Isolation of scorpion (Androctonus amoreuxi) putative alpha neurotoxins and parallel cloning of their respective cDNAs from a single sample of venom

The venoms of buthid scorpions are known to contain basic, single-chain protein toxins (alpha toxins) consisting of 60-70 amino acid residues that are tightly folded by four disulfide bridges. Here we describe isolation and sequencing of three novel putative alpha toxins (AamH1-3) from the venom of the North African scorpion, Androctonus amoreuxi, and subsequent cloning of their precursor cDNAs from the same sample of venom. This experimental approach can expedite functional genomic analyses of the protein toxins from this group of venomous animals and does not require specimen sacrifice for cloning of protein toxin precursor cDNAs.     

Molecular cloning and nucleotide sequence analysis of a cDNA encoding the main beta-neurotoxin from the venom of the South American scorpion Tityus serrulatus.

A cDNA encoding the main Tityus serrulatus beta-neurotoxin was isolated from a venom gland cDNA library by using an oligonucleotide probe. The amino acid sequence deduced from the cDNA nucleotide sequence indicated that the toxin is the processed product of a precursor containing: (i) a signal peptide of 20 residues; (ii) the amino acid sequence of the mature toxin; and (iii) an extra Gly-Lys-Lys tail at the C-terminal end before the termination codon. Thus, in addition to the removal of the signal peptide by a signal peptidase, the generation of the mature toxin requires both a post-translational cleavage by a carboxypeptidase specific for basic residues and the action of an alpha-amidating enzyme. These results also show that the biosynthetic pathway for beta-toxins of 'New World' scorpion venoms is similar to that already described for alpha-toxins of 'Old World' scorpion venoms.     

Amino acid sequence of neurotoxin I from Centruroides sculpturatus Ewing

The further characterization of toxin I from venom of the scorpion Centruroides sculpturatus Ewing (region, Southwestern United States) is reported. Toxin I is a single polypeptide chain of 64 amino acid residues crosslinked by four disulfide bridges. The complete amino acid sequence of toxin I was deduced from the sequence of its tryptic peptides and overlaps provided by its chymotryptic peptides. Toxin I has an amino terminal lysyl residue and a carboxyl terminal threonyl residue.The amino acid sequences of toxin I and neurotoxic variants 1, 2, and 3, likewise isolated from C. sculpturatus venom, differ at 26 positions.The sequences of toxin I from C. sculpturatus and toxins I and II from the North African scorpion, Androctonus australis Hector, are also compared.     

Molecular cloning and nucleotide sequence analysis of encoded anti-insect toxin BotIT2 from the scorpion Buthus occitanus tunetanus venom

Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.     

Cationicity-Enhanced Analogues of the Antimicrobial Peptides,AcrAP1 and AcrAP2, from the Venom of the Scorpion,Androctonus crassicauda,Display Potent Growth Modulation Effects on Human Cancer Cell Lines

The non disulphide-bridged peptides (NDBPs) of scorpion venoms are attracting increased interest due to their structural heterogeneity and broad spectrum of biological activities. Here, two novel peptides, named AcrAP1 and AcrAP2, have been identified in the lyophilised venom of the Arabian scorpion, Androctonus crassicauda, through “shotgun” molecular cloning of their biosynthetic precursor-encoding cDNAs. The respective mature peptides, predicted from these cloned cDNAs, were subsequently isolated from the same venom sample using reverse phase HPLC and their identities were confirmed by use of mass spectrometric techniques. Both were found to belong to a family of highly-conserved scorpion venom antimicrobial peptides - a finding confirmed through the biological investigation of synthetic replicates. Analogues of both peptides designed for enhanced cationicity, displayed enhanced potency and spectra of antimicrobial activity but, unlike the native peptides, these also displayed potent growth modulation effects on a range of human cancer cell lines. Thus natural peptide templates from venom peptidomes can provide the basis for rational analogue design to improve both biological potency and spectrum of action. The diversity of such templates from such natural sources undoubtedly provides the pharmaceutical industry with unique lead compounds for drug discovery.     

ImKTx1, a new Kv1.3 channel blocker with a unique primary structure

Toxins from the venoms of scorpion, snake, and spider are valuable tools to probe the structure-function relationship of ion channels. In this investigation, a new toxin gene encoding the peptide ImKTx1 was isolated from the venom gland of the scorpion Isometrus maculates by constructing cDNA library method, and the recombinant ImKTx1 peptide was characterized physiologically. The mature peptide of ImKTx1 has 39 amino acid residues including six cross-linked cysteines. The electrophysiological experiments showed that the recombinant ImKTx1 peptide had a pharmacological profile where it inhibited Kv1.3 channel currents with IC(50) of 1.70 n± 1.35 μM, whereas 10 μM rImKTx1 peptide inhibited about 40% Kv1.1 and 42% Kv1.2 channel currents, respectively. In addition, 10 μM rImKTx1 had no effect on the Nav1.2 and Nav1.4 channel currents. Multiple sequence alignments showed that ImKTx1 had no homologous toxin peptide, but it was similar with Ca(2+) channel toxins from scorpion and spider in the arrangement of cysteine residues. These results indicate that ImKTx1 is a new Kv1.3 channel blocker with a unique primary structure. Our results indicate the diversity of K(+) channel toxins from scorpion venoms and also provide a new molecular template targeting Kv1.3 channel.     

Novel structural class of four disulfide-bridged peptides from Tityus serrulatus venom

A new structural class of short peptides folded by four disulfide-bridges was found in the venom of the Brazilian scorpion Tityus serrulatus. Peptides were put on evidence independently by means of two different approaches of structurally guided prospection. First, a cDNA sequence was obtained using a degenerate primer constructed according to the C-terminal sequence of kaliotoxin (KTx2), from the Androctonus australis venom. Second, MALDI-TOF mass spectrometry analyses of toxic fraction FIII from T. serrulatus venom revealed a family of molecules ranging approximately from 2900 to 3000 Da. Three new peptides were isolated and named TsPep1, TsPep2, and TsPep3. Biochemical characterization showed that they are 29 amino acids long, constrained by a new pattern of four disulfide-bridges. These results enable us to classify these new molecules as part of a novel structural class of short peptides from scorpion venoms.     

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.     

Structure of the scorpion toxin BmBKTtx1 solved from single wavelength anomalous scattering of sulfur

This report describes the crystal structure of the K(+) channel-blocking toxin, BmBKTx1, isolated recently from the venom of the scorpion Buthus martensi Karsch. This is only the second structure of the short-chain K(+) channel-blocking toxin from scorpion solved by means of X-ray crystallography. Additionally, reductive dimethylation of folded BmBKTx1 employed to induce its crystallization and solution of the structure based on the anomalous signal from the sulfur atoms make this example quite unique. The monomer of BmBKTx1 is formed by 31 amino acid residues, including 6 cysteines connected in 3 disulfide bridges. Crystals of this toxin belong to the space group P2(1) with two molecules present in the asymmetric unit. The unit cell parameters are a = 21.40 A, b=39.70 A, c=29.37 A, and beta-94.13 grades. Based on the high-quality dataset (anomalous signal) collected to the resolution 1.72A using the conventional X-radiation generator (lambda Cu, K alpha = 1.5478 A), the positions of sulfur atoms contributed by 12 cysteine residues have been identified, and subsequent improvement of the experimental phases have allowed structure solution. The final model was refined to the crystallographic R-factor of 0.166. The methyl groups on several lysine residues could be easily modeled into the electron density.     

The amino acid sequence of toxin IV from the Androctonus australis scorpion: differing effects of natural mutations in scorpion alpha-toxins on their antigenic and toxic properties.

The complete amino acid sequence (64 residues) of the AaH IV toxin from the scorpion Androctonus australis Hector was determined by automated Edman degradation and was compared with the sequences of other Androctonus toxins. AaH IV was also tested by radioimmunoassay for binding to antisera raised against other toxins of the same species. The results indicated that AaH IV shares some of the antigenic properties of AaH I and AaH III toxins, but does not cross-react with anti-AaH II antibodies. The structural basis for the observed antigenic relationships can be found in the high degree of homology displayed by AaH IV with regard to AaH I and III, the changes in amino acid residues equally affecting regions included or excluded from the main predicted antigenic sites of AaH IV. The lower biological potency of AaH IV is presumably the result of some of the sequence differences. In particular, substitution affecting the charge and bulkiness of residue 61 could account for the poor receptor binding and consequential weak toxic properties of this molecule.     

Purification and the partial amino acid sequence of an insect neurotoxin from the venom of scorpion Buthus martensi Karsch

1. A neurotoxic peptide was isolated from the venom of the scorpion Buthus martensi Karsch collected in Henan Province, China. 2. This toxin showed the highest neurotoxic potency to crickets amongst all components in the venom examined. 3. The amino acid composition of the toxin was similar to that of insect toxin 1 of Leiurus quinquestriatus quinquestriatus. 4. The partial primary sequence of the toxin at the N-terminal was very similar to that of an insect toxin of Androctonus australis Hector. 5. We conclude that the neurotoxin we isolated is indeed an insect toxin and thus named it as BmK IT.     

Molecular dissection of venom from Chinese scorpion Mesobuthus martensii: identification and characterization of four novel disulfide-bridged venom peptides

Scorpion venom is composed of a large repertoire of biologically active polypeptides. However, most of these peptides remain to be identified and characterized. In this paper, we report the identification and characterization of four novel disulfide-bridged venom peptides (named BmKBTx, BmKITx, BmKKx1 and BmKKx2, respectively) from the Chinese scorpion, Mesobuthus martensii (also named Buthus martensii Karsch). BmKBTx is composed of 58 amino acid residues and cross-linked by three disulfide bridges. The sequence of BmKBTx shows some similarities to that of the toxin, birtoxin, and its analogs. It is likely that BmKBTx is a beta-toxin active on Na+ channels, which is toxic to either insects or mammals. BmKITx is composed of 71 amino acid residues with four disulfide bridges. It is the longest venom peptide identified from M. martensii so far. BmKITx shows little sequence identity with scorpion alpha-toxins toxic to insects. It is likely that BmKITx is a new type of Na+ -channel specific toxin active on both insects and mammals. BmKKx1 contains 38 amino acid residues cross-linked by three disulfide bridges and shows 84% sequence identity with BmTx3, an inhibitor of A-type K+ channel and HERG currents. BmKKx1 has been classified as alpha-KTx-15.8. BmKKx2 is composed of 36 residues and stabilized by three disulfide bridges. BmKKx2 is a new member of the gamma-K+ -channel toxin subfamily (classified as gamma-KTx 2.2). The venoms of scorpions thus continue to provide novel toxins with potential novel actions on targets.     

Amino acid sequence of neurotoxin III of the scorpion Androctonus austrialis 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).     

Tamulustoxin: a novel potassium channel blocker from the venom of the Indian red scorpion Mesobuthus tamulus

We have characterized tamulustoxin, a novel 35-amino-acid peptide found in the venom of the Indian red scorpion (Mesobuthus tamulus). Tamulustoxin was identified through a [125I]toxin I screen, designed to identify toxins that block voltage-activated potassium channels. Tamulustoxin has also been cloned by RT-PCR, using RNA extracted from scorpion venom glands. Tamulustoxin shares no homology with other scorpion venom toxins, although the positions of its six cysteine residues would suggest that it shares the same structural scaffold. Tamulustoxin rapidly inhibited both peak and steady-state currents (18.9 +/- 1.0 and 37 +/- 1.1%, respectively) produced by injecting CHO cells with mRNA encoding the hKv1.6 channel.     

An ERG channel inhibitor from the scorpion Buthus eupeus

The isolation of the peptide inhibitor of M-type K(+) current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996) FEBS Lett. 384, 277-280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K(+) channels was also studied. BeKm-1 inhibited hERG1 channels with an IC(50) of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown to be a novel specific blocker of hERG1 potassium channels.     

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus

The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.     

BTK-2, a new inhibitor of the Kv1.1 potassium channel purified from Indian scorpion Buthus tamulus

A novel inhibitor of voltage-gated potassium channel was isolated and purified to homogeneity from the venom of the red scorpion Buthus tamulus. The primary sequence of this toxin, named BTK-2, as determined by peptide sequencing shows that it has 32 amino acid residues with six conserved cysteines. The molecular weight of the toxin was found to be 3452 Da. It was found to block the human potassium channel hKv1.1 (IC(50)=4.6 microM). BTK-2 shows 40-70% sequence similarity to the family of the short-chain toxins that specifically block potassium channels. Multiple sequence alignment helps to categorize the toxin in the ninth subfamily of the K+ channel blockers. The modeled structure of BTK-2 shows an alpha/beta scaffold similar to those of the other short scorpion toxins. Comparative analysis of the structure with those of the other toxins helps to identify the possible structure-function relationship that leads to the difference in the specificity of BTK-2 from that of the other scorpion toxins. The toxin can also be used to study the assembly of the hKv1.1 channel.     

Primary structure of a short toxin from the venom of a vietnamese scorpion (Buthus sp.)

The complete amino acid sequence of an important toxin (toxin 14) from the venom of a Vietnamese scorpion (Buthus occitanus sp.) has been determined, which includes 35 amino acid residues and three disulfide bridges (molecular weight, 3843 Da). The comparison of the sequence with sequences of short scorpion toxins led us to conclude that toxin 14 belongs to a novel group of toxins affecting the excitability of myelinated nerves.