Structure-function relationships of peptides forming the calcin family of ryanodine receptor ligands.

Calcins are a novel family of scorpion peptides that bind with high affinity to ryanodine receptors (RyRs) and increase their activity by inducing subconductance states. Here, we provide a comprehensive analysis of the structure-function relationships of the eight calcins known to date, based on their primary sequence, three-dimensional modeling, and functional effects on skeletal RyRs (RyR1). Primary sequence alignment and evolutionary analysis show high similarity among all calcins (≥78.8% identity). Other common characteristics include an inhibitor cysteine knot (ICK) motif stabilized by three pairs of disulfide bridges and a dipole moment (DM) formed by positively charged residues clustering on one side of the molecule and neutral and negatively charged residues segregating on the opposite side. [(3)H]Ryanodine binding assays, used as an index of the open probability of RyRs, reveal that all eight calcins activate RyR1 dose-dependently with Kd values spanning approximately three orders of magnitude and in the following rank order: opicalcin1 > opicalcin2 > vejocalcin > hemicalcin > imperacalcin > hadrucalcin > maurocalcin >> urocalcin. All calcins significantly augment the bell-shaped [Ca(2+)]-[(3)H]ryanodine binding curve with variable effects on the affinity constants for Ca(2+) activation and inactivation. In single channel recordings, calcins induce the appearance of a subconductance state in RyR1 that has a unique fractional value (∼20% to ∼60% of the full conductance state) but bears no relationship to binding affinity, DM, or capacity to stimulate Ca(2+) release. Except for urocalcin, all calcins at 100 nM concentration stimulate Ca(2+) release and deplete Ca(2+) load from skeletal sarcoplasmic reticulum. The natural variation within the calcin family of peptides offers a diversified set of high-affinity ligands with the capacity to modulate RyRs with high dynamic range and potency.

Overview of the Knottin scorpion toxin-like peptides in scorpion venoms: Insights on their classification and evolution.

Scorpion venoms include several compounds with different pharmacological activities. Within these compounds, toxins affecting ion channels are among the most studied. They are all peptides that have been classified based on their 3D structure, chain size and function. Usually, they show a spatial arrangement characterized by the presence of a cysteine-stabilized alpha beta motif; most of them affect Na(+) and K(+) ion-channels. These features have been revised in several occasions before, but a complete phylogenetic analysis of the disulfide containing peptides is not been done. In the present contribution, two databases (Pfam and InterPro) including more than 800 toxins from different scorpions were analyzed. Pfam database included toxins from several organisms other than scorpions such as insects and plants, while InterPro included only scorpion toxins. Our results suggest that Na(+) toxins have evolved independently from those of K(+) toxins no matter the length of the peptidic chains. These preliminary results suggest that current classification needs a more detailed revision, in order to have better characterized toxin families, so the new peptides obtained from transcriptomic analyses would be properly classified.

Characterization of the first K⁺ channel blockers from the venom of the Moroccan scorpion Buthus occitanus Paris.

The availability of a large variety of specific blockers, which inhibit different K(+) currents, would help to elucidate their differences in physiological function. Short peptide toxins isolated from scorpion venoms are able to block voltage-dependent or Ca(2+)-activated K(+) channels. Here, we have studied the venom of the Moroccan scorpion Buthus occitanus Paris (BoP) in order to find new peptides, which could enlarge our structure-function relationship knowledge on the Kv1.3 blocker Kaliotoxin (KTX) that belongs to the α-KTx3.1 family. Indeed and since more a decade, KTX is widely used by international investigators because it exhibits a quite sharp specificity and a high-affinity for the Kv1.3 channel, which is not only a neuronal channel but also a therapeutic target for diverse autoimmune diseases such as multiple sclerosis, type 1 diabetes, and rheumatoid arthritis. The BoP venom was first investigated using HPLC and MALDI-TOF/MS. Further, the HPLC fractions were screened by ELISA with antibodies raised against KTX. These antibodies recognized at least three components toxic in mice by intracerebroventricular injection. They were further pharmacologically characterized by competition using (125)I-KTX bound to its specific binding sites on rat brain synaptosomes. A single component (4161 Da) inhibited totally the (125)I-KTX binding and with high-affinity (IC50 = 0.1 nM), while the two other components poorly competed with (IC50 > 100 nM). These toxins were sequenced in full by Edman's degradation. The high-affinity ligand (BoPKTX) shares 86% sequence identity with KTX and was classified as toxin α-KTx3.17. The two others peptides (BoP1 and BoP2, 4093 Da and 4121 Da, respectively) only differ by a Lys/Arg mutation. Their amino acid sequences were related to Martentoxin, which has been characterized from the Chinese scorpion Buthus martenzi Karch and described as both a BKCa and Kv1.3 blocker. Accordingly, they belong to the α-KTx16 family.

Profiling the resting venom gland of the scorpion Tityus stigmurus through a transcriptomic survey.

BACKGROUND: The scorpion Tityus stigmurus is widely distributed in Northeastern Brazil and known to cause severe human envenoming, inducing pain, hyposthesia, edema, erythema, paresthesia, headaches and vomiting. The present study uses a transcriptomic approach to characterize the gene expression profile from the non-stimulated venom gland of Tityus stigmurus scorpion. RESULTS: A cDNA library was constructed and 540 clones were sequenced and grouped into 153 clusters, with one or more ESTs (expressed sequence tags). Forty-one percent of ESTs belong to recognized toxin-coding sequences, with transcripts encoding antimicrobial toxins (AMP-like) being the most abundant, followed by alfa KTx- like, beta KTx-like, beta NaTx-like and alfa NaTx-like. Our analysis indicated that 34% of the transcripts encode "other possible venom molecules", which correspond to anionic peptides, hypothetical secreted peptides, metalloproteinases, cystein-rich peptides and lectins. Fifteen percent of ESTs are similar to cellular transcripts. Sequences without good matches corresponded to 11%. CONCLUSIONS: This investigation provides the first global view of gene expression of the venom gland from Tityus stigmurus under resting conditions. This approach enables characterization of a large number of venom gland component molecules, which belong either to known or non yet described types of venom peptides and proteins from the Buthidae family.

Accelerated evolution and functional divergence of scorpion short-chain K+ channel toxins after speciation.

The α-KTx14 subfamily of scorpion toxins is a group of short-chain polypeptides affecting K(+) channels, including five known members which are restrictedly distributed in Mesobuthus martensii. Here, we describe seven new α-KTx14 peptides from M. martensii and its sibling species Mesobuthus eupeus, two of which (termed MarKTX-3 and MeuKTX-1) were chemically synthesized and refolded for structural and functional studies. Electrophysiological recordings of effects of these two peptides on an array of voltage-gated potassium channels revealed that MarKTX-3 was capable of inhibiting five mammalian K(v)1 isoforms (rK(v)1.1-rK(v)1.5) and the Drosophila Shaker channel with low potency whereas MeuKTX-1 lacks such activity. Circular dichroism spectroscopy analysis combined with homology modeling demonstrates that MarKTX-3 and MeuKTX-1 both adopt a similar cysteine-stabilized α-helical and ß-sheet fold. Evolutionary analysis indicates accelerated amino acid substitutions in the mature-peptide-encoding regions of orthologous α-KTx14 peptides after speciation, thereby providing evidences for adaptive evolution and functional divergence of this subfamily.

Bioinformatic characterizations and prediction of K+ and Na+ ion channels effector toxins.

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.

Chinese-scorpion (Buthus martensi Karsch) toxin BmK alphaIV, a novel modulator of sodium channels: from genomic organization to functional analysis.

In the present study, BmK alphaIV, a novel modulator of sodium channels, was cloned from venomous glands of the Chinese scorpion (Buthus martensi Karsch) and expressed successfully in Escherichia coli. The BmK alphaIV gene is composed of two exons separated by a 503 bp intron. The mature polypeptide contains 66 amino acids. BmK alphaIV has potent toxicity in mice and cockroaches. Surface-plasmon-resonance analysis found that BmK alphaIV could bind to both rat cerebrocortical synaptosomes and cockroach neuronal membranes, and shared similar binding sites on sodium channels with classical AaH II (alpha-mammal neurotoxin from the scorpion Androctonus australis Hector), BmK AS (beta-like neurotoxin), BmK IT2 (the depressant insect-selective neurotoxin) and BmK abT (transitional neurotoxin), but not with BmK I (alpha-like neurotoxin). Two-electrode voltage clamp recordings on rNav1.2 channels expressed in Xenopus laevis oocytes revealed that BmK alphaIV increased the peak amplitude and prolonged the inactivation phase of Na+ currents. The structural and pharmacological properties compared with those of other scorpion alpha-toxins suggests that BmK alphaIV represents a novel subgroup or functional hybrid of alpha-toxins and might be an evolutionary intermediate neurotoxin for alpha-toxins.

CysView: protein classification based on cysteine pairing patterns.

CysView is a web-based application tool that identifies and classifies proteins according to their disulfide connectivity patterns. It accepts a dataset of annotated protein sequences in various formats and returns a graphical representation of cysteine pairing patterns. CysView displays cysteine patterns for those records in the data with disulfide annotations. It allows the viewing of records grouped by connectivity patterns. CysView's utility as an analysis tool was demonstrated by the rapid and correct classification of scorpion toxin entries from GenPept on the basis of their disulfide pairing patterns. It has proved useful for rapid detection of irrelevant and partial records, or those with incomplete annotations. CysView can be used to support distant homology between proteins. CysView is publicly available at http://research.i2r.a-star.edu.sg/CysView/.

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.

Solution structure of BmBKTx1, a new BKCa1 channel blocker from the Chinese scorpion Buthus martensi Karsch.

BmBKTx1 is a 31-amino acid peptide identified from the venom of the Chinese scorpion Buthus martensi Karsch, blocking high-conductance calcium-activated potassium channels. Sequence homology analysis indicates that BmBKTx1 is a new subfamily of short-chain alpha-KTx toxins of the potassium channel, which we term alpha-KTx19. Synthetic BmBKTx1 was prepared by using solid-phase peptide synthesis. Two-dimensional NMR spectroscopy techniques were used to determine the solution structure of BmBKTx1. The results show that the BmBKTx1 forms a typical cysteine-stabilized alpha/beta scaffold adopted by most short-chain scorpion toxins. The structure of BmBKTx1 consists of a two-stranded antiparallel beta-sheet (residues 20-29) and an alpha-helix (residues 5-15). The three-dimensional structure of BmBKTx1 was also compared with those of two function-related scorpion toxins, charybdotoxin (ChTx) and BmTx1, and their structural and functional implications are discussed.

Tetrapandins, a new class of scorpion toxins that specifically inhibit store-operated calcium entry in human embryonic kidney-293 cells.

Venoms from 14 snakes and four scorpions were screened for inhibitory activities toward store-operated Ca2+ entry (SOCE) in human embryonic kidney-293 cells. An inhibitory activity was found in venom from the African scorpion Pandinus imperator. The active agent of this venom was purified by gel filtration and reverse-phase high pressure liquid chromatography methods. Sequence information on the purified fraction, by automatic Edman degradation and mass spectrometry analysis, identified the activity as being contained in two tetrapeptides, which we have named tetrapandins. We demonstrate that synthesized tetrapandins have inhibitory activity for SOCE in human embryonic kidney-293 cells while having no effect on either thapsigargin- or carbachol-stimulated release of Ca2+ stores. These toxins should be extremely useful in future studies to determine downstream events regulated by SOCE as well as to determine whether multiple pathways exist for thapsigargin-stimulated Ca2+ entry.

A large number of novel Ergtoxin-like genes and ERG K+-channels blocking peptides from scorpions of the genus Centruroides.

Twenty-three novel sequences similar to Ergtoxin (ErgTx) were obtained by direct sequencing of peptides or deduced from gene cloned using cDNAs of venomous glands of Centruroides (C.) elegans, C. exilicauda, C. gracilis, C. limpidus limpidus, C. noxius and C. sculpturatus. These peptides have from 42 to 47 amino acid residues cross-linked by four disulfide bridges. They share sequence similarities (60-98% compared with ErgTx1) and were shown to block ERG K(+)-channels of F-11 clone (N18TG-2xrat DRG) cultured cells. An unrooted phylogenetic tree analysis of these peptides showed that they conform at least five different subfamilies, of which three are novel subfamilies.

Characterization of scorpion alpha-like toxin group using two new toxins from the scorpion Leiurus quinquestriatus hebraeus.

Two novel toxins, Lqh6 and Lqh7, isolated from the venom of the scorpion Leiurus quinquestriatus hebraeus, have in their sequence a molecular signature (8Q/KPE10) associated with a recently defined group of alpha-toxins that target Na channels, namely the alpha-like toxins [reviewed in Gordon, D., Savarin, P., Gurevitz, M. & Zinn-Justin, S. (1998) J. Toxicol. Toxin Rev. 17, 131-159]. Lqh6 and Lqh7 are highly toxic to insects and mice, and inhibit the binding of alpha-toxins to cockroach neuronal membranes. Although they kill rodents by intracerebroventricular injection, they do not inhibit the binding of antimammal alpha-toxins (e.g. Lqh2) to rat brain synaptosomes, not even at high concentrations. Furthermore, in voltage-clamp experiments, rat brain Na channels IIA (rNav1.2A) expressed in Xenopus oocytes are not affected by Lqh6 nor by Lqh7 below 3 micro m. In contrast, muscular Na channels (rNav1.4 and hNav1.5) expressed in the same cells respond to nanomolar concentrations of Lqh6 and Lqh7 by slowing of Na current inactivation and a leftward shift of the peak conductance-voltage curve. The structural and pharmacological properties of the new toxins are compared to those of other scorpion alpha-toxins in order to re-examine the hallmarks previously set for the alpha-like toxin group.

Tc1, from Tityus cambridgei, is the first member of a new subfamily of scorpion toxin that blocks K(+)-channels.

A new peptide, Tc1, containing only 23 amino acids closely packed by three disulfide bridges was isolated from the Amazonian scorpion Tityus cambridgei. It blocks reversibly the Shaker B K(+)-channels with a K(d) of 65 nM and displaces binding of noxiustoxin to mouse brain synaptosome membranes. It is the shortest known peptide from scorpion venom that recognizes K(+)-channels and constitutes a new structural subfamily of toxin, classified as alphaKTx 13.1.

[Scorpion toxins]. / Les toxines de scorpion.

Scorpion are known to use a cocktail of toxins to immobilize their prey. So, their venoms constitute a complex mixture of polypeptides exhibiting different pharmacological activities. These polypeptides are small (between 30 and 70 amino acids long), basic and highly reticulated (3 or 4 disulfide bridges). They bind with very high affinities to specific targets, which are different ionic channels of excitable cells. Thus, they constitute usefull tools for the neurobiologist. The scorpion toxins can be divided into: 1) "long chain toxins" (60-70 amino acids residues cross-linked by 4 disulfide bridges) that affect exclusively voltage-dependent Na+ channels of excitable cells; 2) "short chain toxins" (30-40 amino acids residues cross-linked by 3 disulfide bridges) that block several types of K+ channels in different cells (not only potassium channels of excitable cells are affected by these toxins, but also those of erythrocytes or lymphocytes). Specificities or affinities of "long" and "short" chain toxins have been studied using structure-function relationships. Conformational analysis gave the three dimensional structures of an alpha, beta and anti-insect selective toxins. The molecules show a dense core of secondary structure, 2 1/2 turns of alpha-helice, and a short segment of anti-parallel beta-sheet, which exists in all known structures of scorpion toxins, irrespective of their size, sequence and function. From cDNA libraries, full-length cDNAs of about 370 nucleotides encoding precursors of these toxins have been isolated. Sequence analysis of these cDNAs show that the precursors contain signal peptides of about 20 amino acid residues. In addition, precursors of the toxins on mammalian Na+ have extensions at their COOH-terminal ends and have to be processed by specific exopeptidases to give the mature toxins. The cloning and sequencing of their genes revealed that they contain two exons and one intron near the end of the signal peptide sequence of the toxins precursors.

A comparative study of severe scorpion envenomation in children caused by Tityus bahiensis and Tityus serrulatus

No periodo de janeiro de 1984 a maio de 1994, de 239 criancas com ate 15 anos de idade, picadas por escorpioes pertencentes as especies T. bahiensis (84,9 por cento) e T. serrulatus (15,1 por cento), 17 apresentaram envenenamento grave...

Protección contra el alacranismo / Protectión agains scorpionism

En este capítulo se revisan algunos aspectos del problema del alacranismo en México. Se discuten algunos datos obtenidos con una vacuna experimental animal, para la cual el antígeno fue generado por polimerización de extractos solubles de glándulas venenosas de alacranes mexicanos. Se da énfasis a la discusión de los procedimientos y datos bioquímicos más importantes obtenidos en el fraccionamiento del veneno soluble extraído por estimulación eléctrica de telsons de alacranes vivos. También se mencionan algunas propiedades inmunológicas de los extractos de las glándulas, del veneno soluble, de sus fracciones cromatográficas y de sus polipéptidos altamente purificados. Finalmente, se presentan datos sobre la inmunización de ratones con péptidos sintéticos, diseñados con base en la secuencia de aminoácidos de las toxinas de alacranes. Las perspectivas futuras de este trabajo se enfocan a la determinación de los epítopos protectores de las toxinas del veneno y a la clonación y modificación por ingeniería genética de genes que codifican para estos péptidos tóxicos. No obstante el vasto trabajo experimental realizado, todavía no existe una vacuna disponible contra los efectos neurotóxicos del veneno de alacrán. Desde el punto de vista médico, el problema del envenenamiento por piquete de alacrán debe ser tratado mediante el uso del suero antialacrán, única medicina de elección para el tratamiento de los casos severos de accidentes con estos arácnidos

Alacranismo / Scorpionism

El alacranismo es un problema de salud pública originado por la picadura de alacranes venenosos. Afecta a grandes núcleos de población tanto en el medio rural como urbano ocacionando considerables tasas de mortalidad. Históricamente se considera que las zonas de mayor riesgo de este accidente son la ciudad de Durango y el estado de Colima, pero la magnitud de este fenómeno abarca una gran extensión de México. Se pretende dar una visión general del agente agresor y su distribución en México. Se incluyen datos de casos registrados en la ciudad de León, Guanajuato y de los estados de Nayarit y Morelos. Se anotan algunos aspectos bioquímicos del veneno, su mecanismo de acción y fisiopatología. En algunos casos se mencionan especies de otros países, pero como discute más adelante, la homología existente entre toxinas de diferentes especies, sobre todo de Sudamérica, permite hacer analogías respecto al efecto ocasionado por especies mexicanas. Los datos experimentales con veneno de alacranes mexicanos son limitados a la fecha. Finalmente se abordan los factores de riesgo, sintomatología y tratamiento. Se discute su repercusión en la salud de la población y algunas alternativas para abordar este fenómeno