RESUMO
Cysteine-rich secretory proteins (CRISPs) are commonly described as part of the protein content of snake venoms, nevertheless, so far, little is known about their biological targets and functions. Our study describes the isolation and characterization of Bj-CRP, the first CRISP isolated from Bothrops jararaca snake venom, also aiming at the identification of possible targets for its actions. Bj-CRP was purified using three chromatographic steps (Sephacryl S-200, Source 15Q and C18) and showed to be an acidic protein of 24.6kDa with high sequence identity to other snake venom CRISPs. This CRISP was devoid of proteolytic, hemorrhagic or coagulant activities, and it did not affect the currents from 13 voltage-gated potassium channel isoforms. Conversely, Bj-CRP induced inflammatory responses characterized by increase of leukocytes, mainly neutrophils, after 1 and 4h of its injection in the peritoneal cavity of mice, also stimulating the production of IL-6. Bj-CRP also acted on the human complement system, modulating some of the activation pathways and acting directly on important components (C3 and C4), thus inducing the generation of anaphylatoxins (C3a, C4a and C5a). Therefore, our results for Bj-CRP open up prospects for better understanding this class of toxins and its biological actions.
Assuntos
Bothrops , Venenos de Crotalídeos/química , Peptídeos/isolamento & purificação , Sequência de Aminoácidos , Anafilatoxinas/biossíntese , Anafilatoxinas/imunologia , Animais , Coagulação Sanguínea/efeitos dos fármacos , Células Cultivadas , Ativação do Complemento/efeitos dos fármacos , Eletroforese em Gel de Poliacrilamida , Hemorragia/induzido quimicamente , Humanos , Técnicas In Vitro , Masculino , Camundongos Endogâmicos C57BL , Peso Molecular , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , Técnicas de Patch-Clamp , Peptídeos/farmacologia , Peptídeos/toxicidade , Canais de Potássio de Abertura Dependente da Tensão da Membrana/antagonistas & inibidores , Proteínas de Répteis/isolamento & purificação , Proteínas de Répteis/farmacologia , Proteínas de Répteis/toxicidade , Venenos de Víboras/isolamento & purificação , Venenos de Víboras/farmacologia , Venenos de Víboras/toxicidade , Xenopus laevisRESUMO
BACKGROUND: The genus Micrurus, coral snakes (Serpentes, Elapidae), comprises more than 120 species and subspecies distributed from the south United States to the south of South America. Micrurus snake bites can cause death by muscle paralysis and further respiratory arrest within a few hours after envenomation. Clinical observations show mainly neurotoxic symptoms, although other biological activities have also been experimentally observed, including cardiotoxicity, hemolysis, edema and myotoxicity. RESULTS: In the present study we have investigated the action of venoms from seven species of snakes from the genus Micrurus on the complement system in in vitro studies. Several of the Micrurus species could consume the classical and/or the lectin pathways, but not the alternative pathway, and C3a, C4a and C5a were generated in sera treated with the venoms as result of this complement activation. Micrurus venoms were also able to directly cleave the α chain of the component C3, but not of the C4, which was inhibited by 1,10 Phenanthroline, suggesting the presence of a C3α chain specific metalloprotease in Micrurus spp venoms. Furthermore, complement activation was in part associated with the cleavage of C1-Inhibitor by protease(s) present in the venoms, which disrupts complement activation control. CONCLUSION: Micrurus venoms can activate the complement system, generating a significant amount of anaphylatoxins, which may assist due to their vasodilatory effects, to enhance the spreading of other venom components during the envenomation process.
Assuntos
Anafilatoxinas/biossíntese , Ativação do Complemento/efeitos dos fármacos , Venenos Elapídicos/farmacologia , Elapidae/metabolismo , Animais , Proteína Inibidora do Complemento C1/isolamento & purificação , Proteína Inibidora do Complemento C1/metabolismo , Complemento C3/metabolismo , Venenos Elapídicos/metabolismo , Humanos , Proteólise/efeitos dos fármacosRESUMO
Snake venoms are a complex mixture of components, which have a wide range of actions both on prey and human victims. The genus Bothrops causes the vast majority of snakebites in Central and South America, being responsible for 80% of snake envenomations in Brazil. Envenomations are characterized by prominent local effects, including oedema, haemorrhage and necrosis, which can lead to permanent disability. Systemic manifestations such as haemorrhage, coagulopathy, shock and acute renal failure may also occur. In the present study we have investigated the action of venoms from 19 species of snakes from the genus Bothrops, occurring in Brazil, on the complement system in in vitro studies. All venoms were able to activate the classical complement pathway, in the absence of sensitizing antibody. This activation was in part associated with the cleavage of C1-Inhibitor by proteases present in these venoms, which disrupts complement activation control. No modification of the membrane bound complement regulators, such as DAF, CR1 and CD59 was detected, after treatment of human erythrocytes with the snake venoms. Some of the Bothrops venoms were also able to activate alternative and lectin pathways, as measured in haemolytic and ELISA assays. C3a, C4a and C5a were generated in sera treated with the venoms, not only through C-activation, but also by the direct cleavage of complement components, as determined using purified C3 and C4. Metallo- and/or serine-protease inhibitors prevented cleavage of C3 and C4. These results suggest that Bothrops venoms can activate the complement system, generating a large amount of anaphylatoxins, which may play an important role in the inflammatory process presented in humans after snake envenomations, and they may also assist, due to their vasodilatory effects, to enhance the spreading of other venom components.