A simplified procedure for the fractionation of Tityus serrulatus venom: isolation and partial characterization of TsTX-IV, a new neurotoxin

Five toxins from the venom of the Brazilian scorpion Tityus serrulatus were purified to homogeneity by a combination of ion exchange chromatography with ammonium bicarbonate buffer (pH 7.8) on CM-cellulose-52 and rechromatography on the same resin equilibrated with ammonium acetate buffer (pH 4.7). Four of these proteins, obtained in one or two steps in high yield and lethality (named toxins IX3, IX5, and X4 and XIII) were shown to be identical with other toxins already described. A fifth one, TsTX-IV, is reported as a new toxin. Except for IX3, which showed Gly as the sole N-terminal residue, the other four toxins showed Lys. TsTX-IV has an approximate mol. wt of 6880, an i.v. LD50, in mice, of 826 +/- 156 micrograms/kg and an intracisternal LD50 of 11 +/- 9 micrograms/kg, compared to 375 +/- 45 and 4.9 +/- 0.8, respectively, for the whole venom extract. It has 61 amino acid residues and an amino acid composition different from that of any other toxin from Tityus serrulatus venom so far described. Toxins IX5, TsTX-IV and XIII induced a prejunctional type of supersensitivity on the guinea pig vas deferens, probably due to an increased release of noradrenaline.     

Disulfide bridges and blockage of Shaker B K(+)-channels by another butantoxin peptide purified from the Argentinean scorpion Tityus trivittatus.

A peptide was isolated from the venom of the scorpion Tityus trivittatus. It is an isoform of the toxin TsTX-IV earlier described [Toxicon 37 (1999) 651] and identical to butantoxin [Arch. Biochem. Biophys. 379 (2000) 18], both isolated from the Brazilian scorpion Tityus serrulatus. This newly characterized peptide contains 40 amino acid residues with a molecular mass of [M+H(+)] 4507.0, cross-linked by four disulfide bridges, made between the cysteine pairs: Cys2-Cys5, Cys10-Cys31, Cys16-Cys36 and Cys20-Cys38. It blocks in a completely reversible manner the Shaker B K(+)-channels, with a K(d) around 660nM. It belongs to the sub-family 12 and it is now being classified as alpha-KTx 12.2.     

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

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

Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): the first member of a new κ-KTX subfamily

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.     

Isolation of a toxin from Centruroides infamatus infamatus Koch scorpion venom that modifies Na permeability on chick dorsal root ganglion cells

A novel toxin was isolated and characterized from the venom of the Mexican scorpion Centruroides infamatus infamatus. It has an apparent mol. wt of 7600, compatible with the presence of 66 amino acid residues per molecule. The N-terminal amino acid sequence was determined (up to residue 48) and showed approximately 95% similarity with toxins from other Mexican scorpions of the genus Centruroides. Experiments conducted with chick dorsal root ganglion cells showed that toxin 1 is a Na⁺ channel effector, causing a decrease in the peak Na⁺ permeability, similar to decreases observed for typical β-scorpion toxins.     

Novel alpha-KTx peptides from the venom of the scorpion Centruroides elegans selectively blockade Kv1.3 over IKCa1 K+ channels of T cells.

From the venom of the Mexican scorpion Centruroides elegans Thorell five peptides were isolated to homogeneity by chromatographic procedures and their full amino acid sequence was determined by automatic Edman degradation. They all belong to the Noxiustoxin subfamily of scorpion toxins and were given the systematic names alpha-KTx 2.8 to 2.12, with trivial names Ce1 to Ce5, respectively. They have 39 amino acid residues, except for Ce3 which has only 38, but all of them have three disulfide bridges, and have molecular weights of 4255, 4267, 4249, 4295 and 4255 atomic mass units, respectively for Ce1 to Ce5. The C-terminal residues of Ce2, Ce4 and Ce5 were found to be amidated. The electrophysiological assay (whole-cell patch-clamp) showed that out of the five peptides, Ce1 (alpha-KTx 2.8), Ce2 (alpha-KTX2.9) and Ce4 (alpha-KTx 2.11) were effective blockers of Kv1.3 channels of human T lymphocytes, whereas these peptides did not inhibit the Ca2+-activated K+ channels (IKCa1) of the same cells. The equilibrium dissociation constants of these peptides for Kv1.3 were 0.70, 0.25 and 0.98nM for Ce1, Ce2 and Ce4, respectively. Furthermore, toxins Ce1, Ce2 and Ce4 practically did not inhibit the related voltage gated Shaker K+ channels, and rKv2.1 channels of the Shab family. The high affinity blockage of Kv1.3 channels by these peptides and their selectivity for Kv1.3 over IKCa1 may have significance in the development of novel tools for suppressing the function of those T cell subsets whose proliferation critically depends on the activity of Kv1.3 channels.     

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.     

Antigenic polymorphism of the "short" scorpion toxins able to block K+ channels.

BmTX3 is a toxin recently characterised from the venom of the Chinese scorpion Buthus martensi Karch, which specifically blocks a transient A-type K+ current in striatum neurons in culture and binds to rat brain synaptosomes with high affinity. With Aa1 and AmmTX3, it belongs to the new alpha-KTx15 subfamily from "short-chain" scorpion toxins, which specifically block different types of K+ channels. Here, a highly specific polyclonal antiserum was raised in rabbit against a C-terminal deleted BmTX3 analogue (BmTX-del YP). Using liquid-phase radioimmunoassay, we have studied its selectivity for the toxins from the alpha-KTx15 subfamily. We have also demonstrated that no/or poor cross-reactivity was observed with a panel of "short-chain" scorpion toxins representative of other structurally different subfamilies. These results suggest that a wide antigenic polymorphism, similar to that previously observed for "long-chain" scorpion toxins acting as modulators of voltage-activated Na+ channels, is also the rule for the "short-chain" scorpion toxins able to block K+ channels.     

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.     

Anuroctoxin, a new scorpion toxin of the alpha-KTx 6 subfamily, is highly selective for Kv1.3 over IKCa1 ion channels of human T lymphocytes

The physiological function of T lymphocytes can be modulated selectively by peptide toxins acting on Kv1.3 K(+) channels. Because Kv1.3-specific peptide toxins are considered to have a significant therapeutic potential in the treatment of autoimmune diseases, the discovery of new toxins is highly motivated. Through chromatographic procedures and electrophysiological assays, using patch-clamp methodology, the isolation of a novel peptide named anuroctoxin was accomplished using the venom of the Mexican scorpion Anuroctonus phaiodactylus. It has 35 amino acid residues with a molecular weight of 4082.8, tightly bound by four disulfide bridges whose complete covalent structure was determined. It has a pyroglutamic acid at the N-terminal region and an amidated C-terminal residue. Sequence comparison and phylogenetic clustering analysis classifies anuroctoxin into subfamily 6 of the alpha-KTx scorpion toxins (systematic name, alpha-KTx 6.12). Patch-clamp experiments show that anuroctoxin is a high-affinity blocker of Kv1.3 channels of human T lymphocytes with a K(d) of 0.73 nM, and it does not block the Ca(2+)-activated IKCa1 K(+) channels. These two channels play different but important roles in T-lymphocyte activation. Furthermore, the toxin practically does not inhibit Shaker IR, mKv1.1, and rKv2.1 channels, whereas the affinity of anuroctoxin for hKv1.2 is almost an order of magnitude smaller than for Kv1.3. The pharmacological profile and the selectivity of this new toxin for Kv1.3 over IKCa1 may provide an important tool for the modulation of the immune system, especially in cases in which selective inhibition of Kv1.3 is required.     

Amino acid sequence and physiological characterization of toxins from the venom of the scorpion Centruroides limpidus tecomanus Hoffmann

The complete amino acid sequence of the major toxic component (II.20.3.4), named toxin 1, from the venom of the Mexican scorpion C. l. tecomanus is reported. The sequence (66 amino acids) was obtained by direct Edman degradation of reduced and alkylated toxin, followed by sequence determination of selected peptides separated after enzymatic cleavage with S. aureus V8 protease. In cultured chick dorsal root ganglion cells, 0.5 microM toxin 1 slowed down specifically the time course of Na+ current inactivation, while Ca2+ currents from the same preparation were little affected. In neonatal rat ventricular heart cells, toxin 1, at concentrations between 0.1 and 0.5 microM, reduced Na+ currents without changing the kinetics and Ca2+ currents were unaffected. Comparative analysis of the primary structure of this toxin with other scorpion toxins shows a high degree of similarity with the north American scorpion toxins. This analysis suggests that the 'fine tuning' of the molecular mechanism of action of these toxins is related to variations in the primary structure as well as to the type of membrane under study (tissue specificity).     

Scorpion toxins from Tityus cambridgei that affect Na(+)-channels.

By means of high performance liquid chromatography (HPLC) the soluble venom of the Amazonian scorpion Tityus cambridgei was fractionated into over 50 different components. Four toxic and/or lethal peptides to mice were obtained in pure form and sequenced. Mass spectrometry analysis showed molecular weights of 7310, 7151, 7259 and 7405, respectively, for toxins Tc48a, Tc49a, Tc54 and Tc49b. The N-terminal amino acid sequence was obtained for the three first toxins mentioned, whereas the full primary structure was determined for Tc49b. It contains 64 amino acid residues, closely packed by four disulfide bridges. Sequence comparison analysis showed similarities around 50% with other toxins from scorpions of the genus Tityus of Brazil. It is lethal to mice at doses of 20 microg per 20 g mouse. The toxin was shown to affect the Na(+)-currents permeability of rat cerebellum granular cells in culture. Almost a complete elimination of current was observed with 100 nM toxin concentration. This effect was partially reversible. Furthermore, this toxin does not modify the function of the Shaker B K(+)-channels expressed on Sf9 cells, nor does it modify the Na(+)-channel function in a similar manner as those reported for the alpha-scorpion toxins purified from other scorpions.     

A novel conotoxin inhibiting vertebrate voltage-sensitive potassium channels.

Toxins from cone snail (Conus species) venoms are multiple disulfide bonded peptides. Based on their pharmacological target (ion channels, receptors) and their disulfide pattern, they have been classified into several toxin families and superfamilies. Here, we report a new conotoxin, which is the first member of a structurally new superfamily of Conus peptides and the first conotoxin affecting vertebrate K+ channels. The new toxin, designated conotoxin ViTx, has been isolated from the venom of Conus virgo and comprises a single chain of 35 amino acids cross-linked by four disulfide bridges. Its amino acid sequence (SRCFPPGIYCTSYLPCCWGICCSTCRNVCHLRIGK) was partially determined by Edman degradation and deduced from the nucleotide sequence of the toxin cDNA. Nucleic acid sequencing also revealed a prepropeptide comprising 67 amino acid residues and demonstrated a posttranslational modification of the protein by releasing a six-residue peptide from the C-terminal. Voltage clamp studies on various ion channels indicated that the toxin inhibits the vertebrate K+ channels Kv1.1 and Kv1.3 but not Kv1.2. The chemically synthesized product exhibited the same physiological activity and identical molecular mass (3933.7 Da) as the native toxin.     

Amino acid sequence and function of a new alpha-toxin from the Amazonian scorpion Tityus cambridgei.

A toxic peptide earlier denominated Tc48b [Toxicon 40 (2002) 557] was purified to homogeneity and its amino acid sequence determined. It has 64 amino acid residues stabilized by four disulfide bridges with a molecular weight of 7,385.2 atomic mass units (a.m.u.). It affects Na(+)-permeability in pituitary GH3 cells in culture, in a similar fashion as those reported for alpha-scorpion toxins, contrary to most of the New World scorpion toxins that are beta-toxins.     

Interacting sites of scorpion toxin ErgTx1 with hERG1 K+ channels

Peptides purified from scorpion venoms were shown to interact with specific amino acid residues present in the outer vestibule of various sub-types of potassium channels, occluding the pore and causing a decrement of K(+) permeability through the membrane of excitable and non excitable cells. This communication describes the identification of several interacting sites of toxin ErgTx1, a toxin purified from the venom of the scorpion Centruroides noxius, with the human ERG1 K(+) channels, by means of site-directed mutagenesis of specific residues of the toxin. Recombinant mutants of the gene coding for ErgTx1 were expressed heterologously in Escherichia coli, properly folded and their affinities and interactions with hERG1 channels were determined by patch-clamp techniques. Residues in position Y14, Y17 and F37 of the solvent exposed hydrophobic surface, and charged residues at the position K13 and K38 of ErgTx1 were shown to cause a decrement of the affinity from 20 folds to 3 orders of magnitude, thus suggesting that they are certainly participating on the binding surface of this toxin towards the hERG1 channels. Double mutants at positions K13 and F37, Y14 and F37, Y17 and F37 and K13 and K38 were also prepared and assayed, but the results obtained are not much different from the single point mutants of ErgTx1. The results of the present work indicate the most probable surface area of ErgTx1 that makes contact with the hERG channels.     

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.     

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

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

Neurotoxic Proteins in Scorpion Venom

Abstract

Scorpion venoms are complex mixtures of mucous, low molecular weight components (salts and organic compounds) and many basic, neurotoxic proteins (M_R < 8000). These neurotoxins alter the properties of Na channels in excitable cells in three ways. One group of toxins delays inactivation of the Na channel, thereby greatly prolonging the action potential. A second effect, produced by other scorpion toxins, is a transient shift in the voltage dependence of activation, which is demonstrated upon application of a depolarizing pulse. This effect, shown thus far only by toxins from New World species of scorpions, results in an increased tendency of the cell to fire spontaneously and repetitively. The third effect produced by some of the toxins is a reduction of ionic currents (both Na and K currents) with no changes in the kinetics of activation and inactivation. Many observations from pathophysiological studies with scorpion venoms and isolated toxins can be explained on the basis of their effects on Na channels.

The three-dimensional structure of one scorpion toxin (Toxin Var3) is known. Because of homology in the amino acid sequences of many different scorpion toxins, it is believed that the structure proposed for Toxin Var3 from Centruroides sculpturatus (Fig. 2 and 3) is typical of the scorpion toxins in general. Featured in this structure are a dense core of protein secondary structure and a surface hydrophobic patch. Many of the conserved amino acids in the scorpion toxins are associated with this patch thus suggesting that this part of the structure is associated with the biological action of the toxins.     

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.     

Covalent structures of BmK AS and BmK AS-1, two novel bioactive polypeptides purified from Chinese scorpion Buthus martensi Karsch.

Complete amino acid sequences of two novel bioactive polypeptides, each containing 66 amino acid residues, BmK AS and BmK AS-1 purified from the venom of Chinese scorpion Buthus martensi Karsch, have been determined by Edman sequencing and mass spectrometry on native proteins, reduced and S-carboxymethylated proteins and their peptides obtained after cleavage with proteolytic enzymes. Sequence analysis showed 86.4% structural identity between BmK AS and BmK AS-1 and also a high sequence similarity between BmK ASs and AaH IT4, a unique anti-insect toxin and a ligand of Na+ channels obtained from Sahara scorpion A. australis Hector, but poor sequence homology between BmK ASs and those of the known alpha-, beta-type and long-chain insect-selective type scorpion neurotoxins. The positions of four disulfide bridges in BmK AS-1 were established as Cys-12 and Cys-62, Cys-16 and Cys-37, Cys-23 and Cys-44, and Cys-27 and Cys-46, which are the same as those in alpha- and beta-scorpion neurotoxins. These results suggest that BmK ASs and AaH IT4 may form a new group sharing similar structural and functional properties in the family of scorpion neurotoxic polypeptides.