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.     

Analysis by high-performance liquid chromatography of Androctonus mauretanicus mauretanicus (black scorpion) venom

The venom of the black scorpion, Androctonus mauretanicus mauretanicus, was obtained by means of manual stimulation and was analyzed using high-performance liquid chromatography. Starting from 20 mg of venom and using only two chromatographic steps, six toxins were purified to homogeneity. They have been characterized by their amino acid content and compared to those already isolated from a pool of venoms obtained using electric stimulation (Rosso and Rochat, Toxicon 23, 113-125, 1985). The toxins Amm I and Amm II were not found, suggesting either different levels of toxin expression or the existence of Androctonus mauretanicus mauretanicus subspecies. Using rat brain synaptosomes, it was demonstrated that the toxins Amm III, Amm IV and Amm V were alpha-toxins. The toxin Amm VI was neither alpha- or beta-toxin. Unexpectedly, the toxin Amm VII was found to be a beta-toxin, the first one identified in a north African scorpion venom. In addition, some toxins active on mammals exhibited different levels of specificity towards phylogenetically related groups of arthropods.     

New analysis of the toxic compounds from the Androctonus mauretanicus mauretanicus scorpion venom

Scorpion venoms are very complex mixtures of molecules, most of which are peptides displaying different kinds of biological activity. Indeed, these peptides specifically bind to a variety of pharmacological targets, in particular ionic channels located in prey tissues, resulting in neurotoxic effects. Toxins modulating Na+, K+, Ca2+ and Cl(-) currents have been described in scorpion venoms. In this work, we have used several specific antibodies raised against the most lethal scorpion toxins already described to screen the Moroccan scorpion Androctonus mauretanicus mauretanicus venom in order to characterize new compounds. This immunological screening was also implemented by toxicity tests in mice and with mass spectrometry study, providing new informations on the molecular composition of this venom. In fine, we were able to determine the molecular masses of 70-80 different compounds. According to the immunological data obtained, many toxins cross-react with three sera raised against the most lethal alpha-toxins found in North African scorpion venoms, but not at all with those raised against the main beta-toxins from South and North American venoms. Some of the previously described toxins from Androctonus mauretanicus mauretanicus venom could thus be detected by combining immunological tests, toxicity in mice and molecular masses. Among these toxins, one of them, which showed a mild cross-reaction with the serum raised against AaH I (a highly potent toxin from the venom of Androctonus australis), was identified as Amm III and fully sequenced.     

Characterization of ten proteins from the venom of the Moroccan scorpion Androctonus mauretanicus mauretanicus, six of which are toxic to the mouse

When the venom of the scorpion Androctonus mauretanicus mauretanicus was submitted to purification procedures, ten proteins were obtained; six were lethal to mice and four were devoid of toxicity in the biological tests used. The ten molecules were characterized by their amino acid composition, and among them toxin V and polypeptide P2 by their amino acid sequences. Peptide P2 (35 amino acid residues), a structural homologue of the so called Buthus epeus short 'insectotoxins' I1 and I5, was inactive on fly larvae and absent from the 'manual venom' obtained by manual handling and excitation of the scorpions.     

The cDNA sequence of a depressant insect selective neurotoxin from the scorpion Buthotus judaicus.

A 400 nucleotide cDNA clone encoding the depressant insect toxin of the scorpion Buthotus judaicus (BjIT2), was isolated. DNA sequence analysis suggests that the toxin is a processed product of a precursor composed of: (1) a 21 amino acid residue signal peptide; (2) a 61 amino acid region of the mature toxin; and (3) an additional Arg-Lys-Lys tail at the carboxy terminus prior to a termination codon. Comparison between the precursor polypeptides of BjIT2 and another depressant insect toxin derived from the scorpion Leiurus quinquestriatus hebraeus (LqhIT2) shows similarities in their hydropathic profiles.     

Molecular cloning and genomic organization of a K(+) channel toxin from the Chinese scorpion Buthus martensii Karsch.

A full-length cDNA encoding the precursor of a K(+) channel toxin (BmTX2) was first isolated from a venom-gland cDNA library of the Chinese scorpion Buthus martensii Karsch. The precursor is composed of a signal peptide of 21 residues and a mature toxin of 37 residues with three disulfide bridges. The genomic gene of BmTX2 was also cloned and sequenced. It consisted of two exons, disrupted by an intron of 81 bp inserted in the region encoding signal peptide.     

Intraspecific variability and pharmacokinetic characteristics of Androctonus mauretanicus mauretanicus scorpion venom.

We evaluated the degree of venom toxicity and protein content of several specimens of Androctonus mauretanicus mauretanicus. The quantity of protein of individual venom obtained after manual extraction from 31 different scorpions varied from a minimum of 0.15 mg to a maximum of 1.53 mg. We determined the venom toxicity, in mice, by estimating the number of LD(50)s of 20 scorpions chosen randomly among the 31 scorpions. It ranged from less than 40 LD(50)s to a maximum of 272 LD(50)s. The correlation between protein content and venom lethality is not systematic.We also determined the pharmacokinetics of the venom and its specific anti-venom in rabbits to compare their distribution and elimination properties. After a subcutaneous injection, high concentrations of venom were measured by ELISA in the vascular space rapidly after the injection (T(max) = 0.5 h). The terminal half-life was 2.8 h, close the one determined after intravenous injection (t(1/2beta) = 3.2 h). The total volume of distribution (Vd(ss) or Vd(beta)) was between 317 and 380 ml/kg. The total body clearance was 82 ml/kg/h. For scorpion anti-venom, the terminal half-life, after intravenous injection, was 20.25 h; the volume of distribution was 83 ml/kg and the total body clearance was 3 ml/kg/h. After intramuscular administration, T(max) was reached at 36 h.The results show that venom lethality varies from specimen to specimen and that pharmacokinetic parameters of venom and anti-venom are totally different. This must be taken under consideration in anti-venom production (anti-venom titre) as well as in therapeutic protocols (dose, injection route) to improve serotherapy.     

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.     

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.     

Batrachotoxin and alpha-scorpion toxin stabilize the open state of single voltage-gated sodium channels

The combined effects of batrachotoxin (BTX) and either scorpion (Leiurus quinquestriatus quinquestriatus) venom (LqqV) or alpha-scorpion toxin (alpha-LqqTX) purified from LqqV on single voltage-gated Na channels were studied in planar lipid bilayers. In the presence of BTX, LqqV caused the channels to remain open at membrane potentials at least 50 mV more hyperpolarized than with BTX alone. alpha-LqqTX mimicked the effect of LqqV, suggesting that this toxin is the active component of the venom. LqqV did not significantly alter single-channel conductance, voltage-dependent block by saxitoxin, or voltage-dependent block by Ca2+, indicating that the venom preferentially affects gating rather than ion permeation. The results indicate that a cooperative interaction between alpha-LqqTX and BTX strongly favors the open state of the Na channel by causing a large hyperpolarizing shift in the voltage dependence of activation. This effect on activation gating is not predicted from the individual effects of the 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.     

Gene cloning and sequencing of BmK AS and BmK AS-1, two novel neurotoxins from the scorpion Buthus martensi Karsch.

Based on the known amino acid sequences of BmK AS and BmK AS-1, 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' RACE were cloned and sequenced, and the full length cDNA sequences of BmK AS and BmK AS-1 were then completed by overlapping their two partial cDNA sequences, respectively. The predicted amino acid sequences both consist of 85 amino acid residues including a putative signal peptide of 19 residues and a mature toxin of 66 residues. They are different in 17 amino acid residues, among them 11 residues in the mature toxin. The predicted amino acid sequences of BmK AS and BmK AS-1 were almost consistent with those determined and revised (personal communication), only different in one and two residues at their COO-terminal parts, respectively. Based on the determined cDNA sequences, and using the total DNAs isolated from the scorpion venom glands as a template, the genomic DNAs of BmK AS and BmK AS-1 were also amplified by PCR and sequenced. It showed that no intron was inserted in their open reading frames, while in the exon of signal peptide sequences of other Na+, K+ and Cl- channel toxins from the same scorpion, an intron is usually found. However, the Northern blot hybridization results indicated that the sizes of their mRNA should be around 800 bp. Their extra sequences around 400 bp which might function as an intron should be located at their 5' untranslated regions.     

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.     

Characterization of a new family of toxin-like peptides from the venom of the scorpion Leiurus quinquestriatus hebraeus-NMR structure of leiuropeptide II

To extend our knowledge about the structural features of short scorpion toxins, the ion-exchange fractions obtained from Leiurus quinquestriatus hebraeus venom were investigated by plasma desorption mass spectrometry in order to select low molecular mass polypeptides. Three toxin-like peptides with molecular mass close to 3 kDa, named leiuropeptides I, II and III, were purified and found devoid of any significant toxicity against mammals and insects. Their amino acid sequences revealed a cysteine pattern analogous to that of short-chain scorpion toxins. The solution structure of leiuropeptide II was determined by 2D H-NMR spectroscopy and indicated the presence of a helix accomodating a proline, connected to a two-stranded β-sheet by three disulfide bonds. The overall fold of leiuropeptide II is found to be similar to that of leiurotoxin I, a 31-residue toxin present in the same scorpion venom which acts on K⁺ channels. In order to rationalize the absence of toxicity, the electrostatic potential of leiuropeptide II was compared to that of leiurotoxin I. The peptide is characterized by a large negative zone around Glu4, Asp5 and Asp8 residues, beginning in the neighbourhood of the β-turn and extending along the helix. In the same area, leiurotoxin I exhibits a positive surface, around Arg6 and Argl3 basic residues, which are essential for its receptor affinity. © Munksgaard 1997.     

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.     

Purification of contracture-inducing insect toxins from Buthinae scorpion venoms by immunoaffinity and high pressure liquid chromatography

A method of rapid and selective detection and purification of contracture-inducing insect toxins from Buthinae scorpion venoms is described in this paper. It consists of two main steps: the first one is specific as it uses immunoaffinity chromatography with antibodies against Androctonus australis Hector IT; the second, reverse phase high pressure liquid chromatography, allows the final separation of the different toxins present in each of the three venoms studied. Two, three and four insect toxins have been purified, respectively, from the venoms of Androctonus australis Hector, Buthus occitanus mardochei and Leiurus quinquestriatus quinquestriatus. This work demonstrates that, in Buthinae venoms, contracture-inducing insect toxins antigenically related to AaH IT, the first one purified, constitute the most important and, in some cases, the only toxins present.     

Autonomic effects of some scorpion venoms and toxins

1. The autonomic effects of venoms and toxins from several species of scorpions, including the Indian red scorpion Mesobuthus tamulus, the Chinese scorpion Buthus martensi Karsch and the Israeli scorpion Leiurus quinquestriatus quinquestriatus, all belonging to Buthidae, and the Asian black scorpions Heterometrus longimanus and Heterometrus spinifer, belonging to Scorpionidae, are reviewed. 2. The effects of the venoms of M. tamulus and L. q. quinquestriatus on noradrenergic and nitrergic transmission in the rat isolated anococcygeus muscle revealed that both venoms mediated their pharmacological effects via a prejunctional mechanism involving the activation of voltage-sensitive sodium channels with consequent release of neurotransmitters that mediate target organ responses, similar to the effects mediated by other alpha-scorpion toxins. 3. Two new toxins, Makatoxin I and Bukatoxin, were purified to homogeneity from the venom of B. martensi Karsch. Determination of their complete amino acid sequences confirmed that both toxins belonged to the class of alpha-scorpion toxins. The effects of both toxins on noradrenergic and nitrergic transmission in the rat anococcygeus muscle provided firm evidence that their pharmacological actions also closely resembled those mediated by other alpha-scorpion toxins on neuronal voltage-sensitive sodium channels. 4. The venoms of H. longimanus and H. spinifer were found to have high concentrations of noradrenaline (1.8 +/- 0.3 mmol/L) and relatively high concentrations of acetylcholine (79.8 +/- 1.7 micromol/L) together with noradrenaline (146.7 +/- 19.8 micromol/L), respectively, which can account for their potent direct cholinergic and noradrenergic agonist actions in the rat anococcygeus muscle. 5. Our studies confirmed that the rat anococcygeus muscle is an excellent nerve-smooth muscle preparation for investigating the effects of bioactive agents on noradrenergic and nitrergic transmission, as well as the direct agonist actions of these agents on post-synaptic alpha-adrenoceptors and M3 muscarinic cholinoceptors. Although many studies, including our own, have documented that scorpion venoms and toxins mediate their primary effects via a prejunctional mechanism that leads to the marked release of various autonomic neurotransmitters, our studies have shown that there are exceptions to this generally accepted phenomenon. In particular, we have provided firm evidence to show that the venoms from H. longimanus and H. spinifer do not have such a prejunctional site of action but, instead, the venoms mediate their autonomic effects through direct agonist actions on post-junctional muscarinic M3 cholinoceptors and alpha-adrenoceptors.     

A novel short-chain peptide BmKX from the Chinese scorpion Buthus martensi Karsch, sequencing, gene cloning and structure determination.

Scorpion venom is a rich source of bioactive peptides. From the venom of Chinese scorpion Buthus martensi Karsch (BmK), a novel short chain peptide BmKX of 31-amino acid residues was purified, and its amino acid sequence and gene structure were determined. The gene of BmKX was composed of two exons interrupted by an 86-bp intron at the codon-7 upstream of the mature peptide. Although its gene structure is similar to those of other known scorpion toxins, its amino acid sequence, especially the cysteine framework, is different from those of all other known subfamilies of short-chain scorpion toxins. The solution structure of BmKX, determined with two-dimensional NMR spectroscopy, shows that BmKX also forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins, consisting of a short 3(10)-helix and a two-stranded antiparallel beta-sheet, and the short N-terminal segment forms a pseudo-strand of the beta-sheet. However, the orientation between the helix and the beta-sheet is significantly different from the others, which might be the reason for its unique but still unclear physiological function.