Eight linear cationic peptides with cytolytic and insecticidal activity, designated cyto-insectotoxins (CITs), were identified in Lachesana tarabaevi spider venom. The peptides showed antibiotic ...activity towards Gram-positive and Gram-negative bacteria at micromolar concentrations as well as toxicity to insects. The primary structures of the toxins were established by direct Edman sequencing in combination with enzymatic and chemical polypeptide degradation and MS. CITs represent a novel class of cytolytic molecules and spider venom toxins. They are the first example of molecules showing equally potent antimicrobial and insecticidal effects. Analysis of L. tarabaevi venom gland expressed sequence tag database revealed the primary structures of the protein precursors; eight peptides homologous with the purified toxins were additionally predicted. CIT precursors share a conventional prepropeptide structure with an acidic prosequence and a processing motif common to most spider toxin precursors. The most abundant peptide, CIT 1a, was chemically synthesized, and its lytic activity on different bacterial strains, human erythrocytes and lymphocytes, insect cells, planar lipid bilayers and lipid vesicles was characterized. The spider L. tarabaevi is suggested to have evolved to rely on a unique set of linear cytolytic toxins, as opposed to the more common disulfide-containing spider neurotoxins.
Venoms from three poneromorph ant species (Paraponera clavata, Ectatomma quadridens and Ectatomma tuberculatum) were investigated for the growth inhibition of Gram-positive and Gram-negative ...bacteria. It was shown that the venom of E. quadridens and its peptide fraction in particular possess marked antibacterial action. Three linear antimicrobial peptides sharing low similarity to the well-known ponericin peptides were isolated from this ant venom by means of size-exclusion and reversed-phase chromatography. The peptides showed antimicrobial activity at low micromolar concentrations. Their primary structure was established by direct Edman sequencing in combination with mass spectrometry. The most active peptide designated ponericin-Q42 was chemically synthesized. Its secondary structure was investigated in aqueous and membrane-mimicking environment, and the peptide was shown to be partially helical already in water, which is unusual for short linear peptides. Analysis of its activity on different bacterial strains, human erythrocytes and chronic myelogenous leukemia K562 cells revealed that the peptide shows broad spectrum cytolytic activity at micromolar and submicromolar concentrations. Ponericin-Q42 also possesses weak toxic activity on flesh fly larvae with LD50 of ∼105 μg/g.
•Peptides from poneromorph ant venom possess high antimicrobial activity.•Ponericin-Q42 isolated from Ectatomma quadridens shows potent cytolytic effects.•Unlike most linear cationic peptides, it assumes a stable α-helix in water.
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
Modulation of P2X3 receptors by spider toxins Kabanova, Natalia V.; Vassilevski, Alexander A.; Rogachevskaja, Olga A. ...
Biochimica et biophysica acta,
November 2012, 2012-Nov, 2012-11-00, Letnik:
1818, Številka:
11
Journal Article
Recenzirano
Odprti dostop
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 IC50 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.
The spider toxin PT1 enhances the high affinity desensitization of the mouse P2X3 receptor. (A) Time evolution of mP2X3 currents in control (o), in the presence of 50 nM PT1 (●), and in the presence 40 nM CTP and 50 nM PT1 (▲). (B) mP2X3 current elicited by 100 μM CTP versus ambient CTP concentration in control (▲) and in the presence of 50 nM PT1 (Δ). Display omitted
► Peptides, purotoxin-1 and purotoxin-2, were isolated from the venom of the spider Geolycosa sp. ► The sensitivity of recombinant P2X3 receptors to the spider toxins was studied. ► Purotoxin-1 and purotoxin-2 slowed down inactivation of P2X3 currents. ► The spider toxins shifted an inhibitory curve for P2X3 high-affinity desensitization.
The scorpion toxin BeKm-1 is unique among a variety of known short scorpion toxins affecting potassium channels in its selective
action on ether-a-go-go-related gene (ERG)-type channels. BeKm-1 ...shares the common molecular scaffold with other short scorpion
toxins. The toxin spatial structure resolved by NMR consists of a short α-helix and a triple-stranded antiparallel β-sheet.
By toxin mutagenesis study we identified the residues that are important for the binding of BeKm-1 to the human ERG K + (HERG) channel. The most critical residues (Tyr-11, Lys-18, Arg-20, Lys-23) are located in the α-helix and following loop
whereas the âtraditionalâ functional site of other short scorpion toxins is formed by residues from the β-sheet. Thus the
unique location of the binding site of BeKm-1 provides its specificity toward the HERG channel.
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 IC50 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.
AF276623
The wild-type scorpion toxin BeKm-1, which selectively blocks human ether-a-go-go related (hERG) channels, was radiolabeled with iodine at tyrosine 11. Both the mono- and di-iodinated derivatives ...were found to be biologically active. In electrophysiological patch-clamp recordings mono-127I-BeKm-1 had a concentration of half-maximal inhibition (IC50 value) of 27 nM, while wild-type BeKm-1 inhibited hERG channels with an IC50 value of 7 nM. Mono-125I-BeKm-1 was found to bind in a concentration-dependent manner and with picomolar affinity to hERG channel protein in purified membrane vesicles from transfected human embryonic kidney cells (HEK-293). Under optimized conditions the equilibrium dissociation constant ( Kd) values from saturation and kinetic binding analysis were 13 and 14 pM, respectively. Both the association and dissociation of (125)I-BeKm-1 were fast (association rate constant, k(on)=3.6 x 10(7) M(-1)s(-1); dissociation rate constant, k(off)=0.005 s(-1)). Wild-type BeKm-1 displaced binding of 125I-BeKm-1 with half-maximal inhibitory concentrations of 44 pM. In contrast, competition experiments with a BeKm-1 mutant BeKm-1-K18A, in which the toxin interaction site is disrupted, resulted in a drop in affinity by more than 300-fold as compared to the wild-type toxin. Iberiotoxin and apamin, peptide inhibitors of Ca2+-activated K+-channels, had no effect on 125I-BeKm-1 binding. Adding the classical rapid delayed rectifier current (IKr) blocker E-4031 reduced binding of 125I-BeKm-1 to the hERG channel to an IC50 of 7 nM. In autoradiographic studies on rat hearts, binding of 125I-BeKm-1 was dose-dependent and could partially be displaced by the addition of excess amounts of non-radioactive BeKm-1. The density of the radioactive signal was equally distributed in the myocardium of both the ventricle and atria indicating a homogenous expression of hERG channels throughout the heart.
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 °C) in aqueous solution. 3 J N α, 3 J αβ 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 Å for the heavy atoms and 0.42 Å for the backbone heavy atoms. The structures were refined by constrained energy minimization using the SYBYL program. Their analysis indicated the existence of the α-helix (residues 10−21) slightly distorted at the Cys14 residue, two main strands of the antiparallel β-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.
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 110 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
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