Kirill A. Pluzhnikov

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

SummaryTwo-dimensional 1H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 Å for backbone heavy atoms, and 1.25 Å for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.

Cloning and structure of delta-latroinsectotoxin, a novel insect-specific member of the latrotoxin family. Functional expression requires C-terminal truncation

The venom of the black widow spider (BWSV) (Latrodectus mactans tredecimguttatus) contains several potent, high molecular mass (>110 kDa) neurotoxins that cause neurotransmitter release in a phylum-specific manner. The molecular mechanism of action of these proteins is poorly understood because their structures are largely unknown, and they have not been functionally expressed. This study reports on the primary structure of -latroinsectotoxin (-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. -LIT comprises four structural domains: a signal peptide followed by an N-terminal domain that exhibits the highest degree of identity with other latrotoxins, a central region composed of 15 ankyrin-like repeats, and a C-terminal domain. The domain organization of -LIT is similar to that of other latrotoxins, suggesting that these toxins are a family of related proteins. The predicted molecular mass and apparent mobility of the protein (130 kDa) encoded in the -LIT gene differs from that of native -LIT purified from BWSV (110 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native -LIT revealed a molecular ion with m/z+ of 110916 ± 100, indicating that the native -LIT is 991 amino acids in length. When the full-length -LIT cDNA was expressed in bacteria the protein product was inactive, but expression of a C-terminally truncated protein containing 991 residues produced a protein that caused massive neurotransmitter release at the locust neuromuscular junction at nanomolar concentrations. Channels formed in locust muscle membrane and artificial lipid bilayers by the native -LIT have a high Ca permeability, whereas those formed by truncated, recombinant protein do not.

Novel peptide from spider venom inhibits P2X3 receptors and inflammatory pain

P2X3 purinoreceptors expressed in mammalian sensory neurons play a key role in several processes, including pain perception. From the venom of the Central Asian spider Geolycosa sp., we have isolated a novel peptide, named purotoxin‐1 (PT1), which is to our knowledge the first natural molecule exerting powerful and selective inhibitory action on P2X3 receptors. PT1 dramatically slows down the removal of desensitization of these receptors. The peptide demonstrates potent antinociceptive properties in animal models of inflammatory pain. ANN NEUROL 2010;67:680–683

Low molecular weight components from black widow spider venom

Highly purified alpha-latrotoxin from the black widow spider venom (alpha-LTX) consists of two polypeptides with mol. wts of 130,000 and 8000 (LMWP). We have isolated two low mol. wt proteins LMWP and LMWP2 from the low mol. wt fraction of this venom. The chemical properties of these proteins and partial amino acid sequence of novel protein LMWP2 were studied. By means of i.v. or intracerebroventricular injections into mice it was shown that low mol. wt components of the venom at concentrations of 2.3 mg/kg and 0.8 mg/kg, respectively, did not possess any direct toxic effect on vertebrates. Injections of each protein into the third thoracic segment of cockroaches Periplaneta americana (doses up to 80 micrograms/g) did not cause lethality or paralysis of insects.

Modulation of functional activities of the neurotoxin from black widow spider venom.

We have studied the action of an α‐latrotoxin (α‐LTX) complex of two polypeptides (LTX 130 kDa and low molecular weight protein (LMWP) 8 kDa) and the action of a venom fraction containing LTX with excess LMWP on calcium influx into synaptosomes and PC 12 cells as well as on [14C[GABA release from synaptosomes. Both preparations considerably activate calcium influx and stimulate [14C]GABA release from synaptosomes. Preincubation of both preparations with antibodies against a 14 amino acid residue C‐terminal peptide of LMWP differentially modulates these effects. Antibodies inhibit induced calcium influx and enhance induced GABA release.

M-type K + current inhibition by a toxin from the scorpion Buthus eupeus

A number of invertebrate venoms have been tested for effects on M‐type K+ currents (I K(M)) in differentiated mouse neuroblastoma X rat glioma NG108‐15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited I K(M) by ∼ 44% at 50 μg/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrymidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited I K(M) by ∼45% with an IC50 of ∼;33 nM. Neither the crude venom nor the purified polypeptide affected the Ca2+ current or the delayed rectifier K+ current. While the crude venom prolonged the Na+ current, the polypeptide did not. Thus, the 4 kDa Buthus eupeus polypeptide appears to be a selective inhibitor of I K(M) in NG108‐15 cells.

Structure and function of the potassium channel inhibitor from black scorpion venom

A novel inhibitor of K+ channels has been purified from the venom of the Central Asian scorpion Orthochirus scrobiculosus. For this polypeptide toxin (OsK- 1) with molecular mass 4205.7 Da complete amino acid sequence was determined by Edman degradation and C-terminal amino acid analysis, and was confirmed by cloning and sequencing of the toxin cDNA. OsK-1 consists of 38 amino acid residues and possesses high sequence homology with agiotoxin, kaliotoxin and some homology with other known K+-channel blockers from different scorpion venoms. The toxin was shown to block small-conductance Ca++-activated K+-channels in neuroblastomaxglioma NG 10815 hybrid cells (Kd =1.4 x M) which are insensitive to apamin and sensitive to charybdotoxin. The effect of OsK- 1 was reversible and concentration dependent.

Toxic principle of selva ant venom is a pore-forming protein transformer

Ectatomin (Ea) is a newly isolated main toxic component of Ectatomma tuberculatum ant venom. Structural and electrophysiological studies were performed with purified Ea. The protein consists of two homologous polypeptide chains (37 and 34 residues) and forms a four α‐helix bundle in aqueous solution. On insertion into artificial bilayer membranes, two Ea molecules form an ion pore. Our results suggest that the ‘inside‐out’ mechanism of pore formation requires a significant movement of Ea helical parts. The pore formation in the cell membrane might well explain the toxic activity of Ea, not excluding at the same time its intracellular activities.

Modulation of P2X3 receptors by spider toxins.

Recently, the novel peptide named purotoxin-1 (PT1) has been identified in the venom of the spider Geolycosa sp. and shown to exert marked modulatory effects on P2X3 receptors in rat sensory neurons. Here we studied another polypeptide from the same spider venom, purotoxin-2 (PT2), and demonstrated that it also affected activity of mammalian P2X3 receptors. The murine and human P2X3 receptors were heterologously expressed in cells of the CHO line, and nucleotide-gated currents were stimulated by CTP and ATP, respectively. Both PT1 and PT2 negligibly affected P2X3-mediated currents elicited by brief pulses of the particular nucleotide. When subthreshold CTP or ATP was added to the bath to exert the high-affinity desensitization of P2X3 receptors, both spider toxins strongly enhanced the desensitizing action of the ambient nucleotides. At the concentration of 50nM, PT1 and PT2 elicited 3-4-fold decrease in the IC(50) dose of ambient CTP or ATP. In contrast, 100nM PT1 and PT2 negligibly affected nucleotide-gated currents mediated by mP2X2 receptors or mP2X2/mP2X3 heteromers. Altogether, our data point out that the PT1 and PT2 toxins specifically target the fast-desensitizing P2X3 receptor, thus representing a unique tool to manipulate its activity.

Ant polypeptide toxins.

Compared with other insects with social behaviour, ants have no peers. The number of the identified ant species is slightly less than 9,000, but an estimated 11,000 species are yet to be described, mostly from tropical habitats (Blum, 1992). Ants from most subfamilies are capable of stinging. Since stinging ants are particularly abundant in tropical regions, it may be anticipated that a large number of venom species are yet to be described, and the medical importance of these insects will be better appreciated. Ant venoms exhibit greater variability in composition and function than do venoms from any other group of arthropods. The radically different evolutionary and ecological histories of ants as compared with other arthropods would seem to argue against the similarity of the venoms produced in these diverse groups. Unlike social bees and wasps, which use their stings solely for defence, ant stings serve for a variety of additional functions, including prey capture and the elaboration of trail, sex, aggre-gation, and alarm pheromones (Schmidt, 1986). Ant venoms, in general, cause immediate, often intense locomotor activity followed by clonic convulsions, peripheral vasodilitation, and sedation. This is usually followed by cyanosis, shallow breathing and a gradual loss of activity until death (Schmidt et al., 1980).