[Identification and structural analysis of a glycophospholipid component from the venom of ant Paraponera clavata].

The venom of South American ant Paraponera clavata and its low-molecular-mass fraction were shown to possess insectotoxic and pore-forming activities. A number of glycophospholipid components were isolated from this ant venom by means of gel filtration and reversed-phase chromatography. Some of the compounds cause conductivity fluctuations in lipid bilayer membranes within the ranges 3-25 pS and 200-400 pS at concentrations of 10(-6) to 10(-7) M. N-Acetylglucosamine, a fatty acid, and phosphoric acid residues were found in their structures. A full structure, 3-myristoyl-2-acetamido-2-deoxy-alpha-D-glucopyranosyl phosphate, was elucidated for one of the compounds by the use of 1H, 13C, and 31P NMR spectroscopy and mass spectrometry.

[Expression of neurotoxin II from Naja oxiana cobra venom in Escherichia coli in a hybrid form with thioredoxin]. / Ekspressiruiushchaia konstruktsiia dlia narobotki v Escherichia coli neirotoksina i iz iada kobry Naja oxiana v vide gibrida s tioredoksinom.

Neurotoxin II from the venom of cobra Naja oxiana is a short type alpha-neurotoxin, which competitively inhibits nicotinic acetylcholine receptor. The toxin gene was expressed as a construct fused with the thioredoxin gene and the linker encoding the enteropeptidase recognition site and a Met residue between the genes. The fusion protein was mainly cleaved by cyanogen bromide, since enteropeptidase was less effective. The yield of neurotoxin II was 6 mg/l of the bacterial culture. The resulting recombinant protein was identified with native neurotoxin II by its N-terminal analysis, mass spectrometry, and NMR spectroscopy. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 1; see also http://www.maik.ru.

An ERG channel inhibitor from the scorpion Buthus eupeus.

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.

[Molecular cloning and primary structure of cDNA fragment for alpha-latrocrustatoxin from black widow spider venom]. / Molekuliarnoe klonirovanie i opredelenie pervichnoi struktury fragmenta kDNK alpha-latrokrustotoksina iz iada pauka karakurta.

A fragment of the structural gene of alpha-latrocrustotoxin, a new representative of latrotoxins from black widow spider venom, was cloned. The fragment (1191 bp) was obtained by means of PCR based on the data obtained by sequencing tryptic peptides of the toxin. The fragment codes for a 397-aa sequence. The encoded polypeptide is the C-terminal fragment of the toxin central domain that presumably contains a site responsible for the toxin species specificity. The structural similarity of this fragment to the corresponding fragments of other latrotoxins was studied.

[Isolation and partial structural characteristics of major toxic components of Latrodectus pallidus venom]. / Vydelenie i chastichnaia strukturnaia kharakteristika osnovnykh toksicheskikh komponentov iada pauka Latrodectus pallidus.

Toxic components of the Latrodectus pallidus spider venom were isolated and characterized. The venom was shown to contain a toxin specific for mammals and at least one insectospecific toxin. Partial amino acid sequences of both toxins were determined, and their high structural homology with previously studied alpha-latrotoxin and alpha-latroinsectotoxin from L. mactans tredecimguttatus was found.

Three-dimensional structure of toxin OSK1 from Orthochirus scrobiculosus scorpion venom.

A 600 MHz 1H NMR study of toxin OSK1, blocker of small-conductance Ca2+-activated K+ channels, is presented. The unambiguous sequential assignment of all the protons of the toxin was obtained using TOCSY, DQF-COSY, and NOESY experiments at pH 3.0 (10, 30, and 45 degrees C) in aqueous solution. 3J(N alpha), 3J(alphabeta) vicinal spin coupling constants were determined in high-resolution spectra. The cross-peak volumes in NOESY spectra and the coupling constants were used to define the local structure of the protein by the program HABAS and to generate torsion angle and interproton distance constraints for the program DIANA. Hydrogen-deuterium exchange rates of amide protons showed possible locations of hydrogen bonds. The hydrogen bond acceptors and disulfide bridges between residues 8-28, 14-33, and 18-35 were determined when analyzing distance distribution in preliminary DIANA structures. All constraints were used to obtain a set of 30 structures by DIANA. The resulting rms deviations over 30 structures are 1.30 A for the heavy atoms and 0.42 A for the backbone heavy atoms. The structures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the alpha-helix (residues 10-21) slightly distorted at the Cys14 residue, two main strands of the antiparallel beta-sheet (24-29, 32-38), and the extended fragment (2-6). The motif is stabilized by the disulfide bridges in the way, common to all known scorpion toxins. Using the fine spatial toxin structure, alignment of the homologues, mutagenesis analysis, and comparison of scorpion toxin family functions, we delineate some differences significant for the toxin specificity.

[Recombinant proteins containing amino acid sequences of two ectatomin chains]. / Rekombinantnye belki, soderzhashchie aminokislotnye posledovatel'nosti dvukh tsepei éktatomina.

Artificial genes for chains A and B of ectatomin, an Ectatomma tuberculatum ant toxin, were obtained by chemical and enzymic synthesis and cloned into new plasmid vectors. Expression plasmids with the genes of hybrid proteins were constructed containing human interleukin-3 or its terminal 63-mer fragment as well as chains A and B of ectatomin, which are linked via a region containing the cleavage site of specific protease, enterokinase (hybrid proteins IL3ETOXA, IL3ETOXB, ILETOXA, and ILETOXB). Escherichia coli producer strains providing a high yield of IL3ETOXA and IL3ETOXB proteins as inclusion bodies were obtained.

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

A number of invertebrate venoms have been tested for effects on M-type K+ currents (IK(M)) in differentiated mouse neuroblastoma X rat glioma NG108-15 cells. Among the venoms tested, Buthus eupeus scorpion venom reversibly inhibited IK(M) by approximately 44% at 50 microgram/ml. Inhibition was not due to activation of bradykinin or nucleotide (pyrimidine) receptors. On venom fractionation, a polypeptide of 4 kDa was purified that inhibited IK(M) by approximately 45% with an IC50 of approximately 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 IK(M) in NG108-15 cells.

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 delta-latroinsectotoxin (delta-LIT), a novel insect-specific toxin from BWSV, that contains 1214 amino acids. delta-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 delta-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 (approximately 130 kDa) encoded in the delta-LIT gene differs from that of native delta-LIT purified from BWSV (approximately 100 kDa), suggesting that the toxin is produced by proteolytic processing of a precursor. MALDI-MS of purified native delta-LIT revealed a molecular ion with m/z+ of 110916 +/- 100, indicating that the native delta-LIT is 991 amino acids in length. When the full-length delta-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 delta-LIT have a high Ca2+ permeability, whereas those formed by truncated, recombinant protein do not.

[Primary structure of delta-latroinsectotoxin from venom of the Latrodectus mactans tredecimguttatus spider]. / Pervichnaia struktura delta-latroinsektotoksina iz iada pauka Latrodectus mactans tredecimguttatus.

The structural gene of delta-latroinsectotoxin was cloned and its nucleotide sequence was determined. The gene contains an open reading frame of 3642 bp. The deduced amino acid sequence is homologous to the sequences of latrotoxins studied earlier.

[Protein composition of venoms from several species of tropical ants and their effect on mitochondrial H+-ATPase]. / Issledovanie belkovogo sostava iadov nekotorykh vidov tropicheskikh murav'ev i ikh vliianiia na H+-ATPazu mitokhondrii.

Protein compositions of venoms of South-American stinging ants, Ectatomma tuberculatum, Paraponera clavata (subfamily Ponerinae), and "tangarana" were analyzed. The venom of E. tuberculatum displayed the most complex protein composition (more than 15 polypeptides). The water-soluble fraction of the venoms of P. clavata and "tangarana" contained acidic proteins (pI < 3.5 to 5.2), whereas the venom of E. tuberculatum contained predominantly basic proteins (pI 8 to > 9.5). N-Terminal residues and N-terminal sequences of a number of polypeptides were determined. High-molecular-mass polypeptides of the P. clavata venom slightly stimulated the ATPase activity of mitochondrial F1-ATPase. Low-molecular-mass nonprotein components of this venom significantly inhibited the ATPase activity of submitochondrial particles and F1-ATPase from bovine heart mitochondria. The venoms of E. tuberculatum and "tangarana" produced no effect on the ATPase activity.

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.

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom.

Two-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 A for backbone heavy atoms, and 1.25 A for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two alpha-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-alpha-helical bundle structure.

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 alpha-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 functional activities of the neurotoxin from black widow spider venom.

We have studied the action of an alpha-latrotoxin (alpha-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 PC12 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.

[Study of the amino acid sequence of latroinsectotoxin from black widow spider venom]. / Issledovanie aminokislotnoi posledovatel'nosti latroinsektotoksina iz iada pauka karakurta.

The N-terminal amino acid sequence of alpha-latroinsectotoxin from the venom of Latrodectus mactans tredecimguttatus was determined. Then the toxin was digested by trypsin and total or partial amino acid sequences of twenty-six tryptic peptides were established. This resulted in the structural information needed for the construction of probes followed by the cloning of the alpha-latroinsectotoxin structural gene.

[Effect of modification of Naja naja oxiana neurotoxin II on the stoichiometry of its complexes with acetylcholine receptor]. / Vliianie modifikatsii neirotoksina II Naja naja oxiana na stekhiometriiu kompleksov s atsetilkholinovym retseptorom.

It was discovered that illumination of the complex formed by the solubilized acetylcholine receptor from Torpedo marmorata and Lys25-p-azidobenzoyl derivative of neurotoxin II results in the appearance on the receptor of up to 4 additional binding sites. Acetylcholine and neurotoxin II, but not the long-chain neurotoxins bind specifically to these sites. The additional binding sites could be also detected after illuminating the receptor complex with other photoactivable derivatives, provided the latter were displaced from one of the two main binding sites by hexa(trifluoroacetyl)neurotoxin II. A similar, but less pronounced effect, was observed on binding Lys25 (Ac) derivative of neurotoxin II. The formation of the additional binding sites was found to depend on the activity of the receptor preparations as well as on the mutual influence of the two main toxin-binding sites.

[2-D-NMR study of the conformation characteristics of toxin 3 from Naja naja siamensis venom]. / 2-D-OaMR-issledovanie konformatsionnykh osobennostei toksina 3 iz iada kobry Naja naja siamensis.

The spatial structure of "long" toxin 3 Naja naja siamensis in solution has been studied by methods of two-dimensional (2D) 1H NMR spectroscopy. The individual signal assignments for 67 out of 71 residues and analysis of nuclear Overhauser effects between distinct protons of the molecule allowed the comparison of the toxin 3 conformations at different pH values and temperatures. It was shown that the deprotonated imidazole ring of His22 residue (at pH greater than or equal to 7,5) is surrounded by the side chains of Cys17, Pro18, Val23, Cys24, Cys45, Ala46 and Thr48 residues. On the contrary, the protonated imidazole ring of His22 (at pH less than 4,0) is exposed into solvent. Ionization of His22 is accompanied by a change in the Tyr25 aromatic ring orientation and affects the conformational mobility of the Cys17, His22, Cys45 and Ala47 side chains. The revealed conformational features of toxin 3 in solution are discussed in connection with the differences between "long" and "short" neurotoxins in the kinetics of their binding to acetylcholine receptor.

[Relative localization of the bound acetylcholine receptor subunits and neurotoxin]. / Vzaimnoe raspolozhenie sub''edinits atsetilkholinovogo retseptora i sviazannogo s nim neirotoksina.

A series of neurotoxin II (Naja naja oxiana) derivatives, each containing one p-azido-[14C]benzoyl group, have been prepared. Those labeled at Leu1, Lys15, Lys25, Lys26, or Lys46 associate specifically with the acetylcholine receptor from the Torpedo marmorata electric organs and form the crosslinks with it as a result of irradiation. Electrophoresis in polyacrylamide gel and gel chromatography revealed the contacts between the neurotoxins and alpha, beta, gamma and delta subunits of the receptor, modification of a particular subunit being governed by the photoactivable group position in the neurotoxin molecule. The differences of the two neurotoxin binding sites in the receptor were demonstrated by analysis of the photoinduced crosslinks under the conditions of one site being blocked by hexa (trifluoroacetyl) neurotoxin II. The mutual arrangement of the two bound neurotoxin molecules was established. On the basis of data obtained, two models for the acetylcholine receptor subunit topography were proposed.

[1H-NMR study of the Naja naja oxiana neurotoxin II and its spin-labeled derivatives. Conformation of "short" neurotoxins]. / 1H-IaMR-issledovanie neirotoksina II Naja naja oxiana i ego spin-mechenykh proizvodnykh. Konformatsiia "korotkikh" neirotoksinov.

In 1H NMR spectra of neurotoxin II N. n. oxiana the chemical shift pH-dependences in H2O and 2H2O solutions were studied, and also the deuterium exchange rates and chemical shift temperature gradients were measured for the amide protons. The spin probe method was applied to assess the degree of exposure into solvent of the amide and side chain protons. With the purpose of establishing mutual disposition of certain neurotoxin II groupings, nuclear Overhauser effect was studied in the 1H NMR spectra, along with the broadening of proton resonances induced by spin labels selectively attached to epsilon-amino groups of Lys26, Lys27, Lys45 or Lys47. The mobility of these labels was determined from the EPR spectra. The methyl resonances of Val and Leu residues were assigned to a definite position in the amino acid sequence. The following pKa were determined: alpha-NH2 Leu1 (9,2), gamma-COOH Glu2 (3,7), alpha-COOH Asn62 (1,3). The protonation of a carboxyl group(s) in neurotoxin II (alpha-COOH Asn62 seems to be involved) decreases the temperature stability of the neurotoxin II conformation. On the basis of studies on neurotoxin II and some other homologous neurotoxins, the model for the "short" neurotoxin folding in solution was proposed. Comparison of experimental data for the disposition of equivalent groups in homologous neurotoxins and in the X-ray structure of erabutoxin b Laticauda semifasciata revealed that the Val46 side chain in solution might change its orientation by 180 degrees with respect to polypeptide backbone. Binding of spin labeled neurotoxin II derivatives to the acetylcholine receptor was discussed in light of the obtained data.