require specific formulations and may be used topically immediately after extraction.

Caracterización tóxica del veneno de Bothrops (Rhinocerophis) alternatus de diferentes regiones de la provincia de Córdoba (Argentina) / [The toxicity of venom of Bothrops (Rhinocerophris) alternatus in different areas of Cordoba State in Argentina].

Snake venoms can show biochemical and toxicological variability even in specimens from the same specie. The geographical localization of the snakes is one of the factors that can influence those variations. By these reasons the venom from specimens of Bothrops (Rhinocerophis) alternatus ("crucera", "yararágrande"), one of the snakes of highest medical importance in Argentina, from three different regions of Córdoba was studied. Lehtal potency, hemorrhagic, coagulant on plasma and thrombin like activities as well as the electrophoretic patterns of venom from snakes of Calamuchita, Traslasierras and the East of the province were determined. The venom from the snakes of the three regions showed the characteristic activities of the venom of the majority of Bothrops, causing hemorrhage, hemostatic disturbances acting on plasma or directly on fibrinogen with a "thrombin like activity". The different samples were very similar regarding their biochemical characteristics and toxic potencies at difference of previous observations on venoms from the same specie in different regions of other provinces fro Argentina. Bivalent antivenom, the one used by the Provincial Ministry of Health to treat the bothropic accidents, neutralized in all the cases the toxic activities of the venom in very similar range of neutralizing potency.

Bothrops jararacussu venom-induced neuromuscular blockade inhibited by Casearia gossypiosperma Briquet hydroalcoholic extract

The hydroalcoholic extract of Casearia gossypiosperma Briquet (Flacourtiaceae) was standardized for the first time through quality control procedures including pharmacognostic methods, fingerprint chromatograms, defined amounts of marker substances and physicochemical characteristics. The pharmacological activity of C. gossypiosperma (Cg) hydroalcoholic extract was assayed by a traditional in vitro test, which involved irreversible neuromuscular blockade induced by Bothrops jararacussu (Bjssu) venom (60 ìg/mL) in mouse phrenic nerve-diaphragm preparations. Bjssu venom blocked muscle activity for 26 (± 2.0) minutes (n = 6). Cg extract (0.1 mg/mL) induced changes on the baseline muscle activity without impairing the muscle function and inhibited 87.6% (± 1.8) (n = 6) of the Bjssu venom-induced blockade. Both flavonoids (0.624 g%) and polyphenols (4.63 g%) from the extract were spectrophotometrically quantified. Therefore, the present study confirms the antibothropic activity of Cg extract, supporting the ethnomedical use of Casearia sp. in the treatment of snakebite victims.

Aspectos clínico-patológicos e laboratoriais do envenenamento experimental por Bothrops alternatus em bovinos / Clinic and pathological and laboratory aspects of experimental poisoning by Bothrops alternatus venom in cattle

Esse estudo teve como objetivo determinar as alterações clínico-patológicas e os achados laboratoriais em bovinos inoculados com a peçonha de Bothrops alternatus, no intuito de fornecer subsídios para o estabelecimento do diagnóstico e do diagnóstico diferencial, bem como esclarecer pontos obscuros da literatura pertinente. O veneno liofilizado foi diluído em 1 ml de solução fisiológica e administrado a cinco bovinos, por via subcutânea, nas doses de 0,0625, 0,125 e 0,25 mg/kg e a dois outros, por via intramuscular, nas doses de 0,25 e 0,45 mg/kg. Seis bovinos foram a óbito e um que recebeu a dose de 0,0625mg/kg, por via subcutânea, recuperou-se. Os sinais clínicos tiveram início entre 25 minutos a 5 horas 30 minutos após a inoculação. O período de evolução variou de 7 horas 18 minutos a 66 horas 12 minutos. Um animal recuperou-se após 92 horas. O quadro clínico, independentemente das doses, caracterizou-se por aumento de volume (hemorragia/hematoma) no local da inoculação, tempo de sangramento aumentado, mucosas hipocoradas e apatia. O exame laboratorial revelou progressiva anemia normocítica normocrômica, trombocitopenia, redução de fibrinogênio e proteínas plasmáticas totais, hematócrito e hemoglobina diminuídos, além de leve aumento dos níveis de creatinaquinase e desidrogenase lática. Á necropsia, havia, a partir do local da inoculação, extensos hematomas e áreas de hemorragia no tecido celular subcutâneo dos animais que receberam o veneno por via subcutânea; nos animais inoculados por via intramuscular, adicionalmente, havia hemorragia intramuscular. O endocárdio esquerdo apresentava extensas hemorragias e verificaram-se petéquias na serosa do rúmen e do omaso e na mucosa do abomaso e da vesícula biliar. Em cinco animais, o cólon, reto e região perirrenal estavam envoltos por coágulos de sangue. Ao exame histológico observou-se, além do quadro hemorragíparo, necrose muscular coagulativa, acompanhada de hemorragia, no entorno do...

The aim of this study was to determine the clinical-pathological alterations and laboratory findings in cattle inoculated with Bothrops alternatus venom, with the intention of providing information for the establishment of diagnosis and differential diagnosis procedures, as well as to elucidate some obscurities observed in the pertinent literature. The lyophilized venom was diluted in 1 ml of physiologic solution. It was administered to 5 bovines by the subcutaneous route at doses of 0.0625, 0.125 and 0.25mg/kg body weight, and to 2 bovines by the intramuscular route at doses of 0.25 e 0.45mg/kg. Six bovines died and the only animal that survived, who had subcutaneously received the venom at a dose of 0.0625mg/kg, recovered. The first clinical signs were observed from 25min to 5h30min after the inoculation. The clinical evolution time varied from 7 hours 18 minutes to 92 hours. Regardless of the dose, the clinical picture was characterized by swelling (hemorrhage/hematoma) at the site of inoculation, increase in bleeding time and capillary refill time, paleness of mucous membranes and apathy. The laboratory exams revealed progressive normocytic normochromic anemia, thrombocytopenia, reduction in fibrinogen and total plasma proteins, decreased hematocrit and hemoglobin, and slight increase in creatine kinase and lactate dehydrogenase. When subjected to necropsy, the animals that received the venom through the subcutaneous route showed large hematomas and hemorrhagic areas in the subcutaneous tissue, extending from the site of inoculation. The animals in which inoculation was performed intramuscularly exhibited, in addition, intramuscular hemorrhage. The left endocardium showed extensive hemorrhagic lesions, and petechiae were found on the serosae of rumen, omasum, abomasum and gall bladder. The colon, rectum and perirenal areas were surrounded by clotted blood in three animals. Besides hemorrhage, the histological examination revealed...

The defensive strike of five species of lanceheads of the genus Bothrops (Viperidae)

We studied the defensive strike of one species of each of five recognized lineages within the genus Bothrops, namely, B. alternatus, B. jararaca, B. jararacussu, B. moojeni and B. pauloensis. The defensive strike of the studied species was in general similar to that of Crotalus viridis and C. atrox, but some important differences were observed. Bothrops alternatus and B. pauloensis struck preferentially from a tight body posture, whereas B. jararaca and B. moojeni from a loose body posture. Defensive strikes were either true or false (during the latter, the mouth remains closed or partially open). Almost all strikes were successful; only on a few occasions snakes missed their target (flawed strikes). Strike variables were very conservative among the five species, especially strike distance and height, and one possible explanation may be related to constraints imposed on strike variables as a way of increasing strike accuracy.

Estudamos o bote defensivo de uma espécie de cada uma de cinco reconhecidas linhagens do gênero Bothrops, a saber: B. alternatus, B. jararaca, B. jararacussu, B. moojeni e B. pauloensis. O bote defensivo das espécies estudadas foi, em geral, semelhante ao de Crotalus viridis e C. atrox, porém algumas diferenças foram observadas. Bothrops alternatus e B. pauloensis desferiram botes preferencialmente a partir de postura corpórea enrodilhada, ao passo que B. jararaca e B. moojeni desferiram a maioria dos botes a partir de postura corpórea frouxa. Os botes defensivos foram verdadeiros ou falsos (nestes, a boca da serpente permaneceu fechada ou parcialmente aberta). Quase todos os botes foram bem-sucedidos; apenas em alguns casos a serpente errou o alvo (botes falhos). As variáveis relativas aos botes foram bastante conservativas entre as cinco espécies, principalmente distância e altura do bote, e uma possível explicação pode estar relacionada a restrições impostas às variáveis relativas aos botes como forma de aumentar sua acurácia.

Actividades hemorrágicas y coagulante del veneno de Bothrops alternatus de Argentina / Haemorrhagic and clotting activities of Argentina B. alternatus venon

Se trabajó con veneno crudo de Bothrops alternatus para determinar sus actividades hemorrágica y coagulante. Para hallar la actividad hemorrágica se inocularon ratones, en forma intradérmica, con diluciones seriadas de veneno, en un volumen de 0,1 ml de solución salina amortiguada con fosfato, pH 7,2. Luego de 2 horas, se sacrificaron con éter y se midió el área hemorrágica. La Dosis Hemorrágica Mínima (DHM) es la cantidad de veneno que produce un área hemorrágica mínima de 10 mm de diámetro, para el veneno de B. alternatus fue de 3,6 µg. El estudio de la Dosis Coagulante Mínima (DCM) se realizó confrontando diluciones seriadas de veneno con plasma humano. La DMC, capaz de coagular el plasma humano en 60 seg, fue de 14,5 µg/ml. Al igual que otras especies del género Bothrops de Latinoamérica, el veneno de B. alternatus de Argentina posee marcada actividad hemorrágica y coagulante