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The peptide, denominated Ct1a, is a β-toxin of 66 amino acids, isolated from venom of the scorpion, Centruroides tecomanus, collected in Colima, Mexico. This toxin was purified using size exclusion, cationic exchange, and reverse phase chromatography. It is the most abundant toxin, representing 1.7% of the soluble venom. Its molecular mass of 7588.9 Da was determined by mass spectrometry. The amino acid sequence was determined by Edman degradation and confirmed by transcriptomic analysis. Since neurons of the suprachiasmatic nucleus (SCN) maintain a spontaneous firing rate (SFR), we evaluated the physiological effects of toxin Ct1a on these neurons. The SFR exhibited a bimodal concentration-dependent response: 100 nM of Ct1a increased the SFR by 223%, whereas 500 nM and 1000 nM reduced it to 42% and 7%, respectively. Control experiments, consisting of recordings of the SFR during a time similar to that used in Ct1a testing, showed stability throughout the trials. Experiments carried out with denatured Ct1a toxin (500 nM) caused no variation in SFR recordings. Action potentials of SCN neurons, before and after Ct1a (100 nM) showed changes in the time constants of depolarization and repolarization phases, amplitude, and half-time. Finally, recordings of hNav1.6 sodium currents indicated that Ct1a shifts the channel activation to a more negative potential and reduces the amplitude of the peak current. These results all demonstrate that toxin Ct1a affects the SFR of SCN neurons by acting upon sodium channels of sub-type 1.6, implicating them in regulation of the SFR of SCN neurons.

International audience; Cl13 is a toxin purified previously from the venom of the Mexican scorpion Centruroides limpidus. This toxin affects the function of voltage gated Na+-channels, human subtypes Nav1.4, Nav1.5 and Nav1.6 in a similar manner as other known β-toxins from scorpion venoms. Here, we report a correction of the primary structure of Cl13, previously published. The peptide does contain 66 amino acids, but residue 58 is a tryptophan and the last C-terminal amino acid is an amidated lysine, instead of arginine. The main contribution of this communication is the determination of the 3D-structure of Cl13, by solution NMR, showing that Cl13 has the classical cysteine-stabilized α/β (CSα/β) folding. It has a triple stranded antiparallel beta sheet commonly present in scorpion sodium channel β-toxins. In addition, we report and discuss a comparison of Cl13 structure with two other toxins (Cn2 and Css2) from scorpions of the same genus Centruroides, which shows important surface similarities with the structure reported here. Finally, the lack of neutralization of Cl13 toxin by two single-chain antibody fragments (scFvs), named LR and 10FG2, which are capable of neutralizing various toxins from Mexican scorpions, is revised. In particular, 10FG2 is capable of neutralizing toxins Cll1 and Cll2 of the same scorpion C. limpidus. The reasons why LR and 10FG2 are unable of neutralizing Cl13 toxin are discussed.

A fundamental issue of the characterization of single-chain variable fragments (scFvs), capable of neutralizing scorpion toxins, is their cross-neutralizing ability. This aspect is very important in Mexico because all scorpions dangerous to humans belong to the Centruroides genus, where toxin sequences show high identity. Among toxin-neutralizing antibodies that were generated in a previous study, scFv 10FG2 showed a broad cross-reactivity against several Centruroides toxins, while the one of scFv LR is more limited. Both neutralizing scFvs recognize independent epitopes of the toxins. In the present work, the neutralization capacity of these two scFvs against two medically important toxins of the venom of Centruroides sculpturatus Ewing was evaluated. The results showed that these toxins are recognized by both scFvs with affinities between 1.8 × 10−9 and 6.1 × 10−11 M. For this reason, their ability to neutralize the venom was evaluated in mice, where scFv 10FG2 showed a better protective capacity. A combination of both scFvs at a molar ratio of 1:5:5 (toxins: scFv 10FG2: scFv LR) neutralized the venom without the appearance of any signs of intoxication. These results indicate a complementary activity of these two scFvs during venom neutralization.

In this communication the isolation, chemical and physiological characterization of three new toxins from the scorpion Centruroides baergi are reported. Their immunoreactive properties with scFvs generated in our group are described. The three new peptides, named Cb1, Cb2 and Cb3 affect voltage-dependent Na+ channels in a differential manner. These characteristics, explain the toxicity of this venom. Molecular interactions in real-time among these toxins and the best recombinant antibodies generated in our group, revealed that one of them was able to neutralize the main toxin of this venom (Cb1). These results represent an important advance for the neutralization of this venom and serve as the basis for generating new scFvs that will allow the neutralization of similar toxins from other venoms that have no yet been neutralized.

Centruroides hirsutipalpus (Scorpiones: Buthidae) is related to the "striped scorpion" group inhabiting the western Pacific region of Mexico. Human accidents caused by this species are medically important due to the great number of people stung and the severity of the resulting intoxication. This communication reports an extensive venom characterization using high-throughput proteomic and Illumina transcriptomic sequencing performed with RNA purified from its venom glands. 2,553,529 reads were assembled into 44,579 transcripts. From these transcripts, 23,880 were successfully annoted using Trinotate. Using specialized databases and by performing bioinformatic searches, it was possible to identify 147 putative venom protein transcripts. These include α- and β-type sodium channel toxins (NaScTx), potassium channel toxins (KScTx) (α-, β-, δ-, γ- and λ-types), enzymes (metalloproteases, hyaluronidases, phospholipases, serine proteases, and monooxygenases), protease inhibitors, host defense peptides (HDPs) such as defensins, non-disulfide bridge peptides (NDBPs), anionic peptides, superfamily CAP proteins, insulin growth factor-binding proteins (IGFBPs), orphan peptides, and other venom components (La1 peptides). De novo tandem mass spectrometric sequencing of digested venom identificatied 50 peptides. The venom of C. hirsutipalpus contains the highest reported number (77) of transcripts encoding NaScTxs, which are the components responsible for human fatalities.

A proteinic fraction from the skin secretion of Trachycephalus typhonius showed antinociceptive activity in mice when tested by the Hot Plate method. This fraction was purified, and a peptide named Tt7 with a molecular mass of 6,509.7 Da was identified. The primary structure of Tt7 was elucidated. Comparing Tt7 with the structure of other analgesic peptides, it exhibits a strong relationship with the family of anntoxins, which are peptides isolated from the skin secretions of Asian frogs of the genus Hyla, whereas T. typhonius is located only in the Americas. Tt7 contains a disulfide pattern Cys1-Cys4/Cys2-Cys3, a secondary structure defined by a β-hairpin, and a N-terminal α-helix, which are characteristic motifs of Kunitz-type structures. The peptide Tt7 was both chemically and biologically synthesized. The recombinant peptides (rTt7 and HisrTt7) showed analgesic effects. Electrophysiological analyses of Tt7 on the voltage-gated sodium channel (Na v ) subtype 1.7 reduced the Na + current by ca 10%. Further studies showed that Tt7 inhibits the pH gated Ca-fluorescence increase in dorsal root ganglion neurons of the rat, indicating an inhibitory action of Tt7 in the acid gated currents which may account for the observed analgesic action of Tt7, suggesting that this peptide might affect various pain-related receptors.

A previously undescribed toxic peptide named Cl13 was purified from the venom of the Mexican scorpion Centruroides limpidus . It contains 66 amino acid residues, including four disulfide bonds. The physiological effects assayed in 7 different subtypes of voltage gated Na + -channels, showed that it belongs to the β-scorpion toxin type. The most notorious effects were observed in subtypes Nav1.4, Nav1.5 and Nav1.6. Although having important sequence similarities with two other lethal toxins from this scorpion species (Cll1m and Cll2), the recently developed single chain antibody fragments (scFv) of human origin were not capable of protecting against Cl13. At the amino acid sequence level, in 3 stretches of peptide Cl13 (positions 7–9, 30–38 and 62–66) some differences with respect to other similar toxins are observed. Some of these differences coincide with contact points with the human antibody fragments.

The soluble venom of the scorpion Tityus macrochirus was separated by chromatographic procedures and three homogeneous peptides were obtained and their primary structures were determined. They were called: Tma1-Tma3, from the abbreviated name of the scorpion. Tma1 is a peptide containing 65 amino acids with four disulfide linkages and a molecular weight of 7386.2 Da. It is a mammalian toxin, shown to affect human sodium-channels sub-types hNav1.6 and hNav1.4. Tma2 and Tma3 are peptides containing 69 amino acids linked by four disulfide bonds, molecular weights 7819.7 and 7830.0 Da, respectively. They do not affect human sodium-channels but are lethal to insects (crickets). A phylogenic analysis of the three peptides and those of other toxic peptides isolated from the genus Tityus and Centruroides were grouped together and analyzed, permitting to obtain a topology with two main clades, one includes most sodium-channel anti-insect scorpion toxins and others includes mostly sodium-channel scorpion toxins anti-mammalian. Tma1 segregates among a group of well-studied β-class toxins of other Tityus species such as T. discrepans, T. obscurus and T. pachyurus. Tma2 and Tma3 are associated with anti-insect toxins, particularly with one of T. obscurus. This phylogenetic analysis confirms and enforces our experimental results obtained with these three new sodium-channel scorpion toxins.

New approaches aimed at neutralizing the primary toxic components present in scorpion venoms, represent a promising alternative to the use of antivenoms of equine origin in humans. New potential therapeutics developed by these approaches correspond to neutralizing antibody fragments obtained by selection and maturation processes from libraries of human origin. The high sequence identity shared among scorpion toxins is associated with an important level of cross reactivity exhibited by these antibody fragments. We have exploited the cross reactivity showed by single chain variable antibody fragments (scFvs) of human origin to re-direct the neutralizing capacity toward various other scorpion toxins. As expected, during these evolving processes several variants derived from a parental scFv exhibited the capacity to simultaneously recognize and neutralize different toxins from Centruroides scorpion venoms. A sequence analyses of the cross reacting scFvs revealed that specific mutations are responsible for broadening their neutralizing capacity. In this work, we generated a set of new scFvs that resulted from the combinatorial insertion of these point mutations. These scFvs are potential candidates to be part of a novel recombinant antivenom of human origin that could confer protection against scorpion stings. A remarkable property of one of these new scFvs (ER-5) is its capacity to neutralize at least three different toxins and its complementary capacity to neutralize the whole venom from Centruroides suffusus in combination with a second scFv (LR), which binds to a different epitope shared by Centruroides scorpion toxins.

We report the optimization of a family of human single chain antibody fragments (scFv) for neutralizing two scorpion venoms. The parental scFv 3F recognizes the main toxins of Centruroides noxius Hoffmann (Cn2) and Centruroides suffusus suffusus (Css2), albeit with low affinity. This scFv was subjected to independent processes of directed evolution to improve its recognition toward Cn2 (Riaño-Umbarila, L., Juárez-González, V. R., Olamendi-Portugal, T., Ortíz-León, M., Possani, L. D., and Becerril, B. (2005) FEBS J. 272, 2591-2601) and Css2 (this work). Each evolved variant showed strong cross-reactivity against several toxins, and was capable of neutralizing Cn2 and Css2. Furthermore, each variant neutralized the whole venoms of the above species. As far as we know, this is the first report of antibodies with such characteristics. Maturation processes revealed key residue changes to attain expression, stability, and affinity improvements as compared with the parental scFv. Combination of these changes resulted in the scFv LR, which is capable of rescuing mice from severe envenomation by 3 LD(50) of freshly prepared whole venom of C. noxius (7.5 μg/20 g of mouse) and C. suffusus (26.25 μg/20 g of mouse), with surviving rates between 90 and 100%. Our research is leading to the formulation of an antivenom consisting of a discrete number of human scFvs endowed with strong cross-reactivity and low immunogenicity.

Light chain amyloidosis (AL) is a disease that affects vital organs by the fibrillar aggregation of monoclonal light chains. λ3r germ line is significantly implicated in this disease. In this work, we contrasted the thermodynamic stability and aggregation propensity of 3mJL2 (nonamyloidogenic) and 3rJL2 (amyloidogenic) λ3 germ lines. Because of an inherent limitation (extremely low expression), Cys at position 34 of the 3r germ line was replaced by Tyr reaching a good expression yield. A second substitution (W91A) was introduced in 3r to obtain a better template to incorporate additional mutations. Although the single mutant (C34Y) was not fibrillogenic, the second mutation located at CDR3 (W91A) induced fibrillogenesis. We propose, for the first time, that CDR3 (position 91) affects the stability and fiber formation of human λ3r light chains. Using the double mutant (3rJL2/YA) as template, other variants were constructed to evaluate the importance of those substitutions into the stability and aggregation propensity of λ3 light chains. A change in position 7 (P7D) boosted 3rJL2/YA fibrillogenic properties. Modification of position 48 (I48M) partially reverted 3rJL2/YA fibril aggregation. Finally, changes at positions 8 (P8S) or 40 (P40S) completely reverted fibril formation. These results confirm the influential roles of N-terminal region (positions 7 and 8) and the loop 40–60 (positions 40 and 48) on AL. X-ray crystallography revealed that the three-dimensional topology of the single and double λ3r mutants was not significantly altered. This mutagenic approach helped to identify key regions implicated in λ3 AL. Background: λ6a and λ3r are the most implicated germ lines in light chain amyloidosis. Results: Mutagenesis at N-terminal, loop 40–60, and CDR3 regions affected λ3r fibrillogenesis. Conclusion: Changes at residues 7, 48, and 91 increased fibril formation while changes at residues 8 and 40 reverted fibrillogenesis. Significance: Characterization of light chain germ lines helps to identify key regions implicated in amyloidosis.

BCF2, a monoclonal antibody raised against scorpion toxin Cn2, is capable of neutralizing both, the toxin and the whole venom of the Mexican scorpion Centruroides noxius Hoffmann. The single chain antibody fragment (scFv) of BCF2 was constructed and expressed in Escherichia coli. Although its affinity for the Cn2 toxin was shown to be in the nanomolar range, it was non-neutralizing in vivo due to a low stability. In order to recover the neutralizing capacity, the scFv of BCF2 was evolved by error-prone PCR and the variants were panned by phage display. Seven improved mutants were isolated from three different libraries. One of these mutants, called G5 with one mutation at CDR1 and another at CDR2 of the light chain, showed an increased affinity to Cn2, as compared to the parental scFv. A second mutant, called B7 with a single change at framework 2 of heavy chain, also had a higher affinity. Mutants G5 and B7 were also improved in their stability but they were unable to neutralize the toxin. Finally, we constructed a variant containing the changes present in G5 and B7. The purpose of this construction was to combine the increments in affinity and stability borne by these mutants. The result was a triple mutant capable of neutralizing the Cn2 toxin. This variant showed the best affinity constant (KD=7.5x10(-11) M), as determined by surface plasmon resonance (BIAcore). The k(on) and k(off) were improved threefold and fivefold, respectively, leading to 15-fold affinity improvement. Functional stability determinations by ELISA in the presence of different concentrations of guanidinium hydrochloride (Gdn-HCl) revealed that the triple mutant is significantly more stable than the parental scFv. These results suggest that not only improving the affinity but also the stability of our scFv were important for recovering its neutralization capacity. These findings pave the way for the generation of recombinant neutralizing antisera against scorpion stings based on scFvs.

Pi4 is a 38-residue toxin cross-linked by four disulfide bridges that has been isolated from the venom of the Chactidae scorpion Pandinus imperator. Together with maurotoxin, Pi1, Pi7 and HsTx1, Pi4 belongs to the alpha KTX6 subfamily of short four-disulfide-bridged scorpion toxins acting on K+ channels. Due to its very low abundance in venom, Pi4 was chemically synthesized in order to better characterize its pharmacology and structural properties. An enzyme-based cleavage of synthetic Pi4 (sPi4) indicated half-cystine pairings between Cys6-Cys27, Cys12-32, Cys16-34 and Cys22-37, which denotes a conventional pattern of scorpion toxin reticulation (Pi1/HsTx1 type). In vivo, sPi4 was lethal after intracerebroventricular injection to mice (LD50 of 0.2 microg per mouse). In vitro, addition of sPi4 onto Xenopus laevis oocytes heterologously expressing various voltage-gated K+ channel subtypes showed potent inhibition of currents from rat Kv1.2 (IC50 of 8 pm) and Shaker B (IC50 of 3 nm) channels, whereas no effect was observed on rat Kv1.1 and Kv1.3 channels. The sPi4 was also found to compete with 125I-labeled apamin for binding to small-conductance Ca(2+)-activated K+ (SK) channels from rat brain synaptosomes (IC50 value of 0.5 microm). sPi4 is a high affinity blocker of the Kv1.2 channel. The toxin was docked (BIGGER program) on the Kv channel using the solution structure of sPi4 and a molecular model of the Kv1.2 channel pore region. The model suggests a key role for residues Arg10, Arg19, Lys26 (dyad), Ile28, Lys30, Lys33 and Tyr35 (dyad) in the interaction and the associated blockage of the Kv1.2 channel.

SUMMARY 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 Km of 320 nmol l–1, without affecting the Ca2+ 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 Ca2+ currents with Cd2+ 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 μmol 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.

Two novel peptides, named Pi4 and Pi7, were purified from the venom of the scorpion Pandinus imperator , and their primary structures were determined. These peptides have 38 amino acids residues, compacted by four disulfide bridges, instead of the normal three found in most K + -channel specific toxins. Both peptides contain 25 identical amino acid residues in equivalent positions (about 66% identity), including all eight half-cystines. Despite the fact that their C-terminal sequence comprising amino acid residues 27 to 37 are highly conserved (10 out of 11 amino acids are identical), Pi4 blocks completely and reversibly Shaker B K + -channels (a Kv1.1 sub-family type of channel) at 100 nM concentration, whereas Pi7 is absolutely inactive at this concentration. Similar effects were observed in binding and displacement experiments to rat brain synaptosomal membranes using 125 I-Noxiustoxin, a well known K + -channel specific toxin. In this preparation Pi4 displaces the binding of radiolabeled Noxiustoxin with ic 50 in the order of 10 nM, whereas Pi7 is ineffective at same concentration. Comparative analysis of Pi4 and Pi7 sequences with those obtained by site directed mutagenesis of Charybdotoxin, another very well studied K + -channel blocking toxin, shows that the substitution of lysine (in Pi4) for arginine (in Pi7) at position 26, might be one of the important `point mutations' responsible for such impressive variation in blocking properties of both toxins, here described.

Using phage display and directed evolution, our group has progressed in the construction of a second family of human single chain variable fragments (scFv) which bind to scorpion toxins dangerous to mammals. It was observed that scFv C1 only bound initially to toxin Cn2, which constitutes 6.8% of whole venom from the scorpion Centruroides noxius Hoffman. Only a few amino acid changes were necessary to extend its recognition to other similar toxins and without affecting the recognition for its primary antigen (Cn2 toxin). One variant of scFv C1 (scFv 202F) was selected after two cycles of directed evolution against Cll1 toxin, the second major toxic component from the venom of the Mexican scorpion Centruroides limpidus limpidus Karsh (0.5% of the whole venom). scFv 202F is also capable of recognizing Cn2 toxin. Despite not having the highest affinity for toxins Cll1 ( K D = 25.1 × 10 −9 M) or Cn2 ( K D = 8.1 × 10 −9 M), this antibody fragment neutralized one LD 50 of each one of these toxins. Additionally, scFv 202F moderately recognized Cll2 toxin which constitutes 1.5% of the venom from C. limpidus . Based on our previous experience, we consider that these results are promising; consequently, we continue working on generating new optimized variants from scFv C1 that could be part of a recombinant scorpion anti-venom from human origin, that might reach the market in the near future.

Scorpion beta-toxins represent a particular pharmacological group of voltage-gated sodium channel (VGSC) neurotoxins. They typically shift the voltage dependence of activation to more hyperpolarizing potentials and reduce the peak current amplitude by binding to receptor-site 4. Here, we report the purification and functional characterization of the first voltage-gated sodium channel toxins, CeII8 and CeII9, isolated from the scorpion Centruroides elegans (Thorell, 1876), which is responsible for deadly cases of intoxication in Mexico. The soluble venom was fractionated by gel filtration and ion-exchange chromatography, followed by reversed-phase HPLC. The toxins CeII8 and CeII9 were further purified and both their amino acid sequence and molecular weight were determined. Both toxins were electrophysiologically characterized on four mammalian VGSCs (rNa(v)1.2, rNa(v)1.4, hNa(v)1.5 and rNa(v)1.7) expressed heterologously in Xenopus laevis oocytes, using the two-electrode voltage-clamp technique. Although CeII8 has the highest sequence similarity with scorpion alpha-toxins, inhibiting the inactivation of VGSCs, 300 nM toxin had a clear beta-toxin effect and was selective towards Na(v)1.7, involved in short-term and inflammatory pain. To the best of our knowledge, CeII8 is the first beta-toxin active on Na(v)1.7. CeII9, a typical anti-mammalian beta-toxin, selectively modulated Na(v)1.4 at a concentration of 700 nM and was, in contrast to CeII8, found to be lethal to mice. Interestingly, both toxins, despite their differences in amino acid sequence, only altered the biophysical properties of a fraction of the expressed sodium channels. Since these effects have also been reported for the beta-toxin CssIV, the bioactive surfaces of the toxins have been compared to each other.

Seven peptides corresponding to the amino acid sequence of toxin 2 from the scorpion Centruroides noxius were chemically synthesized, purified and assayed in mice for their putative neutralizing properties against scorpion toxins. All the peptides were immunogenic and some produced neutralizing antibodies, as verified by injecting the antisera with toxin into naive animals. However, direct challenge of pre-immunized mice (with the longest synthetic peptides of 27 and 57 amino acid residues) revealed an unexpected sensitization phenomena: the animals did not resist injection of one LD50 of purified toxin 2 (5% survival), but pre-immunization of mice with native toxin protected 100% of the animals. These findings suggest that vaccine preparations with synthetic peptides corresponding to the amino acid sequence of certain toxins should be analyzed cautiously.

Here we describe the basic features of the interaction of K+ channels with Pi1, a recently described 35 amino acid scorpion toxin, which has four disulfide bridges instead of the three commonly found in all the other known scorpion toxins. We found that: (a) Pi1 blocks ShakerB from the outside with a 1:1 stoichiometry, and a Kd of 32 nM in zero external [K+]; (b) extracellular K+, Rb+ and Cs+ but not NH+4 ions strongly impede (destabilize) the block by this toxin; interestingly (c) the destabilizing binding of K+, Rb+, and Cs+ is described by a Hill coefficient n>1; (d) external K+ is more effective than internal K+ to reduce the block by Pi1.