Depressant Toxins Research Articles

Prokaryotically expressed Buthus martensii Karsch insect depressant toxin has insecticidal effects

An insect depressant toxin Buthus martensii Karsch insect toxin 4 (BmK IT 4) cDNA was cloned into the prokaryotic expression vector pSW202 and expressed in HB101 host cells. The authenticity of this in vitro expressed peptide was confirmed by Western blotting, mass spectrometry and N-terminal peptide sequencing. Bioassays using growth media supplemented with BmK IT 4 demonstrated that, at the end of the 5-day experimental period, about 77% of the testing cotton bollworm larvae were killed. Furthermore, the average weight picked up by larvae grown on BmK IT 4 containing media amounts only to 0.7% of that of control group. Our results indicate that BmK IT 4 may be used for biological control of insect damages in place of other traditional insecticides.

Scorpion neurotoxins: structure/function relationships and application in agriculture

Continued use of non-specific chemical insecticides poses potential risks to the environment and to human health resulting from non-target toxicity and increased insect resistance to these agents. Scorpions produce anti-insect selective polypeptide toxins that bind to and modulate voltage-sensitive ion channels in excitable tissues, thus offering alternative, environmentally safer means for insect pest control. Despite this potential, little is known about their structural elements dictating anti-insect preference, which may be useful for the design of selective insecticides. We used a bacterial system for expression and genetic dissection of two pharmacologically distinct scorpion toxins: alpha and excitatory. By exploiting a multi-disciplinary approach consisting of mutagenesis, protein chemistry, electrophysiology, binding and toxicity assays, and structural studies, we elucidated the bioactive surface of two anti-insect toxins, LqhαIT and Bj-xtrIT. In both polypeptides the bioactive surface is composed of residues surrounding the C-terminal region. In addition, a direct, immediate approach in using the toxin genes was demonstrated by engineering baculoviruses with cDNAs encoding LqhIT2 (depressant toxin), and LqhIT1 (excitatory toxin) resulting in viral vectors with significantly improved insecticidal efficacy. © 2000 Society of Chemical Industry

An excitatory scorpion toxin with a distinctive feature: an additional alpha helix at the C terminus and its implications for interaction with insect sodium channels.

Scorpion neurotoxins, which bind and modulate sodium channels, have been divided into two groups, the alpha and beta toxins, according to their activities. The beta-toxin class includes the groups of excitatory and depressant toxins, which differ in their mode of action and are highly specific against insects. The three-dimensional structures of several alpha and beta toxins have been determined at high resolution, but no detailed 3D structure of an excitatory toxin has been presented so far. The crystal structure of an anti-insect excitatory toxin from the scorpion Buthotus judaicus, Bj-xtrIT, has been determined at 2.1 A resolution and refined to an R factor of 0.209. The first 59 residues form a closely packed module, structurally similar to the conserved alpha and beta toxins ('long toxins') affecting sodium channels. The last 17 residues form a C-terminal extension not previously seen in scorpion toxins. It comprises a short alpha helix anchored to the N-terminal module by a disulfide bridge and is followed by a highly mobile stretch of seven residues, of which only four are seen in the electron-density map. This mobile peptide covers part of a conserved hydrophobic surface that is thought to be essential for interaction with the channel in several long toxins. Replacement of the last seven residues by a single glycine abolishes the activity of Bj-xtrIT, strongly suggesting that these residues are intimately involved in the interaction with the channel. Taken together with the partial shielding of the conserved hydrophobic surface and the proximity of the C terminus to an adjacent surface rich in charged residues, it seems likely that the bioactive surface of Bj-xtrIT is formed by residues surrounding the C terminus. The 3D structure and a recently developed expression system for Bj-xtrIT pave the way for identifying the structural determinants involved in the bioactivity and anti-insect specificity of excitatory toxins.

A depressant insect-selective toxin analog from the venom of the scorpion Leiurus quinquestriatus hebraeus--purification and structure/function characterization.

The scorpion venom-derived excitatory and depressant insect-selective polypeptide neurotoxins modify sodium conductance in insect neuronal membranes and differ greatly in their primary structures and symptoms induced in blow fly larvae. We report here the purification and characterization of a new insect selective toxin, LqhIT5. LqhIT5 is more similar to the excitatory toxins in its mode of action and the depressant toxins in its primary structure. This toxin is a single polypeptide composed of 61 amino acids that are cross linked by four disulfide bonds. When LqhIT5 is injected into blow fly larvae, a fast contraction paralysis occurs without depressant activity. No mammalian toxicity was detected by subcutaneous or intracranial injections of this toxin into mice. Sequence comparison of LqhIT5 and known depressant toxins shows a high degree of similarity among the amino acids located on the C-terminus of the toxins. However, there are some clear differences in the amino acids located close to the N-terminus of the toxins. By the aid of homology modeling, we demonstrated that these amino acids have the same orientation in the tertiary structure of the molecule and are exposed to the environment. The change in the mode of action of LqhIT5 (no depressant activity) by substitutions of a few amino acids located on a specific exposed area of the toxin shed a new light on the structure/function relationship of scorpion toxins. These results caution that similarity in the mechanism of action of scorpion toxins does not always follow from an overall similarity in sequence.

Baculovirus-mediated expression of a scorpion depressant toxin improves the insecticidal efficacy achieved with excitatory toxins

The insecticidal efficacy towards Helicoverpa armigera lepidopteran larvae of recombinant Autographa californica M nucleopolyhedroviruses, expressing depressant and excitatory scorpion anti-insect selective toxins, was investigated. The ET 50 (effective paralysis time 50%) values obtained with the recombinant viruses expressing the depressant toxin, LqhIT2, and the excitatory toxin, LqhIT1, were 59 h and 66 h, respectively, whereas the ET 50 value of the wild-type virus was longer, 87 h post infection. The insecticidal effects obtained when using two distinct temporally regulated viral promoters revealed advantage for the very late p10 promoter over the p35 early promoter. The higher insecticidity of the virus expressing the depressant toxin compared to the excitatory toxin suggests that pharmacokinetic factors and/or promoter efficiency may play a role during infection of insect pest larvae by recombinant baculoviruses.

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis.

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 micrograms against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins.

Rapid purification and molecular modeling of AaIT peptides from venom of Androctonus australis

As recombinant viruses expressing scorpion toxins are moving closer toward the market, it is important to obtain large amounts of pure toxin for biochemical characterization and the evaluation of biological activity in nontarget organisms. In the past, we purified a large amount of Androctonus australis anti-insect toxin (AaIT) present in the venom of A. australis with an analytical reversed-phase column by repeated runs of crude sample. We now report 20 times improved efficiency and speed of the purification by employing a preparative reversed-phase column. In just two consecutive HPLC steps, almost 1 mg of AaIT was obtained from 70 mg crude venom. Furthermore, additional AaIT was obtained from side fractions in a second HPLC run. Recently discovered insect selective toxin, AaIT5, was isolated simultaneously from the same venom batch. It shows different biological toxicity symptoms than the known excitatory and depressant insect toxins. AaIT5 gave 100% mortality with a dose of less than 1.3 μg against fourth-instar tobacco budworms Heliothis virescens 24 h after injection. During the purification process, we implemented mass spectrometry in addition to bioassays to monitor the presence of AaIT and AaIT5 in the HPLC fractions. Mass spectrometric screening can unambiguously follow the purification process and can greatly facilitate and expedite the downstream purification of AaIT and AaIT5 eliminating the number of bioassays required. Further, electrospray ionization was compared with matrix-assisted desorption/ionization and evaluated as a method of choice for mass spectrometric characterization of fractions from the venom purification for it provided higher mass accuracy and relative quantitation capability. Molecular models were built for AaIT5, excitatory toxin AaIT4, and depressant toxin LqhIT2. Three-dimensional structure of AaIT5 was compared with structures of the other two toxins, suggesting that AaIT5 is similar to depressant toxins. Arch. Insect Biochem. Physiol. 38:53–65, 1998. © 1998 Wiley-Liss, Inc.

Functional Anatomy of Scorpion Toxins Affecting Sodium Channels

AbstractLong chain scorpion toxins (made of 60 to 70 amino acids) acting on voltage-gated sodium channels in excitable cells are responsible for human envenomation, and comprise a-toxins that inhibit sodium current inactivation and p-toxins, that modify the activation process. These toxins may be further divided according to their pharmacological activities. Thus, a-toxins highly active on mammals or insects, as well as α like toxins may be distinguished within the α-toxin class. The β-toxin class includes toxins active on mammals and, as a separate group, the excitatory and depressant toxins active exclusively on insects. All these toxins possess 4 disulfide bridges and share 15 similar non cystine residues. Accordingly, their 3D structure is highly conserved, comprising an α-helix and a triple stranded β-sheet. The most solvent exposed turns of this structure are prone to insertions or deletions, and accordingly correspond to the most structurally variable regions of the toxins. They have been tested by...

Refined electrophysiological analysis suggests that a depressant toxin is a sodium channel opener rather than a blocker

The effects of a recombinant depressant insect toxin from Leiurus quinquestriatus hebraeus, Lqh IT2-r, have been studied in current and voltageclamp conditions on the isolated axonal and DUM neuron preparations of the cockroach Periplaneta americana. Lqh IT2-r depolarizes the axon, blocks the evoked action potentials, and modifies the amplitude and the kinetics of the sodium current. The inward transient peak current is greatly decreased and is followed by a maintained slow activating-deactivating sodium current. The slow component develops at membrane potentials more negative than the control, and has a time constant of activation of several tens of milliseconds. The flaccid properties of Lqh IT2-r do not correspond to a blockage of the Na + channels, but may be attributed to modified Na + channels which open at more negative potential, activate slowly and do not inactivate normally.

Purification, structure and activity of three insect toxins from Buthus occitanus tunetanus venom

One contractive and two depressant toxins active on insect were purified by high-performance liquid chromatography from the venom of Buthus occitanus tunetanus (Bot). The two depressant toxins, BotIT4 and BotIT5, differ only at position 6 (Arg for Lys) and are equally toxic to insects ( ld 50 to Blatella germanica = 110ng/100mg body weight). They show a strong antigenic cross-reaction with a depressive toxin from Leiurus quinquestriatus quinquestriatus (LqqIT2). The two toxins are able to inhibit with high affinity ( K 0.5 between 2 and 3 nM) the specific binding of the radioiodinated excitatory insect toxin ( 125I-AaHIT) on its receptor site on Periplaneta americana synaptosomal membranes. These toxins depolarize the cockroach axon, irreversibly block the action potential, and slow down and very progressively block the transmembrane transient Na + current. The contracturant toxin BotIT1 is highly toxic to B. germanica ( ld 50 = 60 ng/ 100 mg body weight) and barely toxic to mice ( ld 50 = 1 μg/20 g body weight) when injected intracerebroventricularly. It does not compete with 125I-AaHIT for its receptor site on P. americana synaptosomal membranes. On cockroach axon, BotIT1 develops plateau potentials and slows down the inactivation mechanism of the Na + channels. Thus, BotIT1 belongs to the group of a insect-selective toxins and shows a strong sequence identity (> 90%) with LqhαIT and LqqIII, two insect α-toxins previously purified from the venom of L. q. hebraeus and L. q. quinquestriatus, respectively.

Biochemical and pharmacological characterization of a depressant insect toxin from the venom of the scorpion Buthacus arenicola.

A depressant toxin active on insects, Buthacus arenicola IT2, was isolated from the venom of the North African scorpion B. arenicola and its structural and pharmacological properties were investigated. B. arenicola IT2 is a single polypeptide of 61 amino acid residues, including 8 half-cystines but no methionine and histidine, with a molecular mass of 6835 Da. Its amino acid sequence is 79-95% identical to other depressant toxins from scorpions. When injected into the cockroach Blatella germanica, B. arenicola IT2 induced a slow depressant flaccid paralysis with a LD50 of 175 ng. B. arenicola IT2 has two non-interacting binding sites in cockroach neuronal membranes: one of high affinity (Kd1 = 0.11 +/- 0.04 nM) and low capacity (Bmax1 = 2.2 +/- 0.6 pmol/mg), and one of low affinity (Kd2 = 24 +/- 7 nM) and high capacity (Bmax2 = 226 +/- 92 pmol/mg). Its binding to these two sites was completely inhibited by Leiurus quinquestriatus quinquestriatus IT2, a depressant toxin from L. quinquestriatus quinquestriatus. Reciprocal-binding experiments between B. arenicola IT2 and the excitatory insect-toxin A. australis Hector IT revealed competition between the two toxins for the high-affinity sites of B. arenicola IT2. B. arenicola IT2 has a higher affinity than L. quinquestriatus hebraeus IT2, a depressant toxin from L. quinquestriatus hebraeus. Thus, B. arenicola IT2 represents an interesting tool to study the receptor site for depressant toxins on insect sodium channels.

A new scorpion venom toxin paralytic to insects that affects Na+ channel activation. Purification, structure, antigenicity and mode of action.

A new toxin, BotIT2, with a unique mode of action on the isolated giant axon of the cockroach Periplaneta americana and DUM (dorsal unpaired median) neurons, has been purified from the venom of the scorpion Buthus occitanus tunetanus. Its structural, antigenic and pharmacological properties are compared to those of three other groups of neurotoxins found in Buthidae scorpion venoms. Like excitatory, depressant and alpha-type insect-selective neurotoxins, BotIT2 is toxic to insects, but shows the following common and distinctive characteristics. (a) As alpha-type toxins, BotIT2 lack strict selectivity to insects; they have measurable but low toxicity to mice. (b) As depressant toxins and unlike alpha-type toxins, BotIT2 is able to displace iodinated AaHIT from its binding sites in insect neuronal membranes. This indicates that the binding site for BotIT2 is identical, contiguous or in allosteric interaction with that of AaHIT and depressant toxins. (c) The BotIT2 amino acid sequence shows strong similarity to depressant toxins. However, unexpectedly, despite this high sequence similarity, BotIT2 shares moderate cross-antigenic reactivity with depressant toxins. (d) Voltage and current-clamp studies show that BotIT2 induces limited depolarization concomitantly with the development of depolarizing after potential, repetitive activity and later plateau potentials terminated by bursts. Under voltage-clamp conditions, BotIT2 specifically acts on Na+ channels by decreasing the peak Na+ current and by simultaneously inducing a new current with very slow activation/deactivation kinetics. The voltage dependence of this slow current is not significantly different from that of the control current. These observations indicate that BotIT2 chiefly modifies the kinetics of axonal and DUM neuronal membrane Na(+)-channel activation.

Variability among insect sodium channels revealed by selective neurotoxins

Binding assays with the radioiodinated depressant (LqhIT 2) and excitatory (AaIT) insect selective neurotoxins derived from scorpion venoms to neuronal membrane preparations derived from cockroach, fly and lepidopterous larvae coupled with the employment of sodium channel site-directed antibodies resulted in the following informatio. (1) The two toxins were shown to bind with high affinity to the insect neuronal preparations and revealed similar binding constants in each of the various preparations. (2) Reciprocal competitive displacement assays between the two toxins in the various neuronal preparations revealed symmetrical and asymmetrical binding interactions. The depressant toxin was able to fully displace the excitatory toxin in all preparations with, however, different affinities. The excitatory toxin fully displaced the depressant toxin from its high affinity binding sites in the locust and cockroach preparations; partially in the fly head preparation and no displacement was obtained in the lepidopterous larvae neuronal preparation. (3) The antibody mediated binding inhibition differentially affected the binding of each of the two toxins in both the cockroach and fly neuronal preparations. The above data suggest that sodium channels of various insects possess separate, high affinity binding sites for each of the two groups of insect selective toxins but, however, consist of pharmacologically distinguishable subtypes.

Depressant insect selective neurotoxins from scorpion venom: Chemistry, action, and gene cloning

The present study examines the similarity in the symptoms and binding properties between the depressant and excitatory insect-selective neurotoxins, derived from scorpion venom. A comparison of their primary structures and neuromuscular effects is presented. A new depressant toxin (LqhIT2) was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic its effects on the intact insect, i.e, a brief period of repetitive bursts of regular junction potentials (JPs) is followed by reduced amplitude JPs ending with a block of the neuromuscular transmission. "Loose" patch clamp recordings indicate that the repetitive activity has a presynaptic origin (the motor nerve) and resembles the effect of the excitatory toxin AaIT. The final synaptic block is supposed to be the end result of neuronal membrane depolarization. Such an effect is not caused by an excitatory toxin, which induces long "trains" of repetitive firing. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation indicating a high degree of sequence homology. This conservation differs from those of other groups of scorpion toxins. The opposing pharmacological effects of depressant toxins are discussed in light of the above neuromuscular effects and sequence analysis. A genetic approach in the study of the structure-function relationships of the depressant toxins was initiated by isolating cDNA clones encoding the LqhIT2 and BjIT2 toxins. Their sequence analysis revealed the precursor form of these toxins: A 21 amino acid residue signal peptide followed by a 61 amino acid region of the mature toxin, and three additional amino acids at the carboxy terminus.

Functional duality and structural uniqueness of the depressant insect-selective neurotoxins

Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.

Effects of scorpion venom on squid axon membranes.

Effects of scorpion venom on squid axon membranes.