North African Scorpion Androctonus Australis Research Articles

The Pharmacological and Structural Basis of the AahII-NaV1.5 Interaction and Modulation by the Anti-AahII Nb10 Nanobody.

Scorpion α-toxins are neurotoxins that target the fast inactivation mechanism of voltage-gated sodium (NaV) channels leading to several neuro- and cardiotoxic effects in mammals. The toxin AahII is the most active α-toxin from the North African scorpion Androctonus australis Hector that slows the fast inactivation of NaV channels. To fight scorpion envenomation, an anti-AahII nanobody named NbAahII10 (Nb10) was developed. The efficiency of this nanobody has been evaluated in vivo on mice, but its mechanism of action at the cellular level remains unknown. Here we have shown that AahII toxin slows the fast inactivation of the adult cardiac NaV1.5 channels, expressed in HEK293 cells, in a dose-dependent manner, while current amplitude was not affected. The inactivation of NaV1.5 is slower by a factor of 4, 7, and 35 in the presence of [AahII] at 75, 150, and 300 nM, respectively. The washout partially reversed the toxin effect on inactivation from 8.3 ± 0.9 ms to 5.2 ± 1.2 ms at 75 nM. We have also demonstrated that the highly neutralizing Nb10 can fully reverse the effect of AahII toxin on the channel inactivation kinetics even at the 1:1 M ratio. However, the 1:0.5 M ratio is not able to neutralize completely the AahII effect. Therefore, the application of Nb10 promotes a partial abolishment of AahII action. Bioinformatic analysis and prediction of NaV1.5-driven docking with AahII show that Ala39 and Arg62 of AahII play a crucial role to establish a stable interaction through H-bound interactions with Gln1615 and Lys1616 (S3–S4 extracellular loop) and Asp1553 (S1–S2 loop) from the voltage-sensing domain IV (VSD4) of NaV1.5, respectively. From this, we notice that AahII shares the same contact surface with Nb10. This strongly suggests that Nb10 dynamically replaces AahII toxin from its binding site on the NaV1.5 channel. At the physiopathological level, Nb10 completely neutralized the enhancement of breast cancer cell invasion induced by AahII. In summary, for the first time, we made an electrophysiological and structural characterization of the neutralization potent of Nb10 against the α-scorpion toxin AahII in a cellular model overexpressing NaV1.5 channels.

Subtype-Selective Activation of Kv7 Channels by AaTXKβ(2–64), a Novel Toxin Variant from theAndroctonus australisScorpion Venom

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKβ₂₋₆₄, a novel variant of AaTXKβ₁₋₆₄, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKβ₂₋₆₄, but not AaTXKβ₁₋₆₄, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKβ₂₋₆₄ also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKβ₂₋₆₄-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.

Characterisation of the genes encoding Aa1 isoforms from the scorpion Androctonus australis

Aa1 is a toxin purified from the venom of the North African scorpion Androctonus australis. It blocks fast K + currents in cerebellar granular cells [Biochim. Biophys. Acta 1468 (2000) 203]. Two full-length cDNAs (about 250 bp) encoding the precursors of putative Aa1 isoforms (AaTX1 and AaTX2) were amplified by PCR from a venom gland cDNA library of A. australis. The deduced precursors were composed of 59 amino acid residues including a signal peptide of 22 residues and a mature toxin of 37 residues. The peptides display 94% sequence identity with Aa1. Intron–exon organisation of the gene corresponding to theAaTX1 cDNAs was also depicted.

Use of fusion protein constructs to generate potent immunotherapy and protection against scorpion toxins

We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal α-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major α-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP- AaH I, MBP- AaH II, MBP- AaH I+II and MBP- AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1 nM range. In vitro neutralisation assays indicated that 1 ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD 50 of the toxic fraction ( AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD 50 of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.

Molecular biology of scorpion toxins active on potassium channels

Peptidyl scorpion toxins are known to block diverse types of K+ channels with high affinity and, thus, can be used as powerful tools to study the physiological role of the ionic selectivity, and the architecture of the pore-region of this class of channels. Yet, diversity among K+ channels is large and there has been a profusion of research for new selective ligands in order to elucidate their mechanisms of action and pharmacology significance. Scorpion toxins active on K+ channels are short polypeptides of about 30 to 40 amino acid residues, cross-linked by three or four disulfide bridges. They display a high degree of primary sequence homologies. 1H Nuclear Magnetic Resonance (NMR) analysis has demonstrated that these toxins are composed of an α-helix and a two-stranded antiparallel β-sheet, linked by two disulfide bridges. This structural motif is also found in the insect defensins. A 370 bp cDNA encoding the Kaliotoxin 2 (KTX2) precursor (a 37 amino acid residues peptide purified from the North African scorpion Androctonus australis and acting as a high affinity blocker of K+ channels) was obtained by PCR amplification and the organization of the KTX2 precursor depicted. This precursor is composed of a signal peptide followed by the mature toxin. The transcriptional unit and the promotor region of the gene encoding KTX2 was then amplified from the genomic DNA of Androctonus australis and its sequence determined. A single intron of 87 bp, located close to the region encoding the C-terminal part of the signal peptide, was found. Its A+T content was particularly high (up to 86%). The transcription unit of the gene was 390 bp long. Regulatory consensus sequences were identified. The genes of scorpion ‘short’ toxins active on K+ channels are organized similarly to those of the scorpion ‘long’ toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.

Development of an ELISA for the detection of scorpion venoms in sera of humans envenomed by Androctonus australis garzonii (Aag) and Buthus occitanus tunetanus (Bot): correlation with clinical severity of envenoming in Tunisia

A sandwich ELISA was set up for measuring scorpion venom levels in sera of accidentally envenomed humans with the aim to establish a quantitative relationship between these levels, envenoming severity and clinical symptoms. This assay used equine polyclonal F(ab′) 2, specific to two North African scorpion ( Androctonus australis garzonii: Aag and Buthus occitanus tunetanus: Bot) venoms. The test proved to be simple, reproducible, very sensitive (detection limit=0.9 ng/ml) and linear between 0.5 and 15 ng/ml of venom concentrations. A large survey on scorpion sting envenomings was conducted from 1993 to 1996 in Tunisia to gather accurate epidemiological, clinical and biological data from victims as well as informations on the treatment that they had received. Victims were classified into three grades (GI, GII and GIII) of increasing severity according to clinical signs of envenoming. Blood samples were collected from victims and tested by ELISA for their content of Aag and Bot venoms. A strong correlation was found between clinical symptoms of envenoming and the level of scorpion venom antigens in serum ( r=0.980). Mean serum venom concentrations were: 2.65±0.81 ng/ml in GI envenoming, 9.79±4.08 ng/ml in GII and 21.7±6.51 ng/ml in GIII. The difference between each group was statistically significant ( p<0.01). This ELISA may prove to be helpful to establish a rationale approach of specific antivenom therapy.

Influence of a NH 2-terminal extension on the activity of KTX2, a K + channel blocker purified from Androctonus australis scorpion venom

A cDNA encoding a short polypeptide blocker of K + channels, kaliotoxin 2 (KTX2), from the venom of the North African scorpion Androctonus australis was expressed in the periplasmic space of Escherichia coli. KTX2 was produced as a fusion protein with the maltose binding protein followed by the recognition site for factor Xa or enterokinase preceding the first amino acid residue of the toxin. The fully refolded recombinant KTX2 (rKTX2) was obtained (0.15–0.30 mg/l of culture) and was indistinguishable from the native toxin according to chemical and biological criteria. An N-extended analogue of KTX2 exhibiting three additional residues was also expressed. This analogue had 1000-fold less affinity for the 125I-kaliotoxin binding site on rat brain synaptosomes than KTX2. Conformational models of KTX2 and its mutant were designed by amino acid replacement using the structure of agitoxin 2 from Leiurus quinquestriatus as template, to try to understand the decrease in affinity for the receptor.

Anti-insect toxin 5 (AaIT5) from Androctonus australis.

An insect-selective scorpion toxin (AaIT5) was purified from the venom of the North African scorpion Androctonus australis, and its amino acid sequence was determined by a combination of automated Edman degradation, electrospray-ionization mass spectrometry, and sequence alignment. This insect toxin is very potent against the tobacco budworm, Heliothis virescens (100% lethal dose < 1.8 microg/100 mg body mass) and shows a distinct insect specificity and various symptoms. It is not toxic to mice after subcutaneous injection. The molecular mass of this toxin is 6882 Da and the amino acid sequence is similar to those of Androctonus australis anti-insect toxin 4 (AaIT4), Leiurus quinquestriatus depressant anti-insect toxins (LqhIT2, LqqIT2), and Buthotus judaicus depressant anti-insect toxin (BjIT2).

In vivo protection against Androctonus australis hector scorpion toxin and venom by immunization with a synthetic analog of toxin II

A synthetic peptide mimicking the North African scorpion Androctonus australis hector toxin II was designed and produced by chemical solid-phase synthesis. It contains the entire sequence of toxin II (64 amino acid residues), with each half-cystine being replaced by the isosteric residue a-aminobutyric acid, and was thus devoid of disulfide bridges. This construct was totally nontoxic in mice even if large amounts, equivalent to 1000 times the LD 50 of the original toxin, were injected by the intracerebroventricular route. The synthetic peptide, either as a monomer or polymerized by means of glutaraldehyde, induced the production of antitoxin neutralizing antibodies in immunized mice and rabbits. After three injections with either the monomeric or polymerized synthetic peptide, the immunized mice were protected against several lethal doses of the corresponding native toxin or scorpion venom. Six months after immunization, the mice were completely protected against challenge with eight LD 50 of the original toxin. The protection was better when the polymerized synthetic peptide was used. One month after the start of the immunization program, it showed a good correlation between antibody titer and protection. However, antibody titer decreased with time but protection remained high. This suggests that additional factors other than circulating antibodies play a role in protective activity.

Amino Acid Sequence of an Excitatory Insect-selective Toxin (BmK IT) From Venom of the Scorpion Buthus martensi Karsch

The insect-selective neurotoxin(BmK IT) of scorpion Buthus martensi Karsch was first reduced and S-alkylated, and then digested by TPCK-trypsin and Staphylococcus aureus V-8 Protease. The enzymatic peptides were purified on TLC-plastic sheet and submitted to determine their amino acid compositions and sequences. The sequence of the 70 amino acid residues of BmK IT was established with reference to the primary structure of AaH IT, another excitatory insect-selective toxin from the venom of North African scorpion Androctonus australis Hector. About 75% of the homologous sequence was found in the molecules of BmK IT and AaH IT. It is obvious that the results contribute toward better understanding of the molecular structure characteristics, structure/activity relationship of scorpion insect-selective toxins, and they can serve as the molecular basis for utilizing the toxins as a tool to clarify molecular mechanism involved in channel gating, and to infer the possibility of developing them as new selective bioinsecticides.

An anti-insect toxin purified from the scorpion Androctonus australis Hector also acts on the .alpha.- and .beta.-sites of the mammalian sodium channel: sequence and circular dichroism study

A new anti-insect neurotoxin, AaH IT4, has been isolated from the venom of the North African scorpion Androctonus australis Hector. This polypeptide has a toxic effect on insects and mammals and is capable of competing with anti-insect scorpion toxins for binding to the sodium channel of insects; it also modulates the binding of alpha-type and beta-type anti-mammal scorpion toxins to the mammal sodium channel. This is the first report of a scorpion toxin able to exhibit these three kinds of activity. The molecule is composed of 65 amino acid residues and lacks methionine and, more unexpectedly, proline, which until now has been considered to play a role in the folded structure of all scorpion neurotoxins. The primary structure showed a poor homology with the sequences of other scorpion toxins; however, it had features in common with beta-type toxins. In fact, radioimmunoassays using antibodies directed to scorpion toxins representative of the main structural groups showed that there is a recognition of AaH IT4 via anti-beta-type toxin antibodies only. A circular dichroism study revealed a low content of regular secondary structures, particularly in beta-sheet structures, when compared to other scorpion toxins. This protein might be the first member of a new class of toxins to have ancestral structural features and a wide toxic range.

Topological analysis of hydrogen bonding in protein structure

A recent study has shown that topological stereoisomers exist for the polypeptide chain in disulfide-containing proteins that are represented by non-planar graphs. This topological stereochemistry is demonstrated in the structure of variant 3 toxin in the venom of the North American scorpion Centruroides sculpturatus Ewing and the structure of toxin II from the North African scorpion Androctonus australis Hector. In this report, we found that a similar topological analysis can be applied to the hydrogen bonding in alpha-helices and beta-sheets within protein molecules, and we described the topological characteristics of chiral properties of protein secondary structure elements. Specifically, a closed right-handed alpha-helix of more than six residues long is shown formally to be non-planar and has the L topology. Antiparallel beta-sheets are planar. Two parallel beta-strands each of at least three residues in length, however, constitute a non-planar structural element and can have either L or D topology. The favored right-handed crossover for parallel beta-sheets has the L form, the same as the right-handed alpha-helix. This topological description of the hydrogen bonding in secondary structures may be extended to higher levels of protein structure and may provide a conceptual framework for studying complex protein architecture in general.

Convergent solid phase peptide synthesis. v. synthesis of the 1-4, 32-34, and 53-59 protected segments of the toxin ii of androctonus australis hector.

Two protected peptide segments corresponding to the sequence 32-34 and 53-59 of toxin II of the north African scorpion Androctonus australis Hector have been synthesized on a photolabile Nbb-resin using the TFA-labile Boc α-amino protection and HF-labile side chain protecting groups. A third protected peptide corresponding to segment 1-4 has been synthesized on the same resin but with a t-butyl group for β protection of aspartic acid and a Z group on the lysine side chain. For this last segment a combination of Boc and Fmoc groups for α -amino protection has been used successfully on the Nbb-resin. After photolysis the crude peptides have been treated by solvent extraction and semi-preparative HPLC to yield highly purified segments. These syntheses show the flexibility of the convergent solid phase approach and how segments with different and independent protecting groups can be assembled by solid phase peptide procedure.

Tityus serrulatus toxin VII bears pharmacological properties of both β-toxin and insect toxin from scorpion venoms

Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.

Immunochemistry of scorpion α-toxins: Study with synthetic peptides of the antigenicity of four regions of toxin II of Androctonus australis Hector

Sequences 19–29 and 28–39 of toxin II of the North African scorpion Androctonus australis Hector have been synthesized. These two peptides correspond to the highest peaks in the hydrophilicity profile of toxin II and were thus believed to account for a significant proportion of toxin antigenicity. Affinity chromatography of solid-phase-bonded peptides allowed us to purify two sub-populations from the total IgGs raised against the native toxin. They both still bound to 125I-toxin II and showed a restricted heterogeneity in their specificity. Solid-phase immunoassays confirmed the antigenicity of these synthetic peptides and also that of two other previously described synthetic replicates of the antigenic regions of toxin II: sequences (5–14) S—S (60–64) and 50–59. The location of the four antigenic regions relative to the postulated location of the receptor-binding site of the toxin is discussed.

Large scale purification of toxins from the venom of the scorpion Androctonus australis Hector

M. F. Martin and H. Rochat. Large scale purification of toxins from the venom of the scorpion Androctonus australis Hector. Toxicon 24, 1131 – 1139, 1986. — A large scale procedure for purification of the toxins in the venom of the North African scorpion Androctonus australis has been developed. This procedure optimizes the sequence of the steps, leading to better yields of toxins and shortening of the time. It is possible to use 10–15 g batches of venom and also prevent cross-contamination of the toxins. High pressure liquid chromatography has been used to separate AaH I and I′, two isotoxins which differ by one amino acid residue in position 17: valine or isoleucine. The procedure allows the characterization of a new toxin active in mice, AaH IV, which represents 0.6 and 2.5% of the venom weight and toxicity, respectively. This new component has been characterized as an α toxin made up of 61 amino acid residues and having a neutral isoelectric point.

Immunochemistry of scorpion α-toxins: Purification and characterization of two functionally independent IgG populations raised against toxin II of Androctonus australis Hector

We report the isolation and characterization of two IgG populations specific to two synthetic peptides corresponding to two antigenic sites of toxin II of the North African scorpion Androctonus australis Hector. Firstly, thanks to the use of: (1) antigenic homology studies between toxin II of A. australis Hector and toxin III of Buthus occitanus tunetanus, (2) chemical modification of toxin II of A. australis Hector, and (3) prediction of the localization of the four major antigenic sites of scorpion α-toxins by the method developed by Hopp and Woods [ Proc. natn. Acad. Sci. U.S.A. 78, 3824–3828 (1981)], we have established that the region around the disulfide bridge between cysteines 12 and 63 as well as the stretch of residues 50–59 probably each enclosed an antigenic site. Secondly, the synthetic replicates of these regions linked to Sepharose allowed us to isolate, by immunoaffinity chromatography, two IgG populations from the whole anti-toxin II of A. australis Hector IgG s. Finally, each of these two IgG populations was shown to be specific to one antigenic site as evidenced by the multideterminant effect on the slopes of binding curves developed by Berzofsky et al. [ Biochemistry 15, 2113–2121 (1976)]. Furthermore, these two IgG populations were found to be functionally independent and this could be related to the fact that the two regions carrying the two antigenic sites are not close to each other in space and that there is neither steric hindrance nor cooperative effects between them. The association constant of these site-specific IgG populations was calculated and found to be equal to 1.18–5.14 × 10 9 l/mole for IgG anti-site 1 and 1.16–5.62 × 10 9 l/mole for IgG anti-site 2 respectively by Sips [ J. chem. Phys. 16, 490–495 (1948)], Scatchard [ Am. N.Y. Acad. Sci. 51, 660–772 (1949)] and Steward and Petty [ Immunology 23, 881–887 (1972)] representations. The index of heterogeneity of 0.9 for anti-P 1 and anti-P 2 indicates the purification of essentially homogeneous affinity IgG populations.

Purification and amino acid sequence of toxin I″ from the venom of the North African scorpion Androctonus australis Hector

When a large quantity of the venom of the Scorpion Androctonus australis Hector is submitted to a purification procedure it is possible to purify a new toxin, toxin I″, which is lethal to mouse, and present in very low quantities. The yield is 0.04%. The amino acid sequence of this newly discovered toxin only differs from toxin I of the same scorpion by an extra arginine residue at the C-terminal end provided that the sequence of toxin I, at positions 34 and 35, is in agreement with newer information provided in this paper.

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 diperformic 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 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.

Amino Acid Sequence of Neurotoxin III of the Scorpion Androctonus australis Hector

The amino acid sequence of neurotoxin III, purified from the venom of the North African scorpion Androctonus australis Hector, has been determined by Edman degradation using a liquid-phase sequencer. Carboxypeptidase A hydrolyses confirmed not only the sequence of the five last residues but also the presence of a free alpha-carboxylic group at the C-terminus. Edman degradation was conducted on one hand with the Quadrol [N,N,N',N'-tetrakis(2-hydroxypropyl)ethylene diamine] program and S-alkylated protein before or after coupling with sulfophenylisothiocynate (the first 34 residues were thus identified), on the other hand on tryptic and chymotryptic peptides with a dimethylbenzylamine program (residues 1--23 and 31--34 were confirmed, the positions of residues 35-64 were established). Neurotoxin III was found to belong to the same group of scorpion toxins active on mammals as neurotoxin I purified from the same venom (50 homologous positions exist in the two proteins).