Study of the mechanism of action of snake myotoxins (original) (raw)
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Proceedings of the …, 2010
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. muscle damage | phospholipase A2 | C2C12 cells
Toxicology in Vitro, 2007
Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalyticallyinactive Lys49 phospholipase A 2 homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damage by a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. In this work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phospholipase A 2 homologue. Mt-II induced a dose-and time-dependent cytotoxic effect associated with plasma membrane disruption, evidenced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment in cytosolic Ca 2+ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca 2+ signal was observed in myotube nuclei. Elimination of extracellular Ca 2+ resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca 2+ . Chelation of cytosolic Ca 2+ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca 2+ which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca 2+ influx in myotubes. Extracellular Ca 2+ plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca 2+ influx that occurs after membrane disruption is likely to play a key role in the intracellular degenerative events associated with Mt-II-induced myotube damage.
Isolation and partial characterization of a myotoxin from the venom of the snake Bothrops nummifer
Toxicon, 1986
Papmon Jomnale Ltd. J . M . GurtHaaez, B . Lo>uorrre and L . CP~en~s . Isolation and partial charaaeriration of a myotoxin from the venom of the snake Bothropts nummjja. Taadrnn ?A, [885][886][887][888][889][890][891][892][893][894] 1986 . -A myotoxin from the venom of the make Botbropts memmjja was purified to homoaendty by ionexchange chromatogiaphy on CM-Sephadea . The toxin is a basic dinier with a subunit molecular weight of 16,000, as estimated by SDS-polyacrylamide gel dectrophoreaia . The toxin lacks phaspholipaae A, activity when tested on egg yolk Ixithin and :kdetal muscle homogenates . It induces srdetal muscle damage both In vlvo and In vitro. When igjected i .m. it promotes a drastic increase in serum creative kinase levels; the isozyme CK-MM is responsible for this increment . A rapid release of a~eatIne kinase was observed when mouse gastrocnemius muscle wan incubated with the toxin, suggesting that it induces the formation of relatively large 'lesions' in the plasma membrane of muade odls . Moreover, analysis of the done -response data indicated that the myotoxin afftscxs muscle saroolemma by a 'one hit' mec ar,;arn , Skeletal muscle cells are affected by the toxin when calcium is eliminated from the medium . The myotoxin hes an i .v. Ln of 3 .9 mg/kg body wdght in mice. and induces edema when igjated in the foot pad . On the other hand, it is not diraxly hemolytic, andoo:gulant, hemorr myotoxin show: partial immunol ~c nor cytotoxic for lymphocytes" The ogic identity with a myotoxic phospholipase A, isolated from Botlu+optr~venom . The polyvalent antivenom produced in Coca Rica forms a precipitation arc against B. nummjfa myotoxin on immunoelecrtrophoreais .
Toxicology in Vitro, 2007
Acute muscle tissue damage, myonecrosis, is a typical consequence of envenomations by snakes of the family Viperidae. Catalytically-inactive Lys49 phospholipase A2 homologues are abundant myotoxic components in viperid venoms, causing plasma membrane damage by a mechanism independent of phospholipid hydrolysis. However, the precise mode of action of these myotoxins remains unsolved. In this work, a cell culture model of C2C12 myotubes was used to assess the action of Bothrops asper myotoxin II (Mt-II), a Lys49 phospholipase A2 homologue. Mt-II induced a dose- and time-dependent cytotoxic effect associated with plasma membrane disruption, evidenced by the release of the cytosolic enzyme lactate dehydrogenase and the penetration of propidium iodide. A rapid increment in cytosolic Ca2+ occurred after addition of Mt-II. Such elevation was associated with hypercontraction of myotubes and blebbing of plasma membrane. An increment in the Ca2+ signal was observed in myotube nuclei. Elimination of extracellular Ca2+ resulted in increased cytotoxicity upon incubation with Mt-II, suggesting a membrane-protective role for extracellular Ca2+. Chelation of cytosolic Ca2+ with BAPTA-AM did not modify the cytotoxic effect, probably due to the large increment induced by Mt-II in cytosolic Ca2+ which overrides the chelating capacity of BAPTA-AM. It is concluded that Mt-II induces rapid and drastic plasma membrane lesion and a prominent Ca2+ influx in myotubes. Extracellular Ca2+ plays a dual role in this model: it protects the membrane from the cytolytic action of the toxin; at the same time, the Ca2+ influx that occurs after membrane disruption is likely to play a key role in the intracellular degenerative events associated with Mt-II-induced myotube damage.
Toxicon, 1989
induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer (jumping viper) from Costa Rica . Toxicon 27, 735-745. 1989 .-The mode of action of a basic myotoxin isolated from Bothrops nummifer venom was studied. This myotoxin is a basic polypeptide of 13,000 mol.wt, with a high content of lysine and aspartatc, as well as of hydrophobic amino acids. It lacked phospholipase A2 activity when tested on several substrates at different pH values. Upon i.m . injection into mice, the toxin induced early morphological alterations typified by`delta lesions' in the periphery of muscle fibers, an indication that the plasma membrane was the first cellular structure to be affected . Afterwards, necrotic cells had a clumped appearance, which then changed to a more hyaline histological pattern. Removal of necrotic material by phagocytes was followed by skeletal muscle regeneration, with the presence of myoblasts, myotubes and fully regenerated myofibers. The toxin induced a rapid and drastic drop in muscle creatine and creatine kinase contents of injected muscle, as well as an increase in serum levels of the enzymes lactic dehydrogenase and creatine kinase . Moreover, total muscle calcium increased significantly after toxin administration . Myotoxin induced a dose-dependent release of peroxidase entrapped in liposomes made from muscle phospholipids. The lack of phospholipase A2 activity in this toxin, together with the observation that it behaved as an amphiphilic protein in charge-shift electrophoresis, suggests that it might penetrate and disorganize muscle plasma membrane by means of a hydrophobic interaction .
Why myotoxin-containing snake venoms possess powerful nucleotidases?
2012
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.
… International Journal of …, 2002
In order to analyze its structure-function relationships, the complete amino acid sequence of myotoxin II from Atropoides (Bothrops) nummifer from Costa Rica was determined. This toxin is a Lys49-type phospholipase A 2 (PLA 2 ) homologue, devoid of catalytic activity, structurally belonging to class IIA. In addition to the Asp49 → Lys change in the (inactive) catalytic center, substitutions in the calcium-binding loop suggest that its lack of enzymatic activity is due to the loss of ability to bind Ca 2+ . The toxin occurs as a homodimer of basic subunits of 121 residues. Its sequence has highest similarity to Lys49 PLA 2 s from Cerrophidion, Trimeresurus, Bothrops and Agkistrodon species, which form a subfamily of proteins that diverged early from Asp49 PLA 2 s present in the same species, as shown by phylogenetic analysis. The tertiary structure of the toxin was modeled, based on the coordinates of Cerrophidion godmani myotoxin II. Its exposed C-terminal region 115-129 shows several differences in comparison to the homologous sequences of other Lys49 PLA 2 s, i.e. from Agkistrodon p. piscivorus and Bothrops asper. Region 115-129 of the latter two proteins has been implicated in myotoxic activity, on the basis of the direct membrane-damaging of their corresponding synthetic peptides. However, peptide 115-129 of A. nummifer myotoxin II did not exert toxicity upon cultured skeletal muscle cells or mature muscle in vivo. Differences in several amino acid residues, either critical for toxicity, or influencing the conformation of free peptide 115-129 from A. nummifer myotoxin II, may account for its lack of direct membrane-damaging properties.
Toxicon, 1994
of myotoxin II, a lysine-49 phospholipase A2 of Bothrops asper snake venom. Toxicon 32, 1359Toxicon 32, -1369Toxicon 32, , 1994.-The cytotoxic activity of Bothrops asper myotoxin II, a lysine-49 phospholipase A2 isoform, on different cell types in culture, was investigated . Myotoxin II caused a dose-dependent cytolytic effect on all cell types tested, characterized by rapid release of cytoplasmic lactic dehydrogenase and drastic morphological cell alterations. Quantitative differences in the susceptibility to myotoxin II among cell types fell within a relatively narrow range, and in general, the toxin was cytolytic at concentrations of 50-100 j ug/ml (3-7 uM), when assays were performed using culture medium as a diluent. Toxin activity was markedly enhanced if phosphate-buffered saline was utilized instead of medium. The cytotoxic activity of myotoxin III, an aspartate-49 isoform from the same venom, on both endothelial cells and skeletal muscle myoblasts was higher than that of myotoxin II, suggesting that, although phospholipase A2 activity is clearly not required for the induction of cell damage, it may have an enhancing role . In contrast to B. asper myotoxins, other basic phospholipases A2 with myotoxic activity in vivo (notexin from Notechis scutatus, and two enzymes isolated from Vipera russelli venom) did not affect endothelial cells and myoblasts. Pretreatment of cells with neuraminidase, tunicamycin, or protamine, did not alter their susceptibility to myotoxin II. At low temperatures (2-4°C) myotoxin II was devoid of cytolytic effect . Washing and neutralization experiments using heparin with low affinity for antithrombin or mouse monoclonal antibody MAb-3 suggest that at low temperatures myotoxin II binds very weakly to the cells, and that its normal interaction with the putative target is probably not only based on charge, but that a membrane penetration event may be required .
Specific In Vitro Biological Activity of Snake Venom Myotoxins
Journal of Neurochemistry, 1993
Some snake venoms contain toxins that are reported to be selective for damaging muscle. This specificity can be used to design experiments intended to eliminate muscle. We studied the small myotoxins and fractions IV and V of Bothrops nunmfer venom to evaluate their direct effect on cultured muscle cells, neurons, macrophages, and a fibroblast cell line. The small myotoxins, at 100 pg/ml for 2 h, had no effect in vitro, contrary to the in vivo applications. Fractions IV and V were both myotoxic and, at 100 pg/ml, destroyed all cell types. However, at 10 pg/ml the effects of fraction IV were more selective for muscle. Vacuolation of the sarcoplasmic reticulum and T-tubules was first seen in the poisoned muscles, without initial lesions in the nuclei, sarcolemma, mitochondria, and rough endoplasmic reticulum. Fractions IV and V have different toxic activity in cells other than muscles and are a mixture of two basic proteins (i and ii). Protein ii is predominant in fraction IV and protein i is predominant in fraction V. The toxic effects may be mediated by the formation of nonspecific ionic pores in the sarcolemma and/or T-tubule muscle membrane. Key Words: Myotoxins-Snake venom-Tissue culture-Bolhrops nummifer. Brusks J. L. et al. Specific in vitro biological activity of snake venom myotoxins.