Mexican Scorpion Research Articles

Anuroctoxin, a New Scorpion Toxin of the α-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.

Phaiodotoxin, a novel structural class of insect‐toxin isolated from the venom of the Mexican scorpion Anuroctonus phaiodactylus

A peptide called phaiodotoxin was isolated from the venom of the scorpion Anuroctonus phaiodactylus. It is lethal to crickets, but non toxic to mice at the doses assayed. It has 72 amino acid residues, with a molecular mass of 7971 atomic mass units. Its covalent structure was determined by Edman degradation and mass spectrometry; it contains four disulfide-bridges, of which one of the pairs is formed between cysteine-7 and cysteine-8 (positions Cys63-Cys71). The other three pairs are formed between Cys13-Cys38, Cys23-Cys50 and Cys27-Cys52. Comparative sequence analysis shows that phaiodotoxin belongs to the long-chain subfamily of scorpion peptides. Several genes coding for this peptide and similar ones were cloned by PCR, using cDNA prepared from the RNA of venomous glands of this scorpion. Electrophysiological assays conducted with this toxin in several mammalian cell lines (TE671, COS7, rat GH3 and cerebellum granular cells), showed no effect on Na+ currents. However, it shifts the voltage dependence of activation and inactivation of insect Na+ channels (para/tipE) to more negative and positive potentials, respectively. Therefore, the 'window' current is increased by 225%, which is thought to be the cause of its toxicity toward insects. Phaiodotoxin is the first toxic peptide ever purified from a scorpion of the family Iuridae.

NMR solution structure of Cn12, a novel peptide from the Mexican scorpion Centruroides noxius with a typical beta-toxin sequence but with alpha-like physiological activity.

Cn12 isolated from the venom of the scorpion Centruroides noxius has 67 amino-acid residues, closely packed with four disulfide bridges. Its primary structure and disulfide bridges were determined. Cn12 is not lethal to mammals and arthropods in vivo at doses up to 100 microg per animal. Its 3D structure was determined by proton NMR using 850 distance constraints, 36 phi angles derived from 36 coupling constants obtained by two different methods, and 22 hydrogen bonds. The overall structure has a two and half turn alpha-helix (residues 24-32), three strands of antiparallel beta-sheet (residues 2-4, 37-40 and 45-48), and a type II turn (residues 41-44). The amino-acid sequence of Cn12 resembles the beta scorpion toxin class, although patch-clamp experiments showed the induction of supplementary slow inactivation of Na(+) channels in F-11 cells (mouse neuroblastoma N18TG-2 x rat DRG2), which means that it behaves more like an alpha scorpion toxin. This behaviour prompted us to analyse Na(+) channel binding sites using information from 112 Na(+) channel gene clones available in the literature, focusing on the extracytoplasmic loops of the S5-S6 transmembrane segments of domain I and the S3-S4 segments of domain IV, sites considered to be responsible for binding alpha scorpion toxins.

Antimicrobial peptide induction in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus in response to septic injury.

Antimicrobial peptides (AMPs) are essential components of host defences against infectious microrganisms. In chelicerate organisms they have been implicated in three alternative defensive systems: one is defined by the immediate up-regulation of genes encoding AMPs, another is characterized by the inducible systemic release of AMPs from cellular reservoirs and the third alternative is the systemic constitutive production of AMPs. In this work we used a differential high-performance liquid chromatography and mass spectrometry approach to show that septic injury elicits an immune response in the haemolymph of the Mexican scorpion Centruroides limpidus limpidus. We isolated several haemolymph components, one of which was characterized extensively (amino acid sequence, disulphide pairing, cDNA and genomic clones) and demonstrated to be a novel member of the invertebrate defensin family and consequently named C. limpidus limpidus defensin-like peptide (Cll-dlp). This peptide accumulates in the haemolymph in response to septic injury, independently of transcriptional regulation.

Cobatoxin 1 from Centruroides noxius scorpion venom: chemical synthesis, three-dimensional structure in solution, pharmacology and docking on K+ channels

CoTX1 (cobatoxin 1) is a 32-residue toxin with three disulphide bridges that has been isolated from the venom of the Mexican scorpion Centruroides noxius Hoffmann. Here we report the chemical synthesis, disulphide bridge organization, 3-D (three-dimensional) solution structure determination, pharmacology on K+ channel subtypes (voltage-gated and Ca2+-activated) and docking-simulation experiments. An enzyme-based cleavage of the synthetic folded/oxidized CoTX1 indicated half-cystine pairs between Cys3-Cys22, Cys8-Cys27 and Cys12-Cys29. The 3-D structure of CoTX1 (solved by 1H-NMR) showed that it folds according to the common alpha/beta scaffold of scorpion toxins. In vivo, CoTX1 was lethal after intracerebroventricular injection to mice (LD50 value of 0.5 microg/mouse). In vitro, CoTX1 tested on cells expressing various voltage-gated or Ca2+-activated (IKCa1) K+ channels showed potent inhibition of currents from rat K(v)1.2 ( K(d) value of 27 nM). CoTX1 also weakly competed with 125I-labelled apamin for binding to SKCa channels (small-conductance Ca2+-activated K+ channels) on rat brain synaptosomes (IC50 value of 7.2 microM). The 3-D structure of CoTX1 was used in docking experiments which suggests a key role of Arg6 or Lys10, Arg14, Arg18, Lys21 (dyad), Ile23, Asn24, Lys28 and Tyr30 (dyad) residues of CoTX1 in its interaction with the rat K(v)1.2 channel. In addition, a [Pro7,Gln9]-CoTX1 analogue (ACoTX1) was synthesized. The two residue replacements were selected aiming to restore the RPCQ motif in order to increase peptide affinity towards SKCa channels, and to alter the CoTX1 dipole moment such that it is expected to decrease peptide activity on K(v) channels. Unexpectedly, ACoTX1 exhibited an activity similar to that of CoTX1 towards SKCa channels, while it was markedly more potent on IKCa1 and several voltage-gated K+ channels.

A short-chain peptide toxin isolated from Centruroides sculpturatus scorpion venom inhibits ether-à-go-go-related gene K+ channels

From the venom of the American scorpion Centruroides sculpturatus Ewing we have isolated a minute peptide fraction (named CsEKerg1) which reversibly inhibits the current through ERG (ether-à-go-go-related gene) K+ channels. Isolation was done by CM-cellulose column chromatography and reversed phase high-performance liquid chromatography. To test for an effect on ERG channels we used NG108-15 neuroblastoma×glioma hybrid cells voltage-clamped in the whole-cell mode. CsEKerg1 contains 43 amino acids and has a molecular weight of 4833. Its amino acid sequence is similar but not identical to that of ergtoxin, a peptide isolated recently from the venom of the Mexican scorpion Centruroides noxius [FASEB J. 13 (1999) 953]. Half inhibition of ERG current occurs at a peptide concentration of 1.12μg/ml.

Cn11, the first example of a scorpion toxin that is a true blocker of Na(+) currents in crayfish neurons.

A novel crustacean toxin (Cn11) was isolated and characterized from the venom of the Mexican scorpion Centruroides noxius Hoffmann. It contains 63 amino acid residues and is stabilized by four disulphide bridges. It is lethal to crustaceans (Cambarellus montezumae), less toxic to insects (crickets) and non-toxic to mammals (mice) at the doses assayed. In neurons isolated from the X organ-sinus gland system of the crayfish Procambarus clarkii, it blocks the Na(+) currents with an estimated K(m) of 320 nmol l(-1), without affecting the Ca(2+) and K(+) currents. The voltage-gated tetrodotoxin-sensitive Na(+) current was recorded from X organ neurons in culture 24 h after plating using the whole-cell clamp configuration. The Na(+) current was isolated by blocking Ca(2+) currents with Cd(2+) and Cs(+) and K(+) currents with tetraethylammonium and 4-aminopyridine. Under control conditions, the Na(+) currents were activated at -40 mV with a maximum amplitude at 0 mV. In the presence of 1 micromol l(-1) Cn11, the Na(+) current amplitude was reduced by 75 % without apparent modifications to the gating mechanism. These findings suggest that Cn11 selectively blocks a Na(+) channel. It is the first representative of a new group of scorpion toxins specific for this molecular target.

Mapping of an epitope recognized by a neutralizing monoclonal antibody specific to toxin Cn2 from the scorpion Centruroides noxius, using discontinuous synthetic peptides.

The Na+-channel-affecting toxin Cn2 represents the major and one of the most toxic components of the venom of the Mexican scorpion Centruroides noxius Hoffmann. A monoclonal antibody BCF2 raised against Cn2 has been shown previously to be able to neutralize the toxic effect of Cn2 and of the whole venom of C. noxius. In the present study the epitope was mapped to a surface region comprising the N- and C-terminal segments of Cn2, using continuous and discontinuous synthetic peptides, designed on the basis of the sequence and a three-dimensional model of Cn2. The study of peptides of varying length resulted in the identification of segments 5-14 and 56-65 containing residues essential for recognition by BCF2. The peptide (abbreviated SP7) with the highest affinity to BCF2 (IC50 = 5.1 microM) was a synthetic heterodimer comprising the amino acid sequence from position 3-15 (amidated) of Cn2, bridged by disulfide to peptide from position 54-66, acetylated and amidated. Similar affinity was found with peptide SP1 [heterodimer comprising residues 1-14 (amidated) of Cn2, bridged with synthetic peptide 52-66 (acetylated)]. SP1 and SP7 were used to induce anti-peptide antibodies in mouse and rabbit. Both peptides were highly immunogenic. The sera obtained were able to recognize Cn2 and to neutralize Cn2 in vitro. The most efficient protection (8.3 microgram Cn2 neutralized per mL of serum) was induced by rabbit anti-SP1 serum.

Cobatoxins 1 and 2 from Centruroides noxius Hoffmann constitute a subfamily of potassium-channel-blocking scorpion toxins.

Potassium-channel-blocking scorpion toxins (alpha-K-toxins) have been shown to be valuable tools for the study of potassium channels. Here we report two toxins, cobatoxin 1 and 2, of 32 amino acids, containing three disulphide bridges, that were isolated from the venom of the Mexican scorpion Centruroides noxius. Their primary sequences show less than 40% identity to other alpha-K-toxins. It is therefore proposed that they belong to subfamily 9. The cDNA of cobatoxin 1 encodes a putative signal peptide, a putative short propeptide, the mature peptide and two amino acids that are processed to leave cobatoxin 1 amidated at the C-terminus. In rat brain synaptosomal membranes cobatoxin 1 and cobatoxin 2 bind to a common binding site of alpha-K-toxins with Ki values of 109 pM and 87 pM, respectively. Moreover, they block the Shaker and Kv1.1 K+ channels with moderate affinities, with Kd values of around 0.7 microM and 4.1 microM (Shaker) and 0.5 microM and 1.0 microM (Kv1.1), respectively. A three-dimensional model of cobatoxin 1 was generated and used to interpret the obtained functional data on a structural basis.

Cloning and characterization of the cDNAs encoding Na + channel-specific toxins 1 and 2 of the scorpion Centruroides noxius Hoffmann

Using a cDNA library prepared from venomous glands of the Mexican scorpion Centruroides noxius Hoffmann the genes that encode toxins 1 and 2 were identified, cloned and sequenced. In view of the proposed mechanism for processing the mature peptides coded by these two genes, the corresponding peptide-toxins were sequenced de novo. Mass spectrometric and 1H-NMR analyses of the C-terminal peptide produced by enzymatic digestion of both toxins indicated that the last residue is serine-amide. Sequence comparison revealed that these two genes have a similarity of 56% and 80% at the amino acid and nucleotide levels, respectively. Small corrections to the published primary structures were introduced: Cn toxin 1 has an extra serine residue at position 65 and the residue in position 60 is a proline, while the amino acids at positions 34 and 35 of Cn 2 are, respectively, tyrosine and glycine. Sequence comparison of toxins from the genus Centruroides suggests the presence of at least three classes of distinct peptides in these venoms.

Characterization of a voltage-dependent potassium channel in squid Schwann cells reconstituted in planar lipid bilayers.

An affinity column prepared with noxiustoxin (NTx), a K+ channel blocker from the venom of the Mexican scorpion Centruroides noxius, was used to purify a functional channel from a detergent extract of Schwann cell membrane of the giant axon of the squid Loligo vulgaris. The purified protein was reconstituted as a functional unit in a planar lipid bilayer and tested with a sequence of potentials to obtain information about single-channel amplitude and kinetics. The reconstituted channel showed delayed rectifier behavior with a slope conductance of 10 pS under 5:1 asymmetric KCl concentrations and a clear tendency to open under negative potentials. The zero-current potential was +36mV, which fitted well with the Nernst equation for the CIS/TRANS K(+)-concentration ratio of 5:1. The channel also showed a strong sensitivity to tetraethylammonium and its activity was inhibited by NTx, as expected from the purification procedure. The behavior of this protein in the presence of 0.5 mM ATP (cis side) was also tested, significantly increasing current fluctuations across the membrane. In order to compare the modulation of the Schwann cell K+ channel with that of the axonal K+ channel, a purified protein from the squid axon membrane was also tested in the presence of ATP. This 10-11 pS, delayed rectifier channel from the squid giant axon (Prestipino et al., FEBS Lett. 250:570-574, 1989) was also tested in the presence of ATP and showed a similar rise in activity.

Monoclonal antibodies against noxiustoxin.

Noxiustoxin, a 39-amino acid residue peptide isolated from the venom of the Mexican scorpion Centruroides noxius, has previously been shown to affect voltage-dependent K+ channels. Here we describe the isolation and characterization of monoclonal antibodies (MAbs) against this toxin and their use in structure-function relationship studies. Six hybridoma clones (BNTX4, -12, -14, -16, -18, and -21) producing MAbs against noxiustoxin were isolated. The epitopes defined by the MAbs are overlapping or in close proximity because no MAb pair could bind simultaneously to the toxin. All the MAbs inhibited to various degrees the binding of the toxin to its receptor sites on rat brain synaptosomal membranes. The venom from other Centruroides species was shown to contain components cross-reacting with the MAbs, suggesting the existence of other NTX-like toxins.

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 K+-channel-blocking toxins from the venom of the scorpion Centruroides limpidus limpidus Karsch

Two novel toxins were purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, using an immunoassay based on antibodies raised against noxiustoxin (NTX), a known K(+)-channel-blocker-peptide. The primary structure of C. l. limpidus toxin 1 was obtained by Edman degradation and was shown to be composed of 38 amino acid residues, containing six half-cystines. The first 36 residues of C. l. limpidus toxin 2 were also determined. Both toxins are capable of displacing the binding of radio-labelled NTX to rat brain synaptosomes with high affinity (about 100 pM). These toxins are capable of inhibiting transient K(+)-currents (resembling IA-type currents), in cultured rat cerebellar granule cells. About 50% of the peak currents are reduced by application of a 1.5 microM solution of toxins 1 and 2 The K+ current reduction is partially reversible, under washing but not voltage-dependent. Comparison of the primary structure of C. l. limpidus toxin 1 with other known toxins shows 74% identity with margatoxin, 64% with NTX, 51% with kaliotoxin, 39% with iberiotoxin, 37% with charybdotoxin and Lq2, and 29% with leirutoxin 1. The only invariant amino acids in all these toxins are the six cysteines, a glycine in position 26 and two lysines at positions 28 and 33, respectively. The relevance of these differences in terms of possible structure-function relationships is discussed.

Isolation and characterization of a novel toxin from the venom of the scorpion Centruroides limpidus limpidus Karsch

A novel peptide, toxic to mice, was purified from the venom of the Mexican scorpion Centruroides limpidus limpidus, by means of gel filtration and ion exchange chromatography, followed by high performance liquid chromatography (HPLC). The complete amino acid sequence was determined by automatic Edman degradation of reduced and alkylated toxin, and by overlapping sequences of fragments of the toxin, generated by cleavage with proteinase V8 separated by HPLC. This toxin is composed of 66 amino acid residues, contains eight half-cystine residues, and is highly similar (91%) to the amino acid sequence deduced for toxin 1 of C. limpidus tecomanus and toxin 4 from C. noxius venom (89%). This peptide displaces the binding of radiolabeled toxin 2 of C. noxius from synaptosomal membranes of rat brain with superimposable kinetics, supporting the conclusion that it belongs to the β-scorpion toxin class. Further characterization of C. l. limpidus toxin 1, as we have named it, was performed by means of competition experiments with monoclonal antibodies and various purified scorpion toxins, using an ELISA assay. A panel of six distinct monoclonal antibodies (mAB) against toxin 2 and 3 of C. noxius was used. From these, only three clones, originally named BCF1, BCF8 and BCF9, were able to recognize toxin 1 from C. l. limpidus.

Cloning and characterization of cDNAs that code for Na + -channel-blocking toxins of the scorpion Centruroides noxius Hoffmann

With the purpose of studying the organization and characteristics of the genes that code for toxins present in the venom of the Mexican scorpion Centruroides noxius Hoffmann ( CnH) we prepared a λgt11 cDNA library from the venom glands. Using specific oligodeoxyribonucleotides (oligos) designed according to known amino acid (aa) sequences of CnH toxins (STox) we detected several positive clones determined their nucleotide (nt) sequences and deduced their aa sequences. A comparative analysis of these sequences with previously reported STox revealed that CnH cDNAs code for a family of very similar STox. The cDNA coding for a known STox II-10 was cloned. Additionally three other complete (new) nt sequences were obtained for cDNAs encoding peptides similar to STox 1 from CnH or variants 2 and 3 from Centruroides sculpturatus Ewing. Southern blot genomic DNA analysis showed a minimum size of approximately 600 bp as EcoRI fragments for elements of this family. PCR amplifications of CnH genomic DNA and hybridization of PCR products with specific probes indicated that the genomic structural regions that code for these genes do not contain introns or at least not large introns.

Dendrotoxin-like effects of noxiustoxin

Noxiustoxin from the Mexican scorpion (Centruroides noxius Hoffmann) is known to block neuronal K+ channels. Noxiustoxin facilitated acetylcholine release in chick biventer cervicis nerve-muscle preparations, but not in mouse phrenic nerve-hemidiaphragm preparations. Noxiustoxin displaced binding of a radiolabelled dendrotoxin from synaptosomal membranes from rat brain, with a Ki of 10−10M. It is concluded that noxiustoxin shares some pharmacological properties with the K+ channel blocking dendrotoxins.

Binding affinities of Mexican scorpion toxins to brain synaptosomal membranes: A. N. Ramirez, G. B. Gurrola, H. H. F. Valdivia and L. D. Possani (Department of Biochemistry, CEINGEBI—National University of México, Apto. Postal 510–3, Cuernavaca, 62271, México).

Binding affinities of Mexican scorpion toxins to brain synaptosomal membranes: A. N. Ramirez, G. B. Gurrola, H. H. F. Valdivia and L. D. Possani (Department of Biochemistry, CEINGEBI—National University of México, Apto. Postal 510–3, Cuernavaca, 62271, México).

Systematics of Vaejovis dugesi Pocock, with Descriptions of Two New Related Species (Scorpiones, Vaejovidae)

Vaejovis dugesi Pocock, previously recognized as a subspecies of Vaejovis mexicanus Koch, is redescribed and elevated to specific status. Two new species are described: Vaejovis chisos, from the Chisos Mountains in Big Bend National Park, Texas, and Vaejovis sprousei, from the Mexican states of Tamaulipas and Nuevo Le6n. RESUMEN--Vaejovis dugesi Pocock, reconocido previamente como una subespecie de Vaejovis mexicanus Koch, es redescrito y elevado a estado especifico. Se describen dos nuevas especies: Vaejovis chisos, de Chisos Mountains en el Parque National Big Bend, Texas, y Vaejovis sprousei, de los estados mexicanos de Tamaulipas y Nuevo Le6n. The scorpion Vaejovis mexicanus dugesi Pocock was described from the mountains of Guanajuato (Pocock, 1902), and the type remains the single known specimen referable to this taxon. Vaejovis m. dugesi has rarely been mentioned in subsequent systematic works, and its identity has remained enigmatic. Hoffmann (1931) listed the taxon but indicated he had not seen any material referable to it. He suggested that it was similar to Vaejovis granulatus, especially in color pattern, but did not propose a synonymy. Diaz Naijera (1975) did not mention V. m. dugesi in a revised list of Mexican scorpions. I had the opportunity to study the type of V. m. dugesi. It is clear that its closest relative is not V. mexicanus and that it should be regarded as a valid species. Examination of considerable vaejovid material collected throughout mainland Mexico has uncovered only one additional specimen of this species, but new related species from northeastern M6exico and the Chisos Mountains in the Big Bend area in Texas have been found. These species have obvious affinities with certain members of the V. mexicanus group. They have dusky markings on the dorsum (faint in the species from Big Bend), are similar in metasomal carinal structure, have weak dorsointernal carinae on the pedipalp chelae, and have trichobothrium et of the chela fixed finger situated very close to the third enlarged primary row denticle. They differ from other species in the V. mexicanus group as follows: (1) the pedipalps are relatively long and slender, with chela length: width ratios exc eding 4.0, (2) the chela fingers end in moderately to distinctly enlarged, clawlike terminal denticles. These latter features are suggestive of pecies of the Vaejovis nitidulus group. However, e species of that group have trichobothrium et situated at the level of or distal to the second enlarged primary row denticle and a well developed, crenulate to serrate dorsointernal chela carina. Because Vaejovis dugesi and the new species lack these characters, it seems best to retain them in the V. mexicanus group. Vaejovis dugesi Pocock, new combination

Preparation of a polyvalent antivenom against various Mexican scorpion Centruroides species

G. G. Y Perez, M. F. Martin and H. Rochat. Preparation of a polyvalent antivenom against various Mexican scorpion Centruroides species. Toxicon 26, 1102–1106, 1988.—Antisera were obtained from rabbits injected with four different immunogens from the Mexican scorpion Centruroides suffusus suffusus i.e. the crude venom, a telson extract, a toxic fraction obtained from this telson extract by gel filtration and the same toxic fraction subjected to acetylation. The neutralizing capacity of these antisera are compared: it appears that a telson extract can be used instead of the crude venom to produce an efficient antiserum. The immunological properties of ground telsons obtained from three other species of the Mexican scorpion Centruroides (Centruroides noxius, Centruroides limpidus limpidus, Centruroides limpidus tecomanus) are studied with the antisera raised against Centruroides suffusus suffusus immunogens: an almost total cross-neutralization is observed.