Yuri N. Utkin

Poisonous organisms are represented in many taxa, including kingdom Animalia. During evolution, animals have developed special organs for production and injection of venoms. Animal venoms are complex mixtures, compositions of which depend on species producing venom. The most known and studied poisonous terrestrial animals are snakes, scorpions and spiders. Among marine animals, these are jellyfishes, anemones and cone snails. The toxic substances in the venom of these animals are mainly of protein and peptide origin. Recent studies have indicated that the single venom may contain up to several hundred different components producing diverse physiological effects. Bites or stings by certain poisonous species result in severe envenomations leading in some cases to death. This raises the problem of bite treatment. The most effective treatment so far is the application of antivenoms. To enhance the effectiveness of such treatments, the knowledge of venom composition is needed. On the other hand, venoms contain substances with unique biological properties, which can be used both in basic science and in clinical applications. The best example of toxin application in basic science is α-bungarotoxin the discovery of which made a big impact on the studies of nicotinic acetylcholine receptor. Today compositions of venom from many species have already been examined. Based on these data, one can conclude that venoms contain a large number of individual components belonging to a limited number of structural types. Often minor changes in the amino acid sequence give rise to new biological properties. Change in the living conditions of poisonous animals lead to alterations in the composition of venoms resulting in appearance of new toxins. At the same time introduction of new methods of proteomics and genomics lead to discoveries of new compounds, which may serve as research tools or as templates for the development of novel drugs. The application of these sensitive and comprehensive methods allows studying either of venoms available in tiny amounts or of low abundant components in already known venoms.

Cytotoxins from cobra venom are known to manifest cytotoxicity in various cell types. It is widely accepted that the plasma membrane is a target of cytotoxins, but the mechanism of their action remains obscure. Using the confocal spectral imaging technique, we show for the first time that cytotoxins from cobra venom penetrate readily into living cancer cells and accumulate markedly in lysosomes. Cytotoxins CT1 and CT2 from Naja oxiana, CT3 from Naja kaouthia and CT1 from Naja haje are demonstrated to possess this property with respect to human lung adenocarcinoma A549 and promyelocytic leukaemia HL60 cells. Immobilized plasma membrane binding accompanies the internalization of CT3 from Naja kaouthia in the HL60 cells, but it is very weak for other cytotoxins. Detectable membrane binding is not a property of any of the cytotoxins tested in A549 cells. The kinetics and concentration-dependence of cytotoxin accumulation in lysosomes correlate well with their cytotoxic effects. On the basis of the results obtained, we propose that lysosomes are a primary target of the lytic action of cytotoxins. Plasma membrane permeabilization seems to be a downstream event relative to lysosome rupture. Direct damage to the plasma membrane may be a complementary mechanism, but its relative contribution to the cytotoxic action depends on the cytotoxin structure and cell type.

A novel "weak toxin" (WTX) from Naja kaouthia snake venom competes with [(125)I]alpha-bungarotoxin for binding to the membrane-bound Torpedo californica acetylcholine receptor (AChR), with an IC(50) of approximately 2.2 microm. In this respect, it is approximately 300 times less potent than neurotoxin II from Naja oxiana and alpha-cobratoxin from N. kaouthia, representing short-type and long-type alpha-neurotoxins, respectively. WTX and alpha-cobratoxin displaced [(125)I]alpha-bungarotoxin from the Escherichia coli-expressed fusion protein containing the rat alpha7 AChR N-terminal domain 1-208 preceded by glutathione S-transferase with IC(50) values of 4.3 and 9.1 microm, respectively, whereas for neurotoxin II the IC(50) value was >100 microm. Micromolar concentrations of WTX inhibited acetylcholine-activated currents in Xenopus oocyte-expressed rat muscle AChR and human and rat alpha7 AChRs, inhibiting the latter most efficiently (IC(50) of approximately 8.3 microm). Thus, a virtually nontoxic "three-fingered" protein WTX, although differing from alpha-neurotoxins by an additional disulfide in the N-terminal loop, can be classified as a weak alpha-neurotoxin. It differs from the short chain alpha-neurotoxins, which potently block the muscle-type but not the alpha7 AChRs, and is closer to the long alpha-neurotoxins, which have comparable potency against the above-mentioned AChR types.