Analysis of the subproteomes of proteinases and heparin‐binding toxins of eight Bothrops venoms
Adriana Franco Paes LemeEduardo S. KitanoMaria F. FurtadoRichard H. ValenteAntônio Carlos Martins de CamargoPaulo Lee HoJay W. FoxSolange M.T. Serrano
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Abstract:
Viperid snakes show the most complex snake-venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2-DE, and their subproteomes of proteinases were explored by 2-D immunostaining and 2-D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti-coagulant and anti-inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high-affinity for heparin as composed of mainly serine proteinases and C-type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin-binding toxins. Only the non-bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin-bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.Keywords:
Bothrops jararaca
Bothrops
Viperid snakes show the most complex snake-venom proteomes and offer an intriguing challenge in terms of understanding the nature of their components and the pathological outcomes of envenomation characterized by local and systemic effects. In this work, the venom complexity of eight Bothrops species was analyzed by 2-DE, and their subproteomes of proteinases were explored by 2-D immunostaining and 2-D gelatin zymography, demonstrating the diversity of their profiles. Heparin, a highly sulfated glycosaminoglycan released from mast cells, is involved in anti-coagulant and anti-inflammatory processes. Here, we explored the hypothesis that heparin released upon envenomation could interact with toxins and interfere with venom pathogenesis. We first identified the Bothrops venom subproteome of toxins that bind with high-affinity for heparin as composed of mainly serine proteinases and C-type lectins. Next, we explored the Bothrops jararaca toxins that bind to heparin under physiological conditions and identified a relationship between the subproteomes of proteinases, and that of heparin-binding toxins. Only the non-bound fraction, composed mainly of metalloproteinases, showed lethal and hemorrhagic activities, whereas the heparin-bound fraction contained mainly serine proteinases associated with coagulant and fibrinogenolytic activities. These data suggest that heparin binding to B. jararaca venom components in vivo has a minor protective effect to venom toxicity.
Bothrops jararaca
Bothrops
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Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI‐TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human α 1 B‐glycoprotein, indicating the presence of five immunoglobulin‐like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt‐I/Asp49) and II (mt‐II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA 2 activity of mt‐I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt‐I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA 2 . Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin‐like structure isolated and characterized from animal blood.
Bothrops jararaca
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Myotoxin
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Snake venoms are complex mixtures of proteins of both enzymes and nonenzymes, which are responsible for producing several biological effects. Human envenomation by snake bites particularly those of the viperid family induces a complex pathophysiological picture characterized by spectacular changes in hemostasis and frequently hemorrhage is also seen. The present work reports the ability of six of a series of 1,2,3-triazole derivatives to inhibit some pharmacological effects caused by the venoms of Bothrops jararaca and Lachesis muta. In vitro assays showed that these compounds were impaired in a concentration-dependent manner, the fibrinogen or plasma clotting, hemolysis, and proteolysis produced by both venoms. Moreover, these compounds inhibited biological effects in vivo as well. Mice treated with these compounds were fully protected from hemorrhagic lesions caused by such venoms. But, only the B. jararaca edema-inducing activity was neutralized by the triazoles. So the inhibitory effect of triazoles derivatives against some in vitro and in vivo biological assays of snake venoms points to promising aspects that may indicate them as molecular models to improve the production of effective antivenom or to complement antivenom neutralization, especially the local pathological effects, which are partially neutralized by antivenoms.
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The search for biological antitumor agents has been pursued for over half a century. Snake venom has been shown to possess a wide spectrum of biological activities. The objectives of the present review are to evaluate the existing controversies on this subject published in a number of papers and to propose probable explanations for the phenomena observed. We reported our results obtained in a study, in which we evaluated the action of the venoms of Crotalus durissus terrificus and Bothrops jararaca on Ehrlich ascites tumor cells. We noticed an important antitumor effect, mainly with Bothrops jararaca venom, as well as an increase in the functional activity of macrophages. We also observed an increase in the number of mononuclear and polymorphonuclear cells with Bothrops jararaca venom. Considering these findings, we postulate that both Bothrops jararaca and Crotalus durissus terrificus venoms can act directly on tumor cells. In addition, we propose an indirect mechanism, based on the stimulation of the inflammatory response, to inhibit tumor growth and to promote its rejection.
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Six venoms from snakes of the genus Bothrops were tested for coagulation, platelet aggregation and phospholipase A2 (PLA2) activity. Almost all showed pro-coagulant and PLA2 activities while pro-aggregating properties were found only for some venoms. Bothrops jararaca venom showed different protein peaks associated with these activities. The pro-aggregating activity was inhibited by EDTA, leupeptin and mepacrine while the PLA2 activity was blocked by p-bromophenacyl bromide and 2-mercaptoethanol. Venom screening tests for clotting and platelet aggregation may represent a valuable tool for snake taxonomy and for monitoring the quality of antisera.
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