Nucleotide sequence and structure analysis of a cDNA encoding an alpha insect toxin from the scorpion Leiurus quinquestriatus hebraeus.

A approximately 370 base pair cDNA encoding the alpha insect toxin Lqh alpha IT of the scorpion Leiurus quinquestriatus hebraeus was cloned and sequenced. The deduced amino acid sequence for the putative mature polypeptide is identical to the protein sequence determined chemically (Eitan et al., Biochemistry 29, 5941, 1990). A 19 amino acid signal peptide precedes the 64 amino acid long toxin. Two additional amino acid residues that do not correspond to the purified toxin are found at the COOH-terminus and may imply post-translational modification. The signal peptide region in the present clone differs obviously from that encoding the depressant insect toxin LqhIT2 derived from the same venom, but strongly resembles the leader peptide sequence of an alpha-mammal toxin from the scorpion Androctonus australis.     

Three polymorphic genes encoding a depressant toxin from the Egyptian scorpion Leiurus quinquestriatus quinquestriatus.

Four clones encoding the insect depressant toxin LqqIT2 have been isolated from the Egyptian scorpion Leiurus quinquestriatus quinquestriatus using RT-PCR. The four clones have been sequenced and their deduced amino acid sequences have been compared with the original amino acid sequence determined from the purified LqqIT2 protein and polymorphisms have been shown. This study succeeded in isolating more than one copy of the LqqIT2 gene, although only one amino acid sequence has been identified from the purified LqqIT2 toxin.     

The cDNA sequence of an excitatory insect selective neurotoxin from the scorpion Buthus martensi Karsch.

The full-length cDNA of an excitatory insect selective neurotoxin was amplified from total cDNAs of venomous glands of the scorpion Buthus martensi Karsch (BmK) using the 3'RACE and 5'RACE (rapid amplification of cDNA ends, RACE) method and sequenced. The cDNA encoded a precursor of the insect toxin of 88 amino acid residues, including a signal peptide of 18 residues and a mature toxin of 70 residues. The cDNA deduced sequence of this toxin was homologous with the determined amino acid sequence of BmK IT1, an excitatory insect toxin purified from the scorpion venom, except for three different residues, two at the positions 24-25, and another in the COOH-terminus of the toxin. Among them the COO-terminal residue Gly in the cDNA deduced sequence was predominantly different from the conserved residue Asn found in other known scorpion excitatory insect toxins.     

Purification and cDNA cloning of an insecticidal protein from the venom of the scorpion Orthochirus scrobiculosus.

Injection of crude venom from the scorpion Orthochirus scrobiculosus into larvae of Heliothis virescens (Lepidoptera: Noctuidae) caused trembling and uncoordinated movement before development of a progressive and prolonged flaccid paralysis. The isolation of the toxin (OsI-1) responsible for this effect of O. scrobiclosus venom is described. The molecular mass of OsI-1 toxin was 6994 Da, as determined by desorption mass spectroscopy. The complete primary structure of OsI-1 was deduced from the sequence of cDNA clones obtained by rapid amplification of cDNA ends (RACE) PCR. Comparison of the deduced amino acid sequence of OsI-1 with those of other insecticidal scorpion toxins indicates that it is a sodium (Na+) channel active depressant insect-selective toxin. The analysis of amino acid sequence of the toxin in conjunction with mass spectroscopy data indicates post-translational modification in maturation with the removal of 3 C-terminal amino acids and amidation of the C-terminus.     

Identification of insect-selective and mammal-selective toxins from Parabuthus leiosoma venom.

Venoms were collected from two scorpion species: Parabuthus leiosoma and Parabuthus pallidus from Kenya. Subcutaneous injection and oral toxicity tests of crude and pure fractions of scorpion venoms were done in Mus musculus (mice), Chilo partellus and Busseola fusca. The highest activity against C. partellus was found in P. leiosoma venom (LC(50) 0.689 mg/50mg body weight). Bioassay-guided purification by a combination of cation-exchange (CE) and reverse-phase high-performance liquid chromatography (RP-HPLC) led to the isolation of three toxic peptides. A lepidopteran-selective toxin (P. leiosoma insect toxin, Plit) was isolated, and the partial N-terminal amino acid sequence (-KDGYPVDNANCKYE-) plus the molecular weight (6688.5 Da) determined. A peptide with significant insect toxicity coupled with mild effects on mice (P. leiosoma toxin, Plt) was isolated, and the partial N-terminal amino acid sequence (-LCEKFKVQRLVELNCVD-) plus the molecular weight (6742.5 Da) was determined. Another toxin with anti-mammalian activity (P. leisoma mammal-selective toxin, Plmt), and N-terminal partial amino acid sequence of ADVPGNYPLDKNGNRYY- plus a molecular weight of 7145.5 Da was also isolated. Comparison of the partial N-terminal amino acid sequences with other toxins revealed that Plit shows high homology to other known insect toxins. Similarly, Plmt shows high homology with several birtoxin-like anti-mammalian toxins. Plt does not exhibit homology with any known scorpion toxin with combined anti-insect and anti-mammalian activity.     

长链蝎神经毒素在小鼠体内的组织分布(英文)

用 Iodogen方法对马氏钳蝎的两种神经毒素Bm K 和 Bm K IT2进行了 12 5I放射性标记 .分别将标记产物通过腹腔或静脉注射到小鼠体内 .结果发现肾脏的放射性含量最高 ,血液 ,肝脏 ,脾 ,肺 ,膈肌 ,心脏和大腿肌肉等组织器官中或多或少的含有放射性 ,而在脑中几乎检测不到放射性。结果提示长链蝎神经毒素主要通过肾脏排泄 ,且难以穿过血脑屏障 .     

Cloning and characterization of the cDNA sequences of two venom peptides from Chinese scorpion Buthus martensii Karsch (BmK).

From a cDNA library made from venom glands of Chinese scorpions of Buthus martensii Karsch, full-length cDNAs encoding precursors of two venom peptides have been isolated using a cDNA probe synthesized by polymerase chain reaction. Sequence analysis of the cDNAs revealed that one encoded precursor was 85 amino acid residues long including a signal peptide of 19 residues and a mature peptide (named BmK T) of 66 residues, and another encoded precursor was 84 residues long containing the same length signal peptide and a mature peptide (BmK M4 isoform, named BmK M4') of 64 residues. The analysis of amino acid sequence similarity indicated that the BmK T was homologous with both mammalian and insect toxins from BmK scorpion or other scorpions, and the BmK M4' was highly homologous with the members of the mammalian neurotoxin family of BmK, having two point mutations in amino acid residue sequence compared to BmK M4, a natural toxin from BmK.     

Binding characteristics of BmK I,an α‐like scorpion neurotoxic polypeptide,on cockroach nerve cord synaptosomes

Abstract: In this study, the binding characteristics of BmK I, an α‐like neurotoxic polypeptide purified from the venom of the Chinese scorpion Buthus martensi Karsch, were investigated on rat brain and cockroach nerve cord synaptosomes. The results showed that BmK I can bind to a single class of noninteracting binding sites on cockroach nerve cord synaptosomes with medium affinity (K_d = 16.5 ± 4.4 nm ) and low binding capacity (B_max= 1.05 ± 0.23 pmol/mg protein), but lacks specific binding on rat brain synaptosomes. BmK AS, BmK AS‐1 (two novel sodium channel‐blocking ligands), BmK IT (an excitatory insect‐selective toxin) and BmK IT2 (a depressant insect‐selective toxin) from the same venom were found to be capable of depressing BmK I binding in cockroach nerve cord synaptosomes, which might be attributed to either allosteric modulation of voltage‐gated Na⁺ channels by these toxic polypeptides or partial overlapping between the receptor binding sites of BmK I and these toxins. This thus supported the notion that α‐like scorpion neurotoxic polypeptides bind to a distinct receptor site on sodium channels, which might be similar to the binding receptor site of α‐type insect toxins, and also related to those of BmK AS type and insect‐selective scorpion toxins on insect sodium channels.     

Mammalian skeletal muscle voltage-gated sodium channels are affected by scorpion depressant "insect-selective" toxins when preconditioned

Among scorpion beta- and alpha-toxins that modify the activation and inactivation of voltage-gated sodium channels (Na(v)s), depressant beta-toxins have traditionally been classified as anti-insect selective on the basis of toxicity assays and lack of binding and effect on mammalian Na(v)s. Here we show that the depressant beta-toxins LqhIT2 and Lqh-dprIT3 from Leiurus quinquestriatus hebraeus (Lqh) bind with nanomolar affinity to receptor site 4 on rat skeletal muscle Na(v)s, but their effect on the gating properties can be viewed only after channel preconditioning, such as that rendered by a long depolarizing prepulse. This observation explains the lack of toxicity when depressant toxins are injected in mice. However, when the muscle channel rNa(v)1.4, expressed in Xenopus laevis oocytes, was modulated by the site 3 alpha-toxin LqhalphaIT, LqhIT2 was capable of inducing a negative shift in the voltage-dependence of activation after a short prepulse, as was shown for other beta-toxins. These unprecedented results suggest that depressant toxins may have a toxic impact on mammals in the context of the complete scorpion venom. To assess whether LqhIT2 and Lqh-dprIT3 interact with the insect and rat muscle channels in a similar manner, we examined the role of Glu24, a conserved "hot spot" at the bioactive surface of beta-toxins. Whereas substitutions E24A/N abolished the activity of both LqhIT2 and Lqh-dprIT3 at insect Na(v)s, they increased the affinity of the toxins for rat skeletal muscle channels. This result implies that depressant toxins interact differently with the two channel types and that substitution of Glu24 is essential for converting toxin selectivity.     

Positively selected sites of scorpion depressant toxins: possible roles in toxin functional divergence.

Scorpion depressant toxins represent a distinct pharmacological group of sodium channel neurotoxins, identified by their preferential ability in induction of depressant and flaccid paralysis of insects. However, recent observations that some members in this group exhibit anti-mammal activity raise an interesting evolutionary question of whether it is a consequence of adaptive evolution to the early radiation of mammals on earth. By employing the maximum likelihood method, we provided convincing statistical evidence in favor of positive selection driving the evolution of the depressant toxins, and found that two of three positively selected sites are located on the functional surface of the toxins. A complex model of the scorpion depressant toxin LqhIT2 binding to insect sodium channel alpha-subunit (DmNav1) was constructed by structural bioinformatics approaches which highlights a possible direct interaction between these two sites and insect sodium channels. Our work presented here thus suggests that accelerated substitutions in these site residues could offer an evolutionary advantage for these toxins to adapt different channels from diverse origins.     

Genomic characterisation of the toxin Amm VIII from the scorpion Androctonus mauretanicus mauretanicus.

The genomic DNA sequence encoding the scorpion toxin Amm VIII was amplified from genomic DNA of the scorpion Androctonus mauretanicus mauretanicus from Morocco, subcloned and sequenced. An intron, with a high A+T content (73.5%), split a Gly codon at the end of the precursor signal peptide and the consensus GT/AG splice junction was identified in the Amm VIII gene. This intron of only 166 bp is the smallest intron described so far for a long-chain scorpion toxin gene. In addition, this study led to the identification of three new toxin-related genes. From the deduced amino acid sequences of the encoded precursor proteins, we found that the mature putative toxins were highly similar to the scorpion toxins Leiurus quinquestriatus quinquestriatus IV and Odonthobuthus doriae 1.     

Molecular characterization of a new excitatory insect neurotoxin with an analgesic effect on mice from the scorpion Buthus martensi Karsch.

Besides the neurotoxins active on mammals, a new excitatory insect selective toxin with a mice analgesic activity was found and purified from the venom of the scorpion Buthus martensi Karsch (BmK) (Ji, Y.H., Mansuelle, P., Terakawa, S., Kopeyan, C., Yanaihara, N., Hsu, K., Rochat, H., 1996. Toxicon 34, 987; Luo, M.J., Xiong, Y.M., Wang, M., Wang, D.C., Chi, C.W., 1997. Toxicon 35, 723.). This peptide (designated as BmK IT-AP) is composed of 72 amino acid residues. Its primary structure was determined by automated Edman degradation of the N-terminal part of the reduced and S-carboxamidemethylated protein and its lysylendopeptidase degraded fragments. Based on the determined sequence, the gene specific primers were designed and synthesized for 3' and 5' RACE (rapid amplification of cDNA ends). Their partial cDNA fragments obtained by 3' and 5' RACEwere cloned and sequenced and the full length cDNA sequence of BmK IT-AP was then completed by overlapping their two partial cDNA sequences. It encodes a precursor of 90 amino acid residues: a signal peptide of 18 residues and a mature peptide of 72 residues which are consistent with the determined protein sequence of BmK IT-AP. The genomic DNA of the peptide was also amplified by PCR from the scorpion genomic DNA and sequenced, which is a first report on the genomic structure of a scorpion toxin specific for insects. Its sequence revealed an intron of 590 bp inserted in the end part of the signal peptide. The peptide caused a fast excitatory contraction paralysis on house fly larvae. Furthermore, the peptide also showed an obvious analgesic effect on mice, as assayed by using a twisting test model. This effect of BmK IT-AP well characterized at molecular level is first reported among the known scorpion insect neurotoxins.     

Characterisation of the gene encoding the alpha-toxin Amm V from the scorpion Androctonus mauretanicus mauretanicus.

The full-length cDNA encoding the scorpion alpha-toxin Amm V was amplified from a cDNA library produced from the venom glands of the scorpion Androctonus mauretanicus mauretanicus from Morocco. We deduced the amino acid sequence of the encoded precursor protein and found that the mature toxin was similar to the previously characterised toxin. The genomic DNA sequence encoding the toxin was also amplified, subcloned and sequenced. This also led to the isolation of a new Amm V related-gene. Then, for the first time, we studied changes in the level of toxin mRNA synthesis over time.     

Covalent structure of the insect toxin of the North African scorpion Androctonus australis Hector

The complete covalent structure of the insect toxin purified from the venom of the North-African scorpion Androctonus australis Hector was described. Its amino acid sequence was established by phenylisothiocyanate degradation of several protein derivatives and proteolytic fragments in a liquid protein sequencer using either a “protein” or a “peptide” program. The position of the four disulfide bridges were deduced by analysis of proteolytic peptides before and after performic oxidation, and by partial labeling of the half cystine residues with [¹⁴C]-iodoacetic acid and determining the specific radioactivities of the S-[¹⁴C]-carboxymethylated phenylthiohydantoin cysteines. The sequences of the insect and mammal toxins from scorpions can be aligned with homology with the positions of seven half-cystine residues as registers. The mammal and insect toxins have three disulfide bridges at homologous positions. The fourth bridge is different in that Cys¹² in mammal toxin II is replaced by Cys³⁸ in the insect toxin. It is likely that the position of the disulfide bridges is the same for all scorpion neurotoxins active on mammals. We believe that the shift of one half-cystine residue in the insect toxin may induce a conformational change in the structure of the protein, which, in turn, may partially account for the total specificity of this toxin for insect nervous system.     

The binding of BmK abT,a unique neurotoxin,to mammal brain and insect Na(+) channels using biosensor

The binding properties of BmK abT (a novel neurotoxic polypeptide abT from Chinese scorpion Buthus martensi Karsch), a unique neurotoxin from Chinese scorpion, on mammal brain and insect sodium channels were investigated using the BIAcore assay. Results showed that BmK abT could bind to rat brain synaptosomes with an association rate constant of about 2.49 x 10(6) M(-1) s(-1) and a dissociation rate constant of about 1.57 x 10(-4) s(-1), and to Heliothis nerve cord synaptosomes with an association rate constant of about 1.21 x 10(7) M(-1) s(-1) and a dissociation rate constant of about 0.99 x 10(-3) s(-1). The binding of BmK abT to rat brain synaptosomes could be partially inhibited by increasing the membrane potential, but not by BmK AS (a novel active polypeptide AS from B. martensi Karsch), BmK IT2 (a depressant insect-selective toxin IT2 from B. martensi Karsch), and BmK I (an alpha-like anti-mammal toxin I from B. martensi Karsch). Binding was not modulated by veratridine. In addition, the binding of BmK abT to Heliothis nerve cord synaptosomes was significantly enhanced by increasing the membrane potential and veratridine concentration and was inhibited by BmK IT2, but not by BmK AS or BmK I. The results suggest that BmK abT binds to a distinct receptor site on mammal brain Na(+) channels and associates with a related site for depressant insect-selective toxins on insect sodium channels.     

Purification and characterization of a novel short-chain insecticidal toxin with two disulfide bridges from the venom of the scorpion Liocheles australasiae.

Scorpion venoms contain a variety of peptides toxic to mammals, insects and crustaceans. Most of the scorpion toxins have been isolated from the venoms of scorpions in the family Buthidae, but little interest has been paid to non-Buthidae scorpions. In this study, we isolated a short-chain insecticidal toxin (LaIT1) from the venom of the scorpion Liocheles australasiae belonging to the Hemiscorpiidae family. This toxin showed insect toxicity against crickets at a dose of 1.0 microg/insect, but no toxicity was observed against mice even after injection of 1.0 microg of LaIT1 via the intracerebroventricular route, suggesting that the effect of the toxin is insect-selective. Edman sequencing and mass spectrometric analysis revealed that the toxin is composed of 36 amino acid residues and cross-linked by only two disulfide bridges. The pattern of the disulfide bridges was assigned by LC/MS analysis after enzymatic digestion. LaIT1 shows no sequence homology to any other known toxins, suggesting that this toxin represents a novel structural motif class.     

Two recombinant depressant scorpion neurotoxins differentially affecting mammalian sodium channels

The scorpion depressant toxins are a group of evolutionarily conserved polypeptides targeting sodium channels, which show preferential ability to induce flaccid paralysis in insects, making them attractive candidates for the construction of transgenic plants or viral vectors to control pests. In this study, two new depressant toxin-like peptides (BmKITc and BmKITc2) differing only at position 52 (Lys for Thr) were produced in Escherichia coli. Circular dichroism analysis indicated that these two recombinant peptides display a typical structural feature similar to native scorpion toxins. They both cause a maintained current component at the last phase of inactivation of the insect sodium channel DmNav1/tipE expressed in Xenopus oocytes and interestingly, they do not produce a beta effect despite of their primary structure as beta-toxins. Furthermore, an inhibitory effect with BmKITc but not with BmKITc2 was observed on TTX-R sodium currents in rat DRG neurons. We hypothesize that such differential potency highlights a crucial role of lysine 52 in channel selectivity. Our results therefore indicate that, in spite of the general idea, not all scorpion depressant toxins interact with mammalian and/or insect sodium channels in the same manner.     

BotIT6: a potent depressant insect toxin from Buthus occitanus tunetanus venom.

A new depressant insect toxin Buthus occitanus tunetanus insect-toxin 6 (BotIT6) was purified by high-performance liquid chromatography from Buthus occitanus tunetanus (Bot) venom. BotIT6 is very active against Blatella germanica (LD50=10ng/100mg body mass) thus being one of the most potent anti-insect toxin so far characterised. When compared to other insect toxin sequences, BotIT6 present high similarities with depressant insect toxins with an additional arginine residue at the C-terminus and a methionine at position 27. The calculated net charge of BotIT6 is positive (+3) whereas it is negative for classical depressant toxins: this might be associated with its high toxicity. Voltage current clump studies show that BotIT6 is not a very potent depressant insect toxin despite its high toxicity in vivo. BotIT6 is able to fully inhibit the specific binding of 125I AaHIT and 125I-BotIT2 on Periplaneta americana synaptosomal membrane vesicles with high affinities. Despite its higher toxicity BotIT6 is a weaker competitor with 125I AaHIT and 125I BotIT2 as compared to the other beta toxins.Altogether, these results may suggest that BotIT6 probably defines a novel sub-group of depressant anti-insect toxins for which the receptor site can be overlapping, but not identical to that for classical depressant insect toxins.     

The gene cloning and sequencing of Bm-12, a chlorotoxin-like peptide from the scorpion Buthus martensi Karsch.

According to the known amino acid sequence of Bm-12, a short chain insect neurotoxin from the venom of the scorpion Buthus martensi Karsch (BmK) with considerable primary sequence homology to chlorotoxin, the gene specific primers were designed and synthesized for 3' and 5'RACE (Rapid Amplification of cDNA Ends). The two partial cDNA fragments obtained by 3' and 5'RACE were cloned and sequenced, and the full length cDNA sequence of Bm-12 was then completed by overlapping these two partial cDNA sequences. The predicted amino acid sequence consists of 59 amino acid residues including a putative signal peptide of 24 residues and a mature toxin of 35 residues. The predicted amino acid sequence of Bm-12 was almost consistent with the determined, different only in one residue at position 27, Lys was replaced by Gly. Based on the determined cDNA sequence, and using the total DNA isolated from the scorpion venom glands as a template, the genomic DNA of Bm-12 was also amplified by PCR and sequenced. The genomic DNA sequence revealed an intron of 93 bp present within the signal peptide region.