Myonecrosis induced in mice by a basic myotoxin isolated from the venom of the snake Bothrops nummifer (jumping viper) from Costa Rica (original) (raw)
Related papers
Phospholipase A2 myotoxins from Bothrops snake venoms
Toxicon, 1995
J. M. Guti6rrez and B. Lomonte. Phospholipase A2 myotoxins from Bothrops snake venoms. Toxicon 33, 1405-1424.--Several myotoxins have been isolated from Bothrops snake venoms during the last 10 years. All of them are group II basic phospholipases A2, although some lack enzymatic activity (i.e. Lys-49 variants). These myotoxins appear as an antigenically related family of proteins occurring in many, but not all, Bothrops venoms, bearing a close structural and antigenic relationship to toxins found in other crotalid venoms of the genera Agkistrodon and Trimeresurus. Myotoxins are quantitatively important venom components in some Bothrops species. Intramuscular injection of Bothrops myotoxins leads to a rapid series of drastic degenerative events, probably initiated at the plasma membrane level, which culminate in a selective skeletal muscle necrosis. This in vivo specificity contrasts with the ability of myotoxins to lyse many types of cells in culture. Muscle damage, as well as cytolysis and liposome disruption, occur in conditions where phospholipase A2 activity is inhibited, although enzymatic activity might enhance myotoxin actions.
Cellular and Molecular Life Sciences, 2009
Snake myotoxins have a great impact on human health worldwide. Most of them adopt a phospholipase A2 fold and occur in two forms which often co-exist in the same venom: the Asp49 toxins hydrolyse phospholipids, whilst Lys49 toxins are enzymatically inactive. To gain insights into their mechanism of action, muscle cells were exposed to Bothrops myotoxins, and cytosolic Ca 2+ and cytotoxicity were measured. In both myoblasts and myotubes, the myotoxins induced a rapid and transient rise in cytosolic [Ca 2+ ], derived from intracellular stores, followed, only in myotubes, by a large Ca 2+ influx and extensive cell death. Myoblast viability was unaffected. Notably, in myotubes Asp49 and Lys49 myotoxins acted synergistically to increase the plasma membrane Ca 2+ permeability, inducing cell death. Therefore, these myotoxins may bind to acceptor(s) coupled to intracellular Ca 2+ mobilization in both myoblasts and myotubes. However, in myotubes only, the toxins alter plasma membrane permeability, leading to death.
Cell Biochemistry and Function, 2005
Group II phospholipase A 2 (PLA 2 ) myotoxins isolated from Viperidae/Crotalidae snake venoms induce a rapid cytolytic effect upon diverse cell types in vitro. Previous studies suggested that this effect could be more pronounced on skeletal muscle myotubes than on other cell types, including undifferentiated myoblasts. This study utilized the murine skeletal muscle C2C12 cell line to investigate whether differentiated myotubes are more susceptible than myoblasts, and if this characteristic is specific for the group II myotoxic PLA 2 s. The release of lactic dehydrogenase was quantified as a measure of cytolysis, 3 h after cell exposure to different group II PLA 2 s purified from Bothrops asper, Atropoides nummifer, Cerrophidion godmani, and Bothriechis schlegelii venoms. In addition, susceptibility to lysis induced by synthetic melittin and group III PLA 2 from bee (Apis mellifera) venom, as well as by anionic, cationic, and neutral detergents, was comparatively evaluated on the two cultures. Myotubes were significantly more susceptible to group II PLA 2 myotoxins, but not to the other agents tested, under the same conditions. Moreover, the increased susceptibility of myotubes over myoblasts was also demonstrated with two cytolytic synthetic peptides, derived from the C-terminal region of Lys49 PLA 2 myotoxins, that reproduce the action of their parent proteins. These results indicate that fusion and differentiation of myoblasts into myotubes induce changes that render these cells more susceptible to the toxic mechanism of group II PLA 2 myotoxins, but not to general perturbations of membrane homeostasis. Such changes are likely to involve myotoxin acceptor site(s), which remain(s) to be identified.
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 .
PeerJ, 2014
Viperid venoms often contain mixtures of Asp49 and Lys49 PLA 2 myotoxin isoforms, relevant to development of myonecrosis. Given their difference in catalytic activity, mechanistic studies on each type require highly purified samples. Studies on Asp49 PLA 2 s have shown that enzyme inactivation using p-bromophenacyl bromide (p-BPB) drastically affects toxicity. However, based on the variable levels of residual toxicity observed in some studies, it has been suggested that effector mechanisms independent of catalysis may additionally be involved in the toxicity of these enzymes, possibly resembling those of the enzymatically inactive Lys49 myotoxins. A possibility that Lys49 isoforms could be present in Asp49 PLA 2 preparations exists and, if undetected in previous studies, could explain the variable residual toxicity. This question is here addressed by using an enzyme preparation ascertained to be free of Lys49 myotoxins. In agreement with previous reports, inactivation of the catalytic activity of an Asp49 myotoxin preparation led to major inhibition of toxic effects in vitro and in vivo. The very low residual levels of myotoxicity (7%) and cytotoxicity (4%) observed can be attributed to the low, although detectable, enzyme remaining active after p-BPB treatment (2.7%), and would be difficult to reconcile with the proposed existence of additional catalytic-independent toxic mechanisms. These findings favor the concept that the effector mechanism of toxicity of Asp49 PLA 2 myotoxins from viperids fundamentally relies on their ability to hydrolyze phospholipids, arguing against the proposal that membrane disruption may also be caused by additional mechanisms that are independent of catalysis. How to cite this article Mora-Obando et al. (2014), Role of enzymatic activity in muscle damage and cytotoxicity induced by Bothrops asper Asp49 phospholipase A 2 myotoxins: are there additional effector mechanisms involved? PeerJ 2:e569; DOI 10.7717/peerj.569 Mora-Obando et al. (2014), PeerJ, DOI 10.7717/peerj.569 2/16 Andrião-Escarso SH, Soares AM, Rodrigues VM, Angulo Y, Díaz C, Lomonte B, Gutiérrez JM, Giglio JR. 2000. Myotoxic phospholipases A 2 in Bothrops snake venoms: effect of chemical modifications on the enzymatic and pharmacological properties of bothropstoxins from Bothrops jararacussu. Biochimie 82:755-763 DOI 10.1016/S0300-9084(00)01150-0. Angulo Y, Lomonte B. 2009. Biochemistry and toxicology of toxins purified from the venom of the snake Bothrops asper. Toxicon 54:949-957 (96)00036-0. Bultrón E, Thelestam M, Gutiérrez JM. 1993. Effects on cultured mammalian cells of myotoxin III, a phospholipase A 2 isolated from Bothrops asper (terciopelo) venom. (93)90080-9. Chioato L, de Oliveira AHC, Ruller R, Sá JM, Ward RJ. 2002. Distinct sites for myotoxic and membrane-damaging activities in the C-terminal region of a Lys49-phospholipase A 2 . Biochemical Jounal 366:971-976 DOI 10.1042/BJ20020092. Chioato L, Ward RJ. 2003. Mapping structural determinants of biological activities in snake venom phospholipases A 2 by sequence analysis and site directed mutagenesis. Toxicon 42:869-883
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.
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 .
ACL myotoxin is a protein isolated from the venom of the broad-banded copperhead, Agkistrodon contortrix laticinctus. It is myotoxic, meaning that it has "direct and specific action" on skeletal muscle (Fletcher et al. 1997). Snake venom myotoxins are a large group of toxins that cause several types of necrosis in skeletal muscle cells (Johnson and Ownby 1994). Snake venom myotoxins structurally belong to a larger group of proteins, the PLA2 (phospholipase A2) family (Selistre de Arajo et al. 1995), but ACL myotoxin lacks the properties necessary to be part of that class (Johnson and Ownby 1993). ACL myotoxin has a pI greater than 9, is a single polypeptide (fig 1), having 126 amino acid residues (fig 1 & 2), and has a molecular weight of 14.5 kilodaltons (Johnson and Ownby 1993; Li et al. 1993). ACL myotoxin is a phospholipase A2 like protein. It lacks characteristic PLA2 enzymatic activity, but has similar structure and resultant necrosis after intramuscular injection. PLA2 myotoxins are split into two different groups, based on the presence or absence of an aspartic acid residue at position 49 (Fletcher et al. 1997). Position 49 is highly conserved among the more enzymatically active PLA2 molecules (Selistre de Araujo et al. 1996). When a lysine (K) is replaced for the aspartic acid (D) at position 49 it results in a large reduction, or elimination of the enzymatic activity of the protein (Fletcher et al. 1997). ACL myotoxin has substitution of D by K in position 49 (fig 3). Some other researchers say that this substitution completely stops enzymatic activity, and that any small amount of observed enzymatic activity can be contributed to contamination by D49 PLA2's in the same venom (Fletcher et al. 1997). Ca2+ is not present in the active site of the K49 substituted protein, which is in contrast to the D49 protein. It is also proposed that the K49 substitution hinders Ca2+ from binding to the active site of the protein. This inability to bind may be because of steric hindrance of K49, or the electrostatic repulsion of Ca2+, due to the same positive charge of the K.
Myotoxic class II phospholipases A 2 from snake venoms can be divided into Asp49 and Lys49 types. The latter, including Bothrops asper myotoxin II, exert membrane damage despite lacking catalytic activity. A heparin-binding, hydrophobic/ cationic region, near the C-terminus of myotoxin II (115±129) has been shown to be relevant in its membrane-damaging actions. However, some observations suggest also a potential participation of its N-terminal region. An immunochemical approach was utilized to examine the properties and possible role in toxicity of the N-terminal helix of myotoxin II. Rabbit antibodies raised to a synthetic peptide comprising residues 1±15 recognized the native protein. These antibodies were utilized to compare the antigenic characteristics of the N-terminal helix of several myotoxic phospholipases A 2 , showing generally stronger binding to Lys49 myotoxins, in comparison to Asp49 counterparts. However, three Lys49 myotoxins (Cerrophidion godmani myotoxin II, Atropoides nummifer myotoxin II, and Trimeresurus¯avoviridis basic protein I) were not recognized by the antibodies, revealing a signi®cant antigenic variability of the N-terminal region within this group of toxins. In neutralization experiments, pre-incubation of myotoxin II with af®nity-puri®ed antibodies to the N-terminal helix did not inhibit its myotoxic activity in mice, nor its cytotoxic effect upon cultured muscle cells. These ®ndings argue against a critical role of the N-terminal region of this protein in toxicity. Thus, the precise role of the N-terminal helix of myotoxin II and related Lys49 phospholipases A 2 , regarding their toxic mechanisms, remains controversial, and requires further experimental study to be clari®ed. q
Toxicon, 1987
J . M . GU719RREZ, G. RojAs, B . LomoNTE, J . A . GENé and L . CERDAs . Effects of a myotoxic phospholipase A2 isolated from Bothrops asper venom on skeletal muscle sarcoplasmic reticulum. Taxicon 25, 1244Taxicon 25, -1248Taxicon 25, , 1987 . -The myotoxin from B. asper snake venom inhibited Ca-ATPase activity of rabbit sareoplasmie retieulum after incubation in vitro . Inhibition was non-competitive and albumin enhanced the effect of the toxin . Furthermore, B. riper myotoxin hydrolyzed sarcoplasmic reticulum phospholipids and induced a dose-dependent release of horseradish perolddase that had been trapped in sarcoplasmic reticulum vesicles . Binding studies indicated that myotoxin does not bind to any particular protein of this membrane, suggesting that the toxin might interact with phospholipids. Inhibition of Ca-ATPase is probably a consequence of an alteration in sarcoplasmic reticulum phospholipids .