Lemnitoxin, the major component of Micrurus lemniscatus coral snake venom, is a myotoxic and pro-inflammatory phospholipase A(2)

The venom of Micrurus lemniscatus, a coral snake of wide geographical distribution in South America, was fractionated by reverse-phase HPLC and the fractions screened for phospholipase A(2) (PLA(2)) activity. The major component of the venom, a PLA(2), here referred to as 'Lemnitoxin', was isolated and characterized biochemically and toxicologically. It induces myotoxicity upon intramuscular or intravenous injection into mice. The amino acid residues Arg15, Ala100, Asn108, and a hydrophobic residue at position 109, which are characteristic of myotoxic class I phospholipases A(2), are present in Lemnitoxin. This PLA(2) is antigenically related to M. nigrocinctus nigroxin, Notechis scutatus notexin, Pseudechis australis mulgotoxin, and Pseudonaja textilis textilotoxin, as demonstrated with monoclonal and polyclonal antibodies. Lemnitoxin is highly selective in its targeting of cells, being cytotoxic for differentiated myotubes in vitro and muscle fibers in vivo, but not for undifferentiated myoblasts or endothelial cells. Lemnitoxin is not lethal after intravenous injection at doses up to 2 mu g/g in mice, evidencing its lack of significant neurotoxicity. Lemnitoxin displays anticoagulant effect on human plasma and proinflammatory activity also, as it induces paw edema and mast cell degranulation. Thus, the results of this work demonstrate that Lemnitoxin is a potent myotoxic and proinflammatory class I PLA(2). (C) 2016 Elsevier Ireland Ltd. All rights reserved.

A Lys49-PLA2 myotoxin of Bothrops asper triggers a rapid death of macrophages that involves autocrine purinergic receptor signaling.

Lys49-PLA(2) myotoxins, an important component of various viperid snake venoms, are a class of PLA(2)-homolog proteins deprived of catalytic activity. Similar to enzymatically active PLA(2) (Asp49) and to other classes of myotoxins, they cause severe myonecrosis. Moreover, these toxins are used as tools to study skeletal muscle repair and regeneration, a process that can be very limited after snakebites. In this work, the cytotoxic effect of different myotoxins, Bothrops asper Lys49 and Asp49-PLA(2), Notechis scutatus notexin and Naja mossambica cardiotoxin, was evaluated on macrophages, cells that have a key role in muscle regeneration. Only the Lys49-myotoxin was found to trigger a rapid asynchronous death of mouse peritoneal macrophages and macrophagic cell lines through a process that involves ATP release, ATP-induced ATP release and that is inhibited by various purinergic receptor antagonists. ATP leakage is induced also at sublytical doses of the Lys49-myotoxin, it involves Ca(2+) release from intracellular stores, and is reduced by inhibitors of VSOR and the maxi-anion channel. The toxin-induced cell death is different from that caused by high concentration of ATP and appears to be linked to localized purinergic signaling. Based on present findings, a mechanism of cell death is proposed that can be extended to other cytolytic proteins and peptides.

Calcium imaging of muscle cells treated with snake myotoxins reveals toxin synergism and presence of acceptors.

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.

Cellular pathology induced by snake venom phospholipase A2 myotoxins and neurotoxins: common aspects of their mechanisms of action.

A large variety of snake toxins evolved from PLA(2) digestive enzymes through a process of 'accelerated evolution'. These toxins have different tissue targets, membrane receptors and mechanisms of alteration of the cell plasma membrane. Two of the most commonly induced effects by venom PLA(2)s are neurotoxicity and myotoxicity. Here, we will discuss how these snake toxins achieve a similar cellular lesion, which is evolutionarily highly conserved, despite the differences listed above. They cause an initial plasma membrane perturbation which promotes a large increase of the cytosolic Ca(2+) concentration leading to cell degeneration, following modes that we discuss in detail for muscle cells and for the neuromuscular junction. The different systemic pathophysiological consequences caused by these toxins are not due to different mechanisms of cell toxicity, but to the intrinsic anatomical and physiological properties of the targeted tissues and cells.

Effect of calcineurin inhibitors on myotoxic activity of crotoxin and Bothrops asper phospholipase A2 myotoxins in vivo and in vitro.

Previous studies have shown that calcineurin activity plays a critical role in the myotoxic activity induced by crotoxin (CTX), a group II phospholipase A(2) (PLA(2)) with neurotoxic and myotoxic actions. In order to address whether calcineurin is also important for the activity of non-neurotoxic group II PLA(2) myotoxins we have compared the effects of calcineurin inhibition on the myotoxic capacity of CTX and the non-neurotoxic PLA(2)s, myotoxin II (Mt II) and myotoxin III (Mt III) from Bothrops asper venom. Rats were treated with cyclosporin A (CsA) or FK506, calcineurin inhibitors, and received an intramuscular injection of either CTX, Mt II or Mt III into the tibialis anterior. Animals were killed 24 h after injection of toxins. Tibialis anterior was removed and stored in liquid nitrogen. Myofibers in culture were also treated with CsA or FK506 and exposed to CTX, Mt II and Mt III. It was observed that, in contrast to CTX, CsA and FK506 do not attenuate myotoxic effects induced by both Mt II and Mt III in vivo and in vitro. The results of the present study suggest that calcineurin is not essential for the myotoxic activity of Mt II and Mt III, indicating that distinct intracellular pathways might be involved in myonecrosis induced by neurotoxic CTX and non-neurotoxic Bothrops sp. PLA(2) myotoxins. Alternatively, calcineurin dependent fast fiber type shift might render the muscle resistant to the action of CTX, without affecting its susceptibility to Bothrops sp. myotoxins.

Effect of a recombinant Lys49PLA2 myotoxin and Lys49PLA2-derived synthetic peptides from Agkistrodon species on membrane permeability to water.

The aim of this work was to study the effect of recombinant ACL myotoxin, a Lys49PLA2 from Agkistrodon contortrix laticinctus snake venom and Lys49PLA2-derived synthetic peptides corresponding to the region 115-129 of venom of the two different Agkistrodon species on water permeability in the toad urinary bladder. The water flow through the membrane was measured gravimetrically in bag preparations of the bladder. The addition of recombinant ACL myotoxin-MBP (maltose binding protein) fusion protein (10 nM) to the bathing solution significantly increased (above 60%) the water transport compared with the control hemibladders. The addition of the Lys49PLA2-derived synthetic peptides in several concentrations to the bathing solution did not affect the water transport across membrane. These results suggest that the ACL myotoxin effect on water transport is not related to the cytotoxic C-terminal region.

Snake venom phospholipase A2s (Asp49 and Lys49) induce mechanical allodynia upon peri-sciatic administration Involvement of spinal cord glia, proinflammatory cytokines and nitric oxide

Snakebites constitute a serious public health problem in Central and South America, where species of the lancehead pit vipers (genus Bothrops) cause the majority of accidents. Bothrops envenomations are very painful, and this effect is not neutralized by antivenom treatment. Two variants of secretory phospholipases A2 (sPLA2), corresponding to Asp49 and Lys49 PLA2s, have been isolated from Bothrops asper venom. These sPLA2s induce hyperalgesia in rats following subcutaneous injection. However, venom in natural Bothrops bites is frequently delivered intramuscularly, thereby potentially reaching peripheral nerve bundles. Thus, the present series of experiments tested whether these sPLA2s could exert pain-enhancing effects following administration around healthy sciatic nerve. Both were found to produce mechanical allodynia ipsilateral to the injection site; no thermal hyperalgesia was observed. As no prior study has examined potential spinal mechanisms underlying sPLA2 actions, a series of anatomical and pharmacological studies were performed. These demonstrated that both sPLA2s produce activation of dorsal horn astrocytes and microglia that is more prominent ipsilateral to the site of injection. As proinflammatory cytokines and nitric oxide have each been previously implicated in spinally mediated pain facilitation, the effect of pharmacological blockade of these substances was tested. The results demonstrate that mechanical allodynia induced by both sPLA2s is blocked by interleukin-1 receptor antagonist, anti-rat interleukin-6 neutralizing antibody, the anti-inflammatory cytokine interleukin-10, and a nitric oxide synthesis inhibitor (L-NAME). As a variety of immune cells also produce and release sPLA2s during inflammatory states, the data may have general implications for the understanding of inflammatory pain. © 2003 International Association for the Study of Pain.