Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch.

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Identification of BmKAPi, a novel type of scorpion venom peptide with peculiar disulfide bridge pattern from Buthus martensii Karsch

A novel cDNA sequence encoding a new type of scorpion venom peptide (BmKAPi) was first isolated from the venom gland of Buthus martensiiKarsch by cDNA library screening combined with 5′-race. The encoded precursor of BmKAPi consisted of 89 amino acid residues including a signal peptide of 24 residues, a putative mature peptide of 64 residues (BmKAPi) and an extra basic residue at the C-terminus which might be removed in the post-translational processing. BmKAPi is stabilized by five disulfide bridges, whereas all other disulfide-bridged scorpion toxins described are cross-linked by three or four disulfide bridges. It suggested the three-dimensinal scaffold of BmKAPi might be different from other scorpion toxins. The amino acid sequence of BmKAPi showed no homology with other scorpion venom peptides, but shared a little similarity with some anticoagulant peptides and proteinase inhibitors isolated from hookworm, honeybee or European frog, respectively. RT-PCR analysis showed that BmKAPi mRNA could be induced by venom extraction suggesting BmKAPi might be a component of scorpion venom. These results suggest that BmKAPi is a new type of scorpion venom peptide different from other described scorpion toxins in structural and functional aspects.     

Vejovine, a new antibiotic from the scorpion venom of Vaejovis mexicanus

Multidrug resistant bacterial infections are one of the most important health problems in recent years. Resistance to conventional antibiotics limits the therapeutic options causing increase rate in morbid-mortality in hospitals. Therefore, new antibacterial agents with new bacterial targets have been searched and found in many different sources, including scorpion venom and scorpion hemolymph. Here, we report a new anti-microbial peptide named Vejovine. This peptide was isolated from the venom of the Mexican scorpion Vaejovis mexicanus by two steps of reversed phase high performance liquid chromatography (RP-HPLC). It is composed of 47 amino acid residues with no cysteine residues in its sequence, with a molecular weight of 4873 Da. The chemical synthesis of Vejovine was performed by the solid phase method of Merrifield, using fluoren-9-ylmethoxycarbonyl (Fmoc)-amino acids. Both the native and synthetic peptides were shown to have essentially the same activity. Vejovine inhibits growth of clinical isolates of Gram-negative multidrug resistant (Pseudomonas aeruginosa, Klebsiella pneumoniae, Escherichia coli, Enterobacter cloacae and Acinetobacter baumanii) causing nosocomial infections with a minimum inhibitory concentration (MIC) of 4.4 μM up to 50 μM. This peptide has also hemolytic activity against human erythrocytes with a HC₅₀ value of 100 μM. A cDNA library of the venomous gland of this scorpion provided material for cloning the gene encoding Vejovine. This peptide is a new type of antibiotic, showing less than 50% similarity to other known scorpion peptides. Vejovine is a candidate to be used as a leading compound for future development of an effective peptide against multidrug resistant bacteria.     

Biochemical and molecular characterization of the venom from the Cuban scorpion Rhopalurus junceus

This communication describes the first general biochemical, molecular and functional characterization of the venom from the Cuban blue scorpion Rhopalurus junceus, which is often used as a natural product for anti-cancer therapy in Cuba. The soluble venom of this arachnid is not toxic to mice, injected intraperitoneally at doses up to 200 μg/20 g body weight, but it is deadly to insects at doses of 10 μg per animal. The venom causes typical alpha and beta-effects on Na⁺ channels, when assayed using patch-clamp techniques in neuroblastoma cells in vitro. It also affects K⁺ currents conducted by ERG (ether-a-go-go related gene) channels. The soluble venom was shown to display phospholipase, hyaluronidase and anti-microbial activities. High performance liquid chromatography of the soluble venom can separate at least 50 components, among which are peptides lethal to crickets. Four such peptides were isolated to homogeneity and their molecular masses and N-terminal amino acid sequence were determined. The major component (RjAa12f) was fully sequenced by Edman degradation. It contains 64 amino acid residues and four disulfide bridges, similar to other known scorpion toxins. A cDNA library prepared from the venomous glands of one scorpion allowed cloning 18 genes that code for peptides of the venom, including RjA12f and eleven other closely related genes. Sequence analyses and phylogenetic reconstruction of the amino acid sequences deduced from the cloned genes showed that this scorpion contains sodium channel like toxin sequences clearly segregated into two monophyletic clusters. Considering the complex set of effects on Na⁺ currents verified here, this venom certainly warrant further investigation.     

ImKTx88, a novel selective Kv1.3 channel blocker derived from the scorpion Isometrus maculates

Scorpion toxins are useful in the structure-function research of ion channels and valuable resources for drug design. The Kv1.3 channel is an important pharmacological target for the therapy of T cell-mediated autoimmune diseases, and many toxin peptides targeting Kv1.3 have been identified as good drug candidates in recent years. In this study, a novel toxin gene ImKTx88 was isolated from the venom of the scorpion Isometrus maculates through the construction of the cDNA library method, and the recombinant toxin peptide was purified and characterized physiologically. The mature peptide of ImKTx88 contained 39 amino acid residues including six cysteines and was predicted to be a new member of α-KTx scorpion family by sequence analysis. The electrophysiological experiments further indicated that the rImKTx88 peptide had a novel pharmacological profile: it inhibited Kv1.3 channel current with an IC₅₀ of 91 ± 42 pM, and exhibited very good selectivity for Kv1.3 over Kv1.1 (4200-fold) and Kv1.2 (93000-fold) channels, respectively. All these results suggested that, as a new selective Kv1.3 channel blocker, the ImKTx88 peptide may serve as a potential drug candidate in the therapy of autoimmune diseases.     

Genetic mechanisms of scorpion venom peptide diversification.

The diversity of scorpion venom peptides is well shown by the presence of about 400 such polypeptides with or without disulfide bonds. Scorpion toxins with disulfide bonds present a variety of sequence features and pharmacological functions by affecting different ion channels, while the venom peptides without disulfide bonds represent a new subfamily, having much lower sequence homology among each other and different functions (e.g. bradykinin-potentiating, antimicrobial, molecular cell signal initiating and immune modulating). Interestingly, all scorpion venom peptides with divergent functions may have evolved from a common ancestor gene. Over the lengthy evolutionary time, the diversification of scorpion venom peptides evolved through polymorphism, duplication, trans-splicing, or alternative splicing at the gene level. In order to completely clarify the diversity of scorpion toxins and toxin-like peptides, toxinomics (genomics and proteomics of scorpion toxins and toxin-like peptides) are expected to greatly advance in the near future.     

Three structurally related, highly potent, peptides from the venom of Parabuthus transvaalicus possess divergent biological activity.

The venom of South African scorpion Parabuthus transvaalicus contains a novel group of peptide toxins. These peptides resemble the long chain neurotoxins (LCN) of 60-70 residues with four disulfide bridges; however they are 58 residues long and have only three disulfide bridges constituting a new family of peptide toxins. Here we report the isolation and characterization of three new members of this mammal specific group of toxins. Dortoxin is a lethal peptide, bestoxin causes writhing in mice and altitoxin is a highly depressant peptide. Binding ability of these peptides to rat brain synaptosomes is tested. While the crude venom of P. transvaalicus enhances the binding of [(3)H] BTX to rat brain synaptosomes none of these individual toxins had a positive effect on binding. Although the primary structures of these toxins are very similar to birtoxin, their 3D models indicate significant differences. Dortoxin, bestoxin and altitoxin cumulatively constitute at least 20% of the peptide contained in the venom of P. transvaalicus and contribute very significantly to the toxicity of the venom of this medically important scorpion species. Therefore the amino acid sequences presented here can be used to make more specific and effective antivenins. Possible approaches to a systematic nomenclature of toxins are suggested.     

Covalent structure of toxins I and II from the scorpion Buthus occitanus tunetanus

The amino acid sequences of neurotoxins I and II, which are active on mammals, purified from the venom of Buthus occitanus tunetanus have been determined using standard methods, including mainly automatic phenylisothiocyanate degradation of S-carboxymethylated derivatives of the two proteins and peptides derived by enzymatic hydrolyses. Both toxins are made of sixty-five amino acid residues cross-linked with four disulfide bridges. For toxin II, the complete covalent structure, including the positions of the four disulfide bridges was determined: the positions are similar to those previously found in toxin II of another scorpion from Africa, Androctonus australis Hector. This finding is in favor of a similar structure for all of the scorpion neurotoxins active on mammals.     

AaeAP1 and AaeAP2: Novel Antimicrobial Peptides from the Venom of the Scorpion,Androctonus aeneas: Structural Characterisation,Molecular Cloning of Biosynthetic Precursor-Encoding cDNAs and Engineering of Analogues with Enhanced Antimicrobial and Anticancer Activities

The main functions of the abundant polypeptide toxins present in scorpion venoms are the debilitation of arthropod prey or defence against predators. These effects are achieved mainly through the blocking of an array of ion channel types within the membranes of excitable cells. However, while these ion channel-blocking toxins are tightly-folded by multiple disulphide bridges between cysteine residues, there are additional groups of peptides in the venoms that are devoid of cysteine residues. These non-disulphide bridged peptides are the subject of much research interest, and among these are peptides that exhibit antimicrobial activity. Here, we describe two novel non-disulphide-bridged antimicrobial peptides that are present in the venom of the North African scorpion, Androctonus aeneas. The cDNAs encoding the biosynthetic precursors of both peptides were cloned from a venom-derived cDNA library using 3''- and 5''-RACE strategies. Both translated precursors contained open-reading frames of 74 amino acid residues, each encoding one copy of a putative novel nonadecapeptide, whose primary structures were FLFSLIPSVIAGLVSAIRN and FLFSLIPSAIAGLVSAIRN, respectively. Both peptides were C-terminally amidated. Synthetic versions of each natural peptide displayed broad-spectrum antimicrobial activities, but were devoid of antiproliferative activity against human cancer cell lines. However, synthetic analogues of each peptide, engineered for enhanced cationicity and amphipathicity, exhibited increases in antimicrobial potency and acquired antiproliferative activity against a range of human cancer cell lines. These data clearly illustrate the potential that natural peptide templates provide towards the design of synthetic analogues for therapeutic exploitation.     

Immunological characterization of a non-toxic peptide conferring protection against the toxic fraction (AahG50) of the Androctonus australis hector venom.

KAaH1 and KAaH2 are non-toxic peptides, isolated from the venom of the Androctonus australis hector (Aah) scorpion. In a previous study, we showed these peptides to be the most abundant (approximately 10% each) in the toxic fraction (AahG50) of the Aah venom. KAaH1 and KAaH2 showed high sequence identities (approximately 60%) with birtoxin-like peptides, which likewise are the major peptidic components of Parabuthus transvaalicus scorpion venom. Here, we report the immunological characterization of KAaH1 and KAaH2. These peptides were found to be specifically recognized by polyclonal antibodies raised against AahII, the most toxic peptide of Aah venom, and represents the second antigenic group, including toxins from different scorpion species in the world. Moreover, KAaH1 partially inhibits AahII binding to its specific antibody, suggesting some common epitopes between these two peptides. The identification of possible key antigenic residues in KAaH1 was deduced from comparison of its 3-D model with the experimental structure of AahII. Two clusters of putative antigenically important residues were found at the exposed surface; one could be constituted of V3 and D53, the other of D10, T15 and Y16. Polyclonal antibodies raised against KAaH1 in mice were found to cross-react with both AahII and AahG50, and neutralizing 5LD(50)/ml of the toxic fraction. Mice vaccinated with KAaH1 were protected against a challenge of 2LD(50) of AahG50 fraction. All these data suggest that KAaH1 has clear advantages over the use of the whole or part of the venom. KAaH1 is not toxic and could produce sera-neutralizing scorpion toxins, not only from Aah venom, but also toxins of other venoms from Buthus, Leiurus, or Parabuthus scorpion species presenting antigenically related toxins.     

A covalent peptide inhibitor of RGS4 identified in a focused one-bead, one compound library screen

Background

K⁺ and Na⁺ channel toxins constitute a large set of polypeptides, which interact with their ion channel targets. These polypeptides are classified in two different structural groups. Recently a new structural group called birtoxin-like appeared to contain both types of toxins has been described. We hypothesized that peptides of this group may contain two conserved structural motifs in K⁺ and/or Na⁺ channels scorpion toxins, allowing these birtoxin-like peptides to be active on K⁺ and/or Na⁺ channels.

Results

Four multilevel motifs, overrepresented and specific to each group of K⁺ and/or Na⁺ ion channel toxins have been identified, using GIBBS and MEME and based on a training dataset of 79 sequences judged as representative of K⁺ and Na⁺ toxins. Unexpectedly birtoxin-like peptides appeared to present a new structural motif distinct from those present in K⁺ and Na⁺ channels Toxins. This result, supported by previous experimental data, suggests that birtoxin-like peptides may exert their activity on different sites than those targeted by classic K⁺ or Na⁺ toxins. Searching, the nr database with these newly identified motifs using MAST, retrieved several sequences (116 with e-value < 1) from various scorpion species (test dataset). The filtering process left 30 new and highly likely ion channel effectors. Phylogenetic analysis was used to classify the newly found sequences. Alternatively, classification tree analysis, using CART algorithm adjusted with the training dataset, using the motifs and their 2D structure as explanatory variables, provided a model for prediction of the activity of the new sequences.

Conclusion

The phylogenetic results were in perfect agreement with those obtained by the CART algorithm. Our results may be used as criteria for a new classification of scorpion toxins based on functional motifs.     

Localization of epitopes in the toxins of Tityus serrulatus scorpions and neutralizing potential of therapeutic antivenoms.

Overlapping pentadecapeptides covering the complete amino acid sequence of TsII, TsVII and TsIV toxins from the venom of scorpion Tityus serrulatus (Ts), were prepared by use of the Spot method of multiple peptide synthesis. Horse anti-Ts antisera for therapeutic use were tested for their binding to peptides. All nine antisera tested showed reactivity with several peptides from the three toxins. Three antigenic regions, one in the very N-terminal, the second in the central part and the other in the C-terminal part of the three toxins were frequently, but not constantly recognized, with an intensity that seemed to be related to the neutralizing potency of the tested antivenom. Thus the corresponding peptides (residues 1-15 and 48-62 of TsII; residues 1-15, 16-30 and 48-62 of TsIV and residues 1-15 and 47-61 of TsVII) were synthesized, coupled to KLH and used as antigens to coat the microtitration plates to determine any relationship between their ELISA reactivity with therapeutic horse antivenoms and the neutralizing potential of these antivenoms. The mixture of the N-terminal peptide of TsII, of the N-terminal TsVII peptide and of the C-terminal of TsIV was found to give a linear relationship with the neutralizing titer of horse serum of low neutralizing potency (< or =1 mg/ml). However, high neutralizing antivenoms did not show the expected response in peptide ELISA. This observation is discussed in the context of the occurrence of continuous and discontinuous epitopes on toxins.     

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.     

Turkish scorpion Buthacus macrocentrus: General characterization of the venom and description of Bu1, a potent mammalian Na+-channel α-toxin

The venom of the scorpion Buthacus macrocentrus of Turkey was fractionated by high performance liquid chromatography (HPLC) and its mass finger print analysis was obtained by spectrometry. More than 70 different fractions were obtained, allowing the determination of the molecular masses of at least 60 peptides ranging between 648 and 44,336 Da. The venom is enriched with peptides containing molecular masses between 3200–4500 Da, and 6000–7500 Da. They very likely correspond to K⁺-channel and Na⁺-channel specific peptides, respectively, as expected from venoms of scorpions of the family Buthidae, already determined for other species. The major component obtained from HPLC was shown to be lethal to mice and was further purified and characterized. It contains 65 amino acid residues maintained closely packed by 4 disulfide bridges, and shows a molecular weight of 7263 Da. Additionally, a cDNA from the venomous glands of this scorpion was used in conjunction with sequence data from Edman degradation and mass spectrometry for cloning the gene that codes for Bu1 as we named this toxin. This gene codes for a 67 amino acid residues peptide, where the two last are eliminated post-translationally for production of an amidated C-terminal arginine. Its sequence is closely related to toxins from the species Leiurus quinquestriatus, as revealed by a phylogenetic tree analysis. Electrophysiological results conducted with Bu1 using patch-clamp techniques indicate that it modifies the Na⁺ currents, in a similar way as other well known α-scorpion toxins. These results support the conclusion that this species of scorpions is dangerous to humans, having an epidemiological interest for the country.     

Isolation and characterization of two novel scorpion toxins: The α-toxin-like CeII8, specific for Nav1.7 channels and the classical anti-mammalian CeII9, specific for Nav1.4 channels

Scorpion β-toxins represent a particular pharmacological group of voltage-gated sodium channel (VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. Here, we report the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Thorell, 1876), which is responsible for deadly cases of intoxication in Mexico. The soluble venom was fractionated by gel filtration and ion-exchange chromatography, followed by reversed-phase HPLC. The toxins CeII8 and CeII9 were further purified and both their amino acid sequence and molecular weight were determined. Both toxins were electrophysiologically characterized on four mammalian VGSCs (rNa_v1.2, rNa_v1.4, hNa_v1.5 and rNa_v1.7) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Although CeII8 has the highest sequence similarity with scorpion α-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear β-toxin effect and was selective towards Na_v1.7, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first β-toxin active on Na_v1.7. CeII9, a typical anti-mammalian β-toxin, selectively modulated Na_v1.4 at a concentration of 700 nM and was, in contrast to CeII8, found to be lethal to mice. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the β-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.     

Negative-shift activation, current reduction and resurgent currents induced by β-toxins from Centruroides scorpions in sodium channels

The β-toxins purified from the New World scorpion venoms of the Centruroides species affect several voltage-gated sodium channels (VGSCs) and thus are essential tools not only for the discrimination of different channel sub-types but also for studying the structure-function relationship between channels and toxins. This communication reports the results obtained with four different peptides purified from three species of Centruroides scorpions and assayed on seven distinct isoforms of VGSC (Na_v1.1-Na_v1.7) by specific functional analysis conducted through single cell electrophysiology. The toxins studied were CssII from Centruroides suffusus suffusus, Cll1 and Cll2 from Centruroides limpidus limpidus and a novel toxin from Centruroides noxius, which was characterized for the first time here. It has 67 amino acid residues and four disulfide bridges with a molecular mass of 7626 Da. Three different functional features were identified: current reduction of macroscopic conductance, left shift of the voltage-dependent activation and induction of resurgent currents at negative voltages following brief, strong depolarizations. The isoforms which revealed to be more affected resulted to be Na_v1.6 > 1.1 > 1.2 and, for the first time, a β-toxin is here shown to induce resurgent current also in isoforms different from Na_v1.6. Additionally, these results were analyzed with molecular modelling. In conclusion, although the four toxins have a high degree of identity, they display tri-modal function, each of which shows selectivity among the different sub-types of Na⁺-channels. Thus, they are invaluable as tools for structure-function studies of β-toxins and offer a basis for the design of novel ion channel-specific drugs.     

Electrophysiological Characterization of Ts6 and Ts7, K+ Channel Toxins Isolated through an Improved Tityus serrulatus Venom Purification Procedure

In Brazil, Tityus serrulatus (Ts) is the species responsible for most of the scorpion related accidents. Among the Ts toxins, the neurotoxins with action on potassium channels (α-KTx) present high interest, due to their effect in the envenoming process and the ion channel specificity they display. The α-KTx toxins family is the most relevant because its toxins can be used as therapeutic tools for specific target cells. The improved isolation method provided toxins with high resolution, obtaining pure Ts6 and Ts7 in two chromatographic steps. The effects of Ts6 and Ts7 toxins were evaluated in 14 different types of potassium channels using the voltage-clamp technique with two-microelectrodes. Ts6 toxin shows high affinity for Kv1.2, Kv1.3 and Shaker IR, blocking these channels in low concentrations. Moreover, Ts6 blocks the Kv1.3 channel in picomolar concentrations with an IC₅₀ of 0.55 nM and therefore could be of valuable assistance to further designing immunosuppressive therapeutics. Ts7 toxin blocks multiple subtypes channels, showing low selectivity among the channels analyzed. This work also stands out in its attempt to elucidate the residues important for interacting with each channel and, in the near future, to model a desired drug.     

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.     

The Brazilian scorpion Tityus costatus Karsch: genes, peptides and function.

The venom of the scorpion Tityus costatus contains peptides toxic to humans but scarce information on their structure and function is available. Here, we report the separation of 50 different components by high performance liquid chromatography and the identification of approximately 90 distinct components by mass spectrometry analysis, with molecular weights varying from 413 to 45482 atomic mass units. Four peptides were fully sequenced: (i) a butantoxin-like peptide that blocks Shaker K+ channel; (ii) an insect toxin-like peptide; (iii) a scorpine-like peptide, and a short heptapeptide of unknown function. Fifteen peptides were directly sequenced at the N-terminal region, among which are components toxic to mice. A cDNA library was constructed and 13 clones were isolated and sequenced. Some of these peptides and genes are similar to other known scorpion toxins. Based on these results, stings by scorpions of the species Tityus costatus should be taken with caution by medical doctors.