Snakebites are frequently hazardous accidents that require immediate medical treatment to neutralize the venom’s toxic effects. A feared complication of South American pit viper bites is extended myonecrosis, i.e. large destruction of muscle tissues, which may lead to amputation of the bitten limb and permanent disability. Although the currently available antisera can neutralize the hemotoxic effects of the venom, their success in preventing myotoxic effects is limited. Understanding how the muscle-damaging toxins of the venom act may therefore help to improve current therapies to prevent extended tissue damage. In a recent study published in PLoS ONE, Brazilian scientists led by Márcia Gallacci and Marcos Fontes have analyzed a pit viper myotoxin. They identified the region on the surface of the venomous protein that interacts with an inhibitor to inactivate the protein’s damaging effects, a finding that has important implications for the toxins’ mode of action.
One of the most damaging components of pit viper venoms are catalytically inactive versions of phospholipase A2 (Lys49-PLA2), and it seems that they have a crucial role in myonecrosis. They bind and destabilize the muscle cell membranes, resulting in a collapse of the ionic gradients that are required to transmit electric signals from the nerves to the muscle, which prevents the muscle from responding to nerve signals and results in muscle degeneration. Some compounds, however, appear to be able to prevent membrane destabilization caused by this toxin. One of them is rosmarinic acid, a polyphenolic compound from plants, which inhibits the myotoxic effect by an unknown mechanism.
To provide more insight into myotoxin neutralization by rosmarinic acid, the Brazilian team analyzed the structure and function of PrTX-I, a Lys49-PLA2-type toxin from the venom of the South American Piraja’s lancehead (Bothrops pirajai). First, they characterized the myotoxic effects of PrTX-I on phrenic diaphragm muscles from mice by incubating them in organ-bath chambers and then adding either PrTX-I from the pit viper venom or rosmarinic acid, or both substances. Next they recorded the muscle’s ability to twitch in response to an electric stimulus before preparing histological sections to evaluate the resulting muscle damage.
In the PrTX-I-treated muscles the team observed a dramatic decrease of twitch amplitude (suggesting the loss of neuromuscular signal transmission) and significant muscle damage as indicated by lesions and a loss of myofibrils. However, simultaneous treatment with PrTX-I and rosmarinic acid prevented the loss of nerve signal transmission and muscle damage, while rosmarinic acid alone had no effect. Thus, they demonstrated that rosmarinic acid can efficiently protect the muscle cells from the toxic effects of PrTX-1. Finally, the scientists crystallized the PrTX-I toxin in the presence of rosmarinic acid, hoping that they would be able to visualize the binding site on the toxin’s surface that rosmarinic acid contacts to inhibit the toxin. Indeed, they showed that rosmarinic acid binds to PrTX-I at the entrance of a hydrophobic channel. This finding was unexpected, because the binding site was at a different location from the region that was previously suggested to mediate myotoxicity.
By analysing the structural basis of the neutralizing effect of rosmarinic acid on PrTX-I, Gallacci, Fontes and their colleagues provide new insight into the mode of action of a myotoxin from pit viper venom. They propose a model in which rosmarinic acid impedes the interaction of PrTX-I with muscle cell membranes by blocking a hydrophobic channel that could bind the fatty acid tails of membrane phospholipids. Their discovery may lead to the development of more Lys49-PLA2 myotoxin inhibitors that can be used to improve serum therapy for venomous snakebites.
- © 2012.