Biochemical characterization and insecticidal activity of isolated peptides from the venom of the scorpion Centruroides tecomanus.

The venom of scorpions is a mixture of components that constitute a source of bioactive molecules. The venom of the scorpion Centruroides tecomanus contains peptides toxic to insects, however, to date no toxin responsible for this activity has yet been isolated and fully characterized. This communication describes two new peptides Ct-IT1 and Ct-IT2 purified from this scorpion. Both peptides contain 63 amino acids with molecular weight 6857.85 for Ct-IT1 and 6987.77 Da for Ct-IT2. The soluble venom was separated using chromatographic techniques of molecular size exclusion, cationic exchange, and reverse phase chromatography, allowing the identification of at least 99 components of which in 53 the insecticidal activity was evaluated. The LD50 determined for Ct-IT1 is 3.81 µg/100 mg of cricket weight, but low amounts of peptides (0.8 µg of peptide) already cause paralysis in crickets. The relative abundance of these two peptides in the venom is 2.1% for Ct-IT1 and 1% for Ct-IT2. The molecular masses and N-terminal sequences of both insecticidal toxins were determined by mass spectrometry and Edman degradation. The primary structure of both toxins was compared with other known peptides isolated from other scorpion venoms. The analysis of the sequence alignments revealed the position of a highly conserved amino acid residue, Gly39, exclusively present in anti-insect selective depressant ß-toxins (DBTXs), which in Ct-IT1 and Ct-IT2 is at position Gly40. Similarly, a three-dimensional structure of this toxins was obtained by homology modeling and compared to the structure of known insect toxins of scorpions. An important similarity of the cavity formed by the trapping apparatus region of the depressant toxin LqhIT2, isolated from the scorpion Leiurus quinquestriatus hebraeus, was found in the toxins described here. These results indicate that Ct-IT1 and Ct-IT2 toxins have a high potential to be evaluated on pests that affect economically important crops to eventually consider them as a potential biological control method.

Biochemical characterization of the venom from the Mexican scorpion Centruroides ornatus, a dangerous species to humans.

Every year in Mexico, around 300,000 people suffer from accidents related to scorpion stings. Among the scorpion species dangerous to human is Centruroides ornatus, whose venom characterization is described here. From this venom, a total of 114 components were found using chromatographic separation and mass spectrometry analysis. The most abundant ones have molecular masses between 3000-4000 Da and 6000-8000 Da respectively, similar to other known K+ and Na+-channel specific scorpion peptides. Using intraperitoneal injections into CD1 mice, we were able to identify and fully sequenced three new lethal toxins. We propose to name them Co1, Co2 and Co3 toxins, which correspond to toxins 1 to 3 of the abbreviated species name (Co). Electrophysiology analysis of these peptides using heterologously expressed human Na+-channels revealed a typical ß-toxin effect. Peptide Co52 (the most abundant peptide in the venom) showed no activity in our in vivo and in vitro model assays. A phylogenetic analysis groups the Co1, Co2 and Co3 among other ß-toxins from Centruroides scorpions. Peptide Co52 segregates among peptides of unknown defined functions.

Cn29, a novel orphan peptide found in the venom of the scorpion Centruroides noxius: Structure and function.

A peptide (Cn29) from the venom of the scorpion Centruroides noxius (about 2% of the soluble venom) was purified and its primary and three-dimensional structures were determined. The peptide contains 27 amino acids with primary sequence: LCLSCRGGDYDCRVKGTCENGKCVCGS. The peptide is tightly packed by three disulfide linkages formed between C2-C23, C5-C18 and C12-C25. Since the native peptide was obtained in limited amounts, the full synthetic peptide was prepared using the standard F-moc-based solid phase synthesis method of Merrifield. The native and synthetic peptides were shown to be identical by sequencing, HPLC separation and mass spectrometry. The solution structure of the peptide solved from NMR data shows that it consists of a well-defined N-terminal region without regular secondary structure extending from Leu 1 to Asp 9, followed by a short helical fragment from Tyr10 to Val14 and two short ß strands (Thr17-Glu19 and Lys22-Val24). The primary and tertiary structures of Cn29 are different from all other scorpion peptides described in the literature. Transcriptome analysis of RNA obtained from C. noxius confirmed the expression of a gene coding for Cn29 in its venom gland. Initial experiments were conducted to identify its possible function: lethality tests in mice and insects as well as ion-channel binding using in vitro electrophysiological assays. None of the physiological or biological tests displayed any activity for this peptide, which at present is considered to be another orphan peptide found in scorpion venoms. The peptide is thus the first example of a novel structural component present in scorpion venoms.

Venom characterization of the Amazonian scorpion Tityus metuendus.

The soluble venom from the scorpion Tityus metuendus was characterized by various methods. In vivo experiments with mice showed that it is lethal. Extended electrophysiological recordings using seven sub-types of human voltage gated sodium channels (hNav1.1 to 1.7) showed that it contains both α- and ß-scorpion toxin types. Fingerprint analysis by mass spectrometry identified over 200 distinct molecular mass components. At least 60 sub-fractions were recovered from HPLC separation. Five purified peptides were sequenced by Edman degradation, and their complete primary structures were determined. Additionally, three other peptides have had their N-terminal amino acid sequences determined by Edman degradation and reported. Mass spectrometry analysis of tryptic digestion of the soluble venom permitted the identification of the amino acid sequence of 111 different peptides. Search for similarities of the sequences found indicated that they probably are: sodium and potassium channel toxins, metalloproteinases, hyaluronidases, endothelin and angiotensin-converting enzymes, bradykinin-potentiating peptide, hypothetical proteins, allergens, other enzymes, other proteins and peptides.

Pi5 and Pi6, two undescribed peptides from the venom of the scorpion Pandinus imperator and their effects on K+-channels.

This work reports the isolation, chemical and functional characterization of two previously unknown peptides purified from the venom of the scorpion Pandinus imperator, denominated Pi5 and Pi6. Pi5 is a classical K+-channel blocking peptide containing 33 amino acid residues with 4 disulfide bonds. It is the first member of a new subfamily, here defined by the systematic number α-KTx 24.1. Pi6 is a peptide of unknown real function, containing only two disulfide bonds and 28 amino acid residues, but showing sequence similarities to the κ-family of K-channel toxins. The systematic number assigned is κ-KTx2.9. The function of both peptides was assayed on Drosophila Shab and Shaker K+-channels, as well as four different subtypes of voltage-dependent K+-channels: hKv1.1, hKv1.2, hKv1.3 and hKv1.4. The electrophysiological assays showed that Pi5 inhibited Shaker B, hKv1.1, hKv1.2 and hKv1.3 channels with Kd = 540 nM, Kd = 92 nM and Kd = 77 nM, respectively, other studied channels were not affected. Of the channels tested only hKv1.2 and hKv1.3 were inhibited at 100 nM concentration of Pi6, the remaining current fractions were 68% and 77%, respectively. Thus, Pi5 and Pi6 are high nanomolar affinity non-selective blockers of hKv1.2 and hKv1.3 channels.

Design and expression of recombinant toxins from Mexican scorpions of the genus Centruroides for production of antivenoms.

This manuscript describes the design of plasmids containing the genes coding for four main mammalian toxins of scorpions from the genus Centruroides (C.) of Mexico. The genes that code for toxin 2 of C. noxius (Cn2), toxin 2 from C. suffusus (Css2) and toxins 1 and 2 from C. limpidus (Cll1 and Cll2) were included into individual plasmids carrying the genetic construction for expression of fusion proteins containing a leader peptide (pelB) that directs the expressed protein to the bacterial periplasm, a carrier protein (thioredoxin), the cleavage site for enterokinase, the chosen toxin and a poly-histidine tag (6xHis-tag) for purification of the hybrid protein by immobilized metal ion affinity chromatography after expression in Escherichia coli strain BL21 (DE3). The purified hybrid proteins containing the recombinant toxins (abbreviated Thio-EK-Toxin) were used for immunization of three independent groups of ten mice and four rabbits. Challenging the first group of mice, immunized with recombinant Thio-EK-Css2, with three median lethal doses (LD50) of C. suffusus soluble venom resulted in the survival of all the test animals without showing intoxication symptoms. All control mice (none immunized) died. Similar results were obtained with mice previously immunized with Thio-EK-Cn2 and challenged with C. noxius venom. The third group of mice immunized with both Thio-EK-Cll1 and Thio-EK-Cll2 showed an 80% survival ratio when challenged with only one LD50 of C. limpidus venom, all showing symptoms of intoxication. The sera from rabbits immunized with a combination of the four recombinant toxins were collected separately and used to assess their neutralization capacity in vitro (pre-incubating the serum with the respective scorpion venom and injecting the mixture into mice), using six mice for each serum/venom combination tested. The venoms from the six most dangerous scorpion species of Mexico were assayed: C. noxius, C. suffusus, C. limpidus, C. elegans, C. tecomanus and C. sculpturatus. Two hundred and 50 µL of serum from any of the immunized rabbits were enough to neutralize three LD50 of any of the tested venoms, with mice showing no symptoms of intoxication. These results confirm that the recombinant forms of the main toxins from the most dangerous scorpions of Mexico are excellent immunogens for the production of antivenoms to treat scorpion intoxications.

Serum level of scorpion toxins, electrolytes and electrocardiogram alterations in Mexican children envenomed by scorpion sting.

The scorpion Centruroides limpidus limpidus (C.l.l.) is endemic in México, producing hundreds of accidents in humans; children being one of the most susceptible targets. Few studies reported that severe envenoming by scorpion venom induces cardiac damage and electrolytes abnormalities in children, but the relationship of envenoming severity and toxic blood levels is unknown. The aim of this study was to determine the relationship among clinical status of envenoming, serum electrolyte, electrocardiographic abnormalities, and serum toxin levels in 44 children stung by scorpion over a period of 6 months in the State of Morelos, Mexico. The patients were said to be asymptomatic, when they presented just local symptoms, and were said to be symptomatic when showing local symptoms and at least one systemic symptom. The clinical status was evaluated at the admission at the emergency room of the Hospital, and 30 min after the administration of polyspecific F(ab')2 anti-scorpion therapy to symptomatic children. Forty-one percent of the children were asymptomatic and 59% symptomatic. Potassium and sodium imbalance and an elongation of the QT interval were detected; the rate of hypokalemia was higher in symptomatic than on asymptomatic children (50% and 6%, respectively). Hypokalemia persisted in 19% in symptomatic patients, whereas sodium reached normal levels 30 min after anti-venom therapy. The hypokalemia statistically correlated with elongation of the QT interval. The concentration of the toxic components of C.l.l in serum was significantly higher in symptomatic than asymptomatic children, and the serum levels of the toxic component significantly decreased to undetectable levels after the application of anti-venom therapy. Despite the small size of the sample, this study establishes that severity of envenoming was statistically related to potassium imbalance in serum, QT interval and the concentration of toxic components in serum, which decreased at undetectable levels after specific treatment with the anti-scorpion venom, correlating with clinical disappearance or greatly reduction of symptoms of envenomation.

Comprehensive analysis of venom from the scorpion Centruroides tecomanus reveals compounds with antimicrobial, cytotoxic, and insecticidal activities.

Centruroides tecomanus is a medically important scorpion of the state of Colima (Mexico). This communication reports the identification of venom components of this scorpion with biological activity over insects/crickets (Acheta domestica), crustaceans/fresh water shrimps (Cambarellus montezumae), and mammalians/mice (Mus musculus, strain CD1). It also describes the pharmacological effects on cell lines in culture (L5178Y cells, HeLa cells, HuTu cells and Jurkat E6-1 cells), as well as on several types of bacteria (see below). The soluble venom of this scorpion was fractionated by high-performance liquid chromatography (HPLC) and collected separately in twelve independent fractions collected over 60 min run (5 min time apart each other). The HPLC components of fraction VII were lethal to all three species used for assay. The IVth fraction had a toxic effect on freshwater shrimps. In this species, fractions VI, VII and VIII were all lethal. For crickets, fractions V and VI were toxic and fraction VII was lethal. In mouse, the lethal components were found in fraction VII, whereas fraction VIII was toxic, but not lethal, at the doses assayed. The molecular weight of peptides from the various group of fractions were identified by mass spectrometry determination. Components lethal to mice showed molecular weights from 7013 to 7487 Da. Two peptides were obtained in homogeneous form and shown to be lethal to the three species of animal used for assay. The soluble venom tested on L5178Y cell line survival was shown to be cytotoxic, at 10-100 µg/mL concentration, when compared to control murine splenocytes (p = 0.007). The soluble venom applied to Hela, Hutu and Jurkat cell lines did not show cytotoxic effects at these concentrations. On the contrary, it seems to have a proliferative effect. However the HPLC fractions I, III, VI and XII do have a cytotoxic effect on Jurkat E06-1 cells in culture at 200 µg/mL concentration. The antimicrobial activity of the venom fractions on Staphylococcus aureus (gram-positive), Escherichia coli, Pseudomonas aeruginosa y Salmonella spp (gram-negative) was measured, using the liquid inhibition growth system. The four strains of bacteria used were susceptible to fractions III and IV, affecting all four bacterial strains at concentrations below 5 µg/mL.

Scorpion venom components that affect ion-channels function.

The number and types of venom components that affect ion-channel function are reviewed. These are the most important venom components responsible for human intoxication, deserving medical attention, often requiring the use of specific anti-venoms. Special emphasis is given to peptides that recognize Na(+)-, K(+)- and Ca(++)-channels of excitable cells. Knowledge generated by direct isolation of peptides from venom and components deduced from cloned genes, whose amino acid sequences are deposited into databanks are nowadays in the order of 1.5 thousands, out of an estimate biodiversity closed to 300,000. Here the diversity of components is briefly reviewed with mention to specific references. Structural characteristic are discussed with examples taken from published work. The principal mechanisms of action of the three different types of peptides are also reviewed. Na(+)-channel specific venom components usually are modifier of the open and closing kinetic mechanisms of the ion-channels, whereas peptides affecting K(+)-channels are normally pore blocking agents. The Ryanodine Ca(++)-channel specific peptides are known for causing sub-conducting stages of the channels conductance and some were shown to be able to internalize penetrating inside the muscle cells.

Toxin modulators and blockers of hERG K(+) channels.

The K(+) channel encoded by the Ether-á-go-go-Related Gene (ERG) is expressed in different tissues of different animal species. There are at least three subtypes of this channel, being the sub-type 1 (ERG1) crucial in the repolarization phase of the cardiac action potential. Mutations in this gene can affect the properties of the channel producing the type II long QT syndrome (LQTS2) and many drugs are also known to affect this channel with a similar side effect. Various scorpion, spider and sea anemone toxins affect the ERG currents by blocking the ion-conducting pore from the external side or by modulating channel gating through binding to the voltage-sensor domain. By doing so, these toxins become very useful tools for better understanding the structural and functional characteristics of these ion channels. This review discusses the interaction between the ERG channels and the peptides isolated from venoms of these animals. Special emphasis is placed on scorpion toxins, although the effects of several spider venom toxins and anemone toxins will be also revised.

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.

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.

Scorpion and spider venom peptides: gene cloning and peptide expression.

This communication reviews most of the important findings related to venom components isolated from scorpions and spiders, mainly by means of gene cloning and expression. Rather than revising results obtained by classical biochemical studies that report structure and function of venom components, here the emphasis is placed on cloning and identification of genes present in the venomous glands of these arachnids. Aspects related to cDNA library construction, specific or random ESTs cloning, transcriptome analysis, high-throughput screening, heterologous expression and folding are briefly discussed, showing some numbers of species and components already identified, but also shortly mentioning limitations and perspectives of research for the future in this field.

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.

Expression systems of human ß-defensins: vectors, purification and biological activities.

Human beta-defensins are 2-5 kDa, cationic, microbicidal peptides, which represent the first-line host defense against several Gram-negative and Gram-positive bacteria, fungi and viruses. They contain a conserved disulfide-bridge pattern of three pairs of intramolecular cystine bonds. The well-known public health problem related with the growing number of multiresistant bacteria has driven research to look for novel antibiotics, such beta-defensins and a feasible way to produce them. Heterologous expression of beta-defensins could be one way to generate large quantities of beta-defensins for clinical research; however, heterologous expression of beta-defensins has some biochemical problems, such toxicity toward the host cell, peptide degradation by proteolytic cell enzymes, size, folding constrains and low recombinant peptide yields. In this communication, several heterologous systems for producing human beta-defensins are reviewed.

Amino acid substitutions in an alpha-helical antimicrobial arachnid peptide affect its chemical properties and biological activity towards pathogenic bacteria but improves its therapeutic index.

Four variants of the highly hemolytic antimicrobial peptide Pin2 were chemically synthesized with the aim to investigate the role of the proline residue in this peptide, by replacing it with the motif glycine-valine-glycine [GVG], which was found to confer low hemolytic activity in a spider antimicrobial peptide. The proline residue in position 14 of Pin2 was substituted by [V], [GV], [VG] and [GVG]. Only the peptide variant with the proline substituted for [GVG] was less hemolytic compared to that of all other variants. The peptide variant [GVG] kept its antimicrobial activity in Muller-Hilton agar diffusion assays, whereas the other three variants were less effective. However, all Pin2 antimicrobial peptide variants, were active when challenged against a Gram-positive bacteria in Muller-Hilton broth assays suggesting that chemical properties of the antimicrobial peptides such as hydrophobicity is an important indication for antimicrobial activity in semi-solid environments.

Recombinant expression of the toxic peptide ErgTx1 and role of Met35 on its stability and function.

Ergtoxin 1 (ErgTx1) is a 42 amino acid peptide purified from the venom of the Mexican scorpion Centruroides noxius Hoffmann, capable of blocking specifically human potassium channels of the ether-á-go-go-related gene family (hERG). This peptide binds to a partially overlapping site on the channel outer mouth, in which residues of the S5-P linker are critically involved. Here we describe results of site directed mutagenesis of the ErgTx1 gene and its heterologous expression in Escherichia coli. The recombinant products show the fundamental role played by methionine in position 35 (Met35) of the primary structure. Naturally oxidized Met35 decreases by three orders of magnitude the affinity of the peptide for the hERG1 channels. This result is quite relevant, because it shows two possible situations: either Met35 is involved in the proper folding of the molecule or it plays a direct role in the interaction with the channel, i.e., constitutes part of the interacting surfaces. These two situations were evaluated by preparing heterologously expressed ErgTx1 gene and a mutant containing alanine in position 35. Additionally circular dichroism measurements of both native and recombinant peptides were performed. The electrophysiological recordings and the structural values obtained by optical measurements, strongly support the idea that Met35 is indeed a key residue on the interacting surfaces of the toxin with the channels.

Rational design of synthetic peptides to generate antibodies that recognize in situ CD11c(+) putative dendritic cells in horse lymph nodes.

A three-dimensional model of the alphaX I-domain of the horse integrin CD11c from dendritic cells provided information for selecting two segments of the primary structure for peptide synthesis. Peptide 1 contains 20 amino acids and peptide 2 has 17 amino acid residues. The first spans from position Thr229 to Arg248 of an alpha-helix segment of the structure, whereas peptide 2 goes from Asp158 to Phe174 and corresponds to an exposed segment of the loop considered to be the metal ion-dependent adhesion site. Murine polyclonal antisera against both peptides were generated and assayed in peripheral blood cell suspensions and in cryosections of horse lymph nodes. Only the serum against peptide 2 was capable of identifying cells in suspension and in situ by immunohistochemistry, some with evident dendritic morphology. Using this approach, an immunogenic epitope exposed in CD11c was identified in cells from horse lymph node in situ.

Antidotes against venomous animals: state of the art and prospectives.

This communication revises the state of the art concerning antivenoms against snakes, spiders and scorpions. An overview of the historical facts that preceded the therapeutic use of antibodies is mentioned. A brief list of the major protein components of these venomous animals is revised with a short discussion of what is known on the proteomic analysis of their venoms, but the emphasis is placed on the type of antivenoms available commercially, including pertinent literature and addresses of the companies that prepare these antivenoms. The final section revises and discusses current research on the field and new potential applications that are being developed geared at obtaining new therapeutic antibodies or fragments of antibodies for neutralization of toxic components of venomous animals.