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ABSTRACT: The venom of the snake Bothrops asper causes muscle necrosis, pain and inflammation. This venom contains myotoxins which cause an increase in intracellular Ca(2+) concentration and release of K(+) and ATP from myotubes. ATP is a key danger molecule that triggers a variety of reactions, including activation of the innate immune response. Here, using ATP-luciferase bioluminescence imaging technique, we show for the first time in vivo, that the purified myotoxins induce rapid release of ATP, whilst the complete venom of B. asper does at a very small extent. This apparent contradiction is explained by the finding that the venom contains powerful nucleotidases that in vivo convert ATP into ADP, AMP, and adenosine. These findings indicate that high concentrations of adenosine are generated by the double action of the venom and provide the experimental basis to the suggestion that in situ generated adenosine plays an important role in envenomation via its hypotensive, paralyzing and anti-coagulant activities.
Biochemical and Biophysical Research Communications 12/2012; · 2.48 Impact Factor
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ABSTRACT: Phospholipases A2 (PLA2s) are enzymes responsible for membrane disruption through Ca2+-dependent hydrolysis of phospholipids. Lys49-PLA2s are well-characterized homologue PLA2s that do not show catalytic activity but can exert a pronounced local myotoxic effect. These homologue PLA2s were first believed to present residual catalytic activity but experiments with a recombinant toxin show they are incapable of catalysis. Herein, we present a new homologue Asp49-PLA2 (BthTX-II) that is also able to exert muscle damage. This toxin was isolated in 1992 and characterized as presenting very low catalytic activity. Interestingly, this myotoxic homologue Asp49-PLA2 conserves all the residues responsible for Ca2+ coordination and of the catalytic network, features thought to be fundamental for PLA2 enzymatic activity. Previous crystallographic studies of apo BthTX-II suggested this toxin could be catalytically inactive since a distortion in the calcium binding loop was observed. In this article, we show BthTX-II is not catalytic based on an in vitro cell viability assay and time-lapse experiments on C2C12 myotube cell cultures, X-ray crystallography and phylogenetic studies. Cell culture experiments show that BthTX-II is devoid of catalytic activity, as already observed for Lys49-PLA2s. Crystallographic studies of the complex BthTX-II/Ca2+ show that the distortion of the calcium binding loop is still present and impairs ion coordination even though Ca2+ are found interacting with other regions of the protein. Phylogenetic studies demonstrate that BthTX-II is more phylogenetically related to Lys49-PLA2s than to other Asp49-PLA2s, thus allowing Crotalinae subfamily PLA2s to be classified into two main branches: a catalytic and a myotoxic one. Proteins 2010. © 2010 Wiley-Liss, Inc.
Proteins Structure Function and Bioinformatics 09/2010; 79(1):61 - 78. · 3.39 Impact Factor
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ABSTRACT: Myotoxins play a major role in the pathogenesis of the envenomations caused by snake bites in large parts of the world where this is a very relevant public health problem. We show here that two myotoxins that are major constituents of the venom of Bothrops asper, a deadly snake present in Latin America, induce the release of large amounts of K(+) and ATP from skeletal muscle. We also show that the released ATP amplifies the effect of the myotoxins, acting as a "danger signal," which spreads and causes further damage by acting on purinergic receptors. In addition, the release of ATP and K(+) well accounts for the pain reaction characteristic of these envenomations. As Bothrops asper myotoxins are representative of a large family of snake myotoxins with phospholipase A(2) structure, these findings are expected to be of general significance for snake bite envenomation. Moreover, they suggest potential therapeutic approaches for limiting the extent of muscle tissue damage based on antipurinergic drugs.
Proceedings of the National Academy of Sciences 08/2010; 107(32):14140-5. · 9.68 Impact Factor
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ABSTRACT: Myotoxins are abundant components of snake venoms, being a significant public health problem worldwide. Among them, Lys49 phospholipase A(2) homologue myotoxins cause extensive necrosis in skeletal muscle tissue. Their mechanisms of action are still poorly understood, but there is evidence that the C-terminal region is involved in membrane damage leading to myotoxicity. To investigate the effect of the C-terminal peptide 115-129 of Agkistrodon contortrix laticinctus myotoxin on the plasma membrane of myoblasts and myotubes, the entry of Ca(2+) was monitored by fluorescence imaging, and the ensuing cytotoxicity was determined. The myotoxin synthetic peptide was found to act selectively on myotubes, which were rapidly overloaded with Ca(2+) with ensuing necrosis. The profile of intracellular Ca(2+) increase induced by the C-terminal peptide, but not by its scrambled version control, reproduces the second, prominent wave of the biphasic response documented in previous studies using whole Lys49 myotoxins. These observations provide relevant insights into the mechanism of action of this family of toxins, with implications for the understanding of their structure-function relationships.
Toxicon 10/2009; 55(2-3):590-6. · 2.51 Impact Factor
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Mariana Cintra-Francischinelli
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ABSTRACT: Envenoming resulting from snake bites is a relevant important world public health hazard, especially in tropical and subtropical countries. Particularly in Latin America, the Bothrops genus is responsible for 50–90% of the snakebites. Serum therapy reverses all systemic manifestations of envenoming being efficient against the lethal effect of toxins. In contrast, antivenoms are not very effective in the reversal toxicity induced by phospholipases A2 group toxins, the main components of Bothrops snake venoms. The PLA2 myotoxins can be divided into two principal groups, those with an aspartic acid residue at position 49 endowed with catalytic activity (Asp49 PLA2) and those in which a lysine residue substitutes for aspartic acid at this same position losing the ability to hydrolyze phospholipids (Lys49 PLA2). Even the high amount of studies, little is know about the mechanism of action of myotoxins and this was the main goal of this work. We used C2C12 muscle cells as our cellular model and we focused on calcium-imaging and cell vitality determination techniques.
In the first chapter, we showed that the two groups of myotoxins (Asp49 and Lys49 PLA2s) bind to acceptors coupled to intracellular calcium stores; that the PLA2 inactive Lys49 and the PLA2 active Asp49 myotoxins act via different mechanisms in allowing calcium entry into the differentiated muscle cells. This thesis shows for the first time their kinetics of action; and the very important biological finding that the non enzymatically active PLA2 myotoxin synergizes with the active PLA2 myotoxin providing the evolutionary significance for the presence of the two toxin types in the same snake venom.
The second chapter shows news clues for the essential role of C-terminal of the Lys49 PLA2 myotoxins. In fact, this peptide region is strongly involved in the membrane permeabilization. Thus, C-terminal region of Lys49 myotoxins is able to cause an extensive and rapid calcium influx leading to muscle cell death within few minutes.
In the third chapter the atypical myotoxin from Bothrops jararacussu, bothropstoxin-II, is studied. Although it belongs to the Asp49 group, its PLA2 activity is much lower than other Asp49 PLA2s and it is comparable with Lys49 PLA2 homologues. Beside this, BthTX-II has the ability of act also in absence of calcium in medium, differently from all Asp49 studied until here; demonstrating, for the first time, an atypical Asp49 myotoxin that can disrupt the plasma membrane of myotubes leading to cell death, both in presence or absence of extracellular calcium ions.
In the fourth chapter, we described unpublished results regarding the release of intracellular substances by toxin-damaged cells, that alert other cells about a danger situation. We show that B. asper myotoxin-II is able to induce a release of high amounts of ATP from injured cells that is responsible for the amplification of the myonecrosis. At the same time, we also observed a rapid and large potassium efflux that could be involved in pain generation and in some proinflammatory mechanisms. All these results could help to understand the inflammatory pattern also observed in Bothrops envenomations.
