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A Recombinant Insect‐Specific α‐Toxin of Buthus occitanus tunetanus Scorpion Confers Protection Against Homologous Mammal Toxins
Abstract
We have constructed a cDNA library from venom glands of the scorpion Buthus occitanus tunetanus and cloned a DNA sequence that encodes an alpha-toxin. This clone was efficiently expressed in Escherichia coli as a fusion protein with two Ig-binding (Z) domains of protein A from Staphylococcus aureus. After CNBr treatment of the fusion protein and HPLC purification, we obtained approximately 1 mg recombinant apha-toxin/l bacterial culture. The toxin, called Bot XIV, displays no toxicity towards mammals but is active towards insects as shown by its paralytic activity against Blatella germanica cockroach and by electrophysiological studies on Periplaneta americana cockroaches. The Bot XIV protein fused to two Z domains is highly immunogenic in mice and induces production of antisera that specifically recognize and neutralize highly toxic components that had been injected into mice. This fusion protein could be very useful for development of potent protective antisera against scorpion venoms.
Supplementary resource (1)
... Recently, we cloned and characterized a new insect a-toxin from the venom of the scorpion Buthus occitanus tunetanus called BotXIV [19]. We showed that BotXIV shows 49.25 and 52.23% identities with LqhaIT and LqqIII, respectively, and is not toxic on mice even at high concentration [up to 2.5 lg per 20 g of body weight at intracerebroventricularly (i.c.v.) route]. ...
... Site-directed mutagenesis assays were performed according to Kunkel et al. [22] using a Bio-Rad kit. The cDNA encoding the precursor of BotXIV [19] was modified as follows: a KpnI/ BamHI fragment carrying the sequence encoding BotXIV was inserted into the corresponding restriction sites of M13mp19 to produce M13mp19-BotXIV template for mutagenesis. Phages from an individual lysis plaque were used to re-infect fresh host cells to produce high-titer phage stock. ...
... Bacteria E. coli HB101 transformed by the expression vectors pEZZ-M8-10, pEZZ-M19-22 or pEZZ-M38-43 were grown in a 5-L fermentor (LSL Biolafitte, Saint Germain en Lay, France) with an initial culture volume of 4-L of tryptic soy broth (TSB) medium (Difco) supplemented with 5 gAEL )1 of glucose and 200 lgAEmL )1 ampicillin. Conditions of production were performed as previously described [19]. Hybrid recombinant proteins contained in extracted periplasmic fractions and in the culture medium were purified by affinity chromatography on an IgG-Sepharose column according to Ducancel et al. [26], then, lyophilized. ...
BotXIV and LqhalphaIT are two structurally related long chain scorpion alpha-toxins that inhibit sodium current inactivation in excitable cells. However, while LqhalphaIT from Leiurus quinquestriatus hebraeus is classified as a true and strong insect alpha-toxin, BotXIV from Buthus occitanus tunetanus is characterized by moderate biological activities. To assess the possibility that structural differences between these two molecules could reflect the localization of particular functional topographies, we compared their sequences. Three structurally deviating segments located in three distinct and exposed loops were identified. They correspond to residues 8-10, 19-22, and 38-43. To evaluate their functional role, three BotXIV/LqhalphaIT chimeras were designed by transferring the corresponding LqhalphaIT sequences into BotXIV. Structural and antigenic characterizations of the resulting recombinant chimera show that BotXIV can accommodate the imposed modifications, confirming the structural flexibility of that particular alpha/beta fold. Interestingly, substitution of residues 8-10 yields to a new electrophysiological profile of the corresponding variant, partially comparable to that one of alpha-like scorpion toxins. Taken together, these results suggest that even limited structural deviations can reflect functional diversity, and also that the structure-function relationships between insect alpha-toxins and alpha-like scorpion toxins are probably more complex than expected.
... Two most dangerous species, Buthus occitanus tunetanus and A. australis Hector, have been characterized thoroughly and formed structural-antigenic groups that have been described in detail [6,42,[50][51][52][53]. Two experimental lines have been conducted. ...
... Two experimental lines have been conducted. In one of them the authors' goal was to produce a recombinant toxin "that includes toxoids structurally and antigenically related to the potent toxins of A. australis Hector and B. occitanus tunetanus to progressively substitute the usual use of the venoms as immunogens for serotherapy production and to confer immunoprevention against scorpion venoms" ( [53], p. 659). A cDNA library of scorpion gland genes was screened with hybridization probes from the conserved regions of the Bot I group toxins, and a recombinant gene for a toxin, designated Bot XIV, was cloned, expressed in Escherichia coli and used finally to generate antibodies in mice [53]. ...
... In one of them the authors' goal was to produce a recombinant toxin "that includes toxoids structurally and antigenically related to the potent toxins of A. australis Hector and B. occitanus tunetanus to progressively substitute the usual use of the venoms as immunogens for serotherapy production and to confer immunoprevention against scorpion venoms" ( [53], p. 659). A cDNA library of scorpion gland genes was screened with hybridization probes from the conserved regions of the Bot I group toxins, and a recombinant gene for a toxin, designated Bot XIV, was cloned, expressed in Escherichia coli and used finally to generate antibodies in mice [53]. The antibody induced by this novel toxin cross-reacts with the entire Bot I group but does not recognize other groups. ...
Scorpions and other venomous animals contain concentrates of biologically active substances developed to block vital physiological and biochemical functions of the victims. These have contrasting human health concerns, provide important pharmacological raw material and pose a serious threat to human life and health in tropical and subtropical regions. Because only occasional and minor quantities of venom are introduced into the human organism with a scorpion sting and their mortal effect is an acute phenomenon these substances are unknown to the immune defense system and thus no immunity has appeared against them during evolution. Antidotes prepared from animal anti-sera are effective against some species of scorpions but depend on the manufacturer and the availability of product to the medical community. Although significant progress has been made in immunological studies of certain groups of toxins, few centers are dedicated to this research. Information is still insufficient to generate a comprehensive picture of the subject and to propose vaccines against venoms. A novel approach based on mimotopes selected from phage-displayed random peptide libraries show potential to impel further progress of toxin immunological studies and to provide putative vaccine resources. In this report we revise the "state of the art" in the field.
... Increasing amounts of the toxin/antibody mixture were brought to a volume of 5 l, incubated for 1 h at 37°C, and i.c.v. injected in cohorts of 4 mice (19, 20, 25). The survival of mice was recorded 24 h after injection. ...
... Evidently, the strongest toxin neutralizing Nb needs to be identified for future immunotherapy. We applied the standard neutralization protocol (19, 20, 25) that is used to test the neutralizing capacity of the polyclonal Fab 2 before its release as immunotherapeutic. According to this approach, various Nbs are mixed at a 2:1 M ratio with the AahI toxin. ...
Envenoming following scorpion sting is a common emergency in many parts of the world. Our aim was to ameliorate the current 100-kDa horse plasma antivenom serum (PAS)-derived Fab'(2) to more quickly reach the highly diffusible scorpion toxins (7 kDa). We immunized dromedaries with toxins from Androctonus australis hector (Aah) scorpions and cloned the single-domain antibody fragments or nanobodies (15 kDa) from their B cells. Nanobodies against AahI' toxin (with AahII the most toxic compound of the venom) were retrieved from the libraries, and their AahI'-toxin neutralization was monitored in mice. Remarkably, the NbAahI'F12 fully protected mice against 100 LD(50) of AahI' administered intracerebroventricularly. Moreover, where PAS failed completely to neutralize 2 LD(50) of crude venom injected subcutaneously, the designed bispecific NbF12-10 against AahI'/AahII toxins succeeded in neutralizing 5 LD(50). Finally, in a challenge assay in which mice were subcutaneously injected with a lethal dose of scorpion venom, the subsequent intravenous injection of 85 microg of NbF12-10 protected all mice, even if the whole procedure was repeated 3 times. Furthermore, the NbF12-10 remained fully protective when mice with severe signs of envenoming were treated a few minutes before the untreated mice died.
... Several approaches have been developed to improve serotherapy against scorpion stings [1,[7][8][9][10]. Here, we describe a new procedure for induction of neutralising antibodies against scorpion toxins. ...
... In our experiments, the yields of the fusion proteins were satisfactory, but not increased by prolonging induction of expression beyond 3 h. Other studies based on developing vaccines have focused on recombinant molecules and provide significant but, only initial data [8,10]. Injecting mice with high concentrations of our fusion proteins produced no signs of paralysis. ...
We report the use of recombinant scorpion toxins in the form of fusion proteins as antigens for immunisation in rabbits and mice: the aim was to produce in these animal models protective antisera against the most lethal alpha-type toxins in the venom from the North African scorpion Androctonus australis. The cDNAs encoding AaH I, AaH II and AaH III (the three major alpha-type toxins acting on voltage-sensitive sodium channels) were fused to the sequence encoding the maltose binding protein (MBP). The constructs (MBP-AaH I, MBP-AaH II, MBP-AaH I+II and MBP-AaH III) were expressed in Escherichia coli, and resulting fusion proteins were translocated to the periplasmic space. The recombinant fusion proteins were characterised and used as antigens to generate antibodies in rabbits. These antibodies raised specifically recognised their corresponding radiolabelled-toxin with affinities in the 0.1nM range. In vitro neutralisation assays indicated that 1ml of serum raised against a mixture of fusion proteins was able to neutralise 15 LD(50) of the toxic fraction (AaH-G50) purified from the crude venom by molecular filtration through Sephadex G50. In vivo, the fusion proteins induced a long-term protection in mice against the lethal effects of AaH-G50 or of the native toxins. Ten weeks after the beginning of the immunisation programme, mice were challenged with various toxins or AaH-G50 doses. Mice were fully protected against three LD(50) of AaH-G50. Our work shows that fusion protein constructs can be used as a vaccine providing efficient immune protection against A. australis venom.
... ELISA was used to assess specific titres of immune sera and isolated polyclonal IgG fractions against AahG50 toxic fraction and AahII toxin coated with 1 mg/ml in maxisorb plates (NUNC, Rochester, NY, USA). The assays were conducted according to an optimized previously described procedure (Chavez-Olortegui et al., 1991;Bouhaouala-Zahar et al., 1996). Residual protein binding sites were blocked by adding 200 ml PBS, 0.5% milk powder per well, for 1 h at 37 8C. ...
... Neutralizing capacity of immune sera and IgG fractions were evaluated after subcutaneous (s.c.) and intracerebroventricular (i.c.v.) injections. Toxic doses (2 or 3 LD 50 ) of AahII toxin or AahG50 fraction were incubated for 1 h 30 min at 37 8C and 30 min at 4 8C with a constant volume of immune serum (150 ml) according to the previously described procedure (Bouhaouala-Zahar et al., 1996). The new LD 50 was estimated as previously described (Martin and Rochat, 1986) after 24 h delay. ...
Scorpion envenoming is a real health problem. The only specific treatment is immunotherapy with antibodies from immunized horses. The severity of scorpion envenoming and the rapid diffusion of the toxins into the blood compartment require an improvement of the present antivenom therapy. In this study, we report successful immunization of dromedaries (Camelus dromedarius) against the small weakly antigenic neurotoxins of Androctonus australis hector scorpion. Camel immune sera was tested for its specific antigenic reactivity and neutralizing capacity against Aah toxic fraction and AahII toxin. We demonstrate that a substantial proportion of polyclonal heavy chain antibodies bind to Aah toxins and in particular to AahII, the most toxic one scorpion venom component. Furthermore, we show that both dromedary sera and heavy chain antibody subclasses are capable of neutralizing the toxicity of Aah toxins in mice.
... Animal immunization can be performed with: attenuated crude venom to increase the life expectancy of the animal; venom toxins, to obtain better specific and neutralizing antibodies; anatoxins, which are detoxified and therefore not toxic to mammals but immunogenic, inducing the same immune response than toxins, and with recombinant toxins or synthetic peptides conjugated with a carrier protein, produced in E. coli (Bouhaouala-Zahar et al. 1996;Legros et al. 2001;Carmo et al. 2015). ...
Scorpion envenomation is a serious health problem in tropical and subtropical zones. The access to scorpion antivenom is sometimes limited in availability and specificity. The classical production process is cumbersome, from the hyper-immunization of the horses to the IgG digestion and purification of the F(ab)′2 antibody fragments. The production of recombinant antibody fragments in Escherichia coli is a popular trend due to the ability of this microbial host to produce correctly folded proteins. Small recombinant antibody fragments, such as single-chain variable fragments (scFv) and nanobodies (VHH), have been constructed to recognize and neutralize the neurotoxins responsible for the envenomation symptoms in humans. They are the focus of interest of the most recent studies and are proposed as potentially new generation of pharmaceuticals for their use in immunotherapy against scorpion stings of the Buthidae family. This literature review comprises the current status on the scorpion antivenom market and the analyses of cross-reactivity of commercial scorpion anti-serum against non-specific scorpion venoms. Recent studies on the production of new recombinant scFv and nanobodies will be presented, with a focus on the Androctonus and Centruroides scorpion species. Protein engineering-based technology could be the key to obtaining the next generation of therapeutics capable of neutralizing and cross-reacting against several types of scorpion venoms.
Key points
• Commercial antivenoms consist of predominantly purified equine F(ab)′2fragments.
• Nanobody-based antivenom can neutralize Androctonus venoms and have a low immunogenicity.
• Affinity maturation and directed evolution are used to obtain potent scFv families against Centruroides scorpions.
... Da (16-100% death in crickets). They are assumed to be Na v toxins and are widely reported in the range of 6-8.5 kDa by many authors (Arnon et al., 2005;Bouhaouala-Zahar et al., 1996;Froy et al., 1999;Guerrero-Vargas et al., 2012;Kawachi et al., 2013;Nakagawa et al., 1997;Possani et al., 1999;Valdez-Velázquez et al., 2013;Weinberger et al., 2010). ...
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 β-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.
... Scorpion toxins used for immunization purposes can be produced in Escherichia coli with little to no effect of toxicity on the immunized animal (Table I-10). The recombinant toxin BotXIV can be used to produce an anti-BotXIV serum, capable of neutralizing Buthus occitanus tunetanus scorpion venom in the same capacity than the commercially available equine-derived antivenom without inducing toxic effects in mice [155]. Furthermore, the chimera BotXIVM8-10, produced from BotXIV and the LqhαIT Leiurus quinquestriatus hebraeus scorpion toxin, showed properties comparable to α-toxins [156]. ...
Scorpionism is a serious public health problem in tropical areas, especially in Africa, southern India, the Middle East, and Latin America. There are about 12 species of scorpions that are dangerous to humans and all belong to the Buthidae family. In particular, venom on scorpion Androctonus australis hector is particularly toxic to humans. Serotherapy uses antibodies or fragments of antibodies to target the neurotoxins of the scorpion venom. Nanobodies, camelid-derived antibodies, are more effective than the conventional fragments of antibodies due to their low molecular weight (15 kDa). The Laboratoire des Venins et Molécules Thérapeutiques of the Pasteur Institute of Tunis (LVMT-IPT, Tunis, Tunisia) has produced the bispecific nanobodies against the neurotoxins of the Androctonus australis hector scorpion.In this doctoral project, the production of the bispecific nanobodies NbF12-10 and CH10-12 as recombinant proteins in Escherichia coli was studied. Two clones of the NbF12-10 strain were used (NN and NO), and strain E. coli WK6 was used as reference. The four strains were characterized in rich medium (TB) and defined minimal medium (MM) in shake-flask cultures. In TB, all strains grow at an average µ=0.4h-1. The strains WK6 and CH10-12 had a µmax=0.6h-1 in MM. The clone NbF12-10 NO could not grow in MM, and the clone NbF12-10 NN had a µmax=0.2h-1 in MM. The yield YX/S was 0.4g/g in MM for all strains and the yield of acetate on glucose was between 0.06 and 0.14g/g. The periplasmic extraction of the nanobodies NbF12-10 and CH10-12 was tested in TB, an improvement of 300% in the release of periplasmic proteins was achieved, compared to conventional methods. The strain CH10-12 produced 20-fold higher nanobody than the strain NbF12-10 NN (1.58 vs 0.08mg/L). The quantification of the nanobody was made through a densitometry protocol developed during this thesis. The protocol uses a processing image program (ImageJ) to quantify the optical density profiles of electrophoresis gels. The band of 50kDa of the molecular weight marker was used as reference for 750ng of recombinant protein. This was the subject of an article published in MicrobiologyOpen (Wiley).The production of the nanobodies CH10-12 and NbF12-10 were tested in bioreactor cultures in high cell density cultures in MM (>25gcdw/L) through a specific fed-batch strategy. The effect of the temperature (28–37°C) during protein expression and the duration of the induction phase (6–38h) were studied. The lower temperatures (28°C, 30°C) produced the highest titer of nanobody CH10-12 (2.3mg/L) after 10h and 12h of induction, respectively. In high temperature cultures (33°C, 37°C) the production of the nanobody was lower (0.4, 0.2mg/L). In cultures where the induction phase was longer (>30h), the production of the nanobody CH10-12 was hampered and reached a plateau after 10h (32°C, 0.7mg/L) to 25h (29°C, 1.4mg/L). The nanobody NbF12-10 also reached a plateau after 10h of induction (0.7mg/L). The metabolic burden was lowered by decreasing the temperature of induction, allowing the production of higher titers of recombinant nanobody. The effect of the low specific growth rate (0.02h-1) and the long exposure to the inducer (IPTG, >30h) could have produced a stringent response in the strain. High maintenance metabolism was found during the induction phase, and the consumption of citrate could hint an inadequate composition of the culture medium for the induction phase. Two non-specific proteins were found in the purified nanobody samples: a low molecular weight (11kDa), degradation product of the nanobody caused by a high temperature, and a high molecular weight protein (84kDa), protein aggregate produced as a response to an inadequate medium composition and the low specific growth rate. The generation of unknown proteins in the purified samples of nanobody could be a response of the strain to the low specific growth rate and the long exposure time to the inducer.
... At this time, the recombinant protein expression was induced with 1 mM isopropyl-thiogalactopyranoside and incubation for at least 16 h at 28°C. From the periplasmic compartment, proteins were extracted by osmotic shock [39] and loaded on a Ni-NTA super-flow Sepharose column (Qiagen). The purity of His-6 tagged NbF12-10 was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the final yield was monitored from the UV absorption at 280 nm and the theoretical extinction coefficient of the Nb, calculated from its amino acid content, as previously described [35]. ...
... At this time, the recombinant protein expression was induced with 1 mM isopropyl-thiogalactopyranoside and incubation for at least 16 h at 28°C. From the periplasmic compartment, proteins were extracted by osmotic shock [39] and loaded on a Ni-NTA super-flow Sepharose column (Qiagen). The purity of His-6 tagged NbF12-10 was checked by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and the final yield was monitored from the UV absorption at 280 nm and the theoretical extinction coefficient of the Nb, calculated from its amino acid content, as previously described [35]. ...
... The first scorpion recombinant toxins used in the immunization of animals were expressed as N-or C-terminal fusion proteins that increased protein solubility and facilitated their purification. Zahar et al. (1996) expressed the a-toxin ...
... The first subgroup of the anti-insect scorpion toxins is depressant toxins that induce a slow progressive onset of flaccid paralysis in insect larvae, while the second one is classified by excitatory toxins that induce a fast, spastic and excitatory contraction paralysis resulting from repetitive action potentials [13]. These scorpion neurotoxins are toxic to insects but not mammals [14][15][16]. Consequently, anti-insect scorpion toxins have great potential for increasing pest resistance in plants. ...
... Deaths within each cohort of four mice were monitored 24 h after injection. The LD 50 was determined as the amount of toxin whereby 50 % of the mice survived the injection [17,29]. All assays were repeated three times to assess the reproducibility. ...
Envenoming following scorpion sting is a common emergency in many parts of the world. During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim causing serious medical problems. The exploration of toxin structure-function relationship would benefit from the generation of soluble recombinant scorpion toxins in Escherichia coli. We developed an in vitro wheat germ translation system for the expression of the highly toxic Androctonus australis hector (AahII) protein that requires the proper formation of four disulfide bonds. Soluble, recombinant N-terminal GST-tagged AahII toxin is obtained in this in vitro translation system. After proteolytic removal of the GST-tag, purified recombinant AahII toxin, which contains two extra amino acids at its N terminal relative to the native AahII, is highly toxic after intracerebroventricular injection in Swiss mice. An LD50-value of 10 ng (or 1.33 pmol), close to that of the native toxin (LD50 of 3 ng) indicates that the wheat germ in vitro translation system produces properly folded and biological active recombinant AahII. In addition, NbAahII10, a camel single domain antibody fragment, raised against the native AahII toxin, recognizes its cognate conformational epitope on the recombinant toxin and neutralizes the toxicity of purified recombinant AahII upon injection in mice.
... In summary, after the clones had grown to a logarithmic phase, expression was induced by 1 mM IPTG at 37°C, overnight. The periplasmic proteins were obtained by osmotic shock (23). Then, they were loaded on a His-Select column (Sigma-Aldrich) for immobilized-metal affinity chromatography (IMAC). ...
Hemiscorpius lepturus scorpionism poses one of the most dangerous health problems in many parts of the world. The common therapy consists of using antivenom antibody fragments derived from a polyclonal immune response raised in horses. However, this immunotherapy creates serious side effects, including anaphylactic shock sometimes even leading to death. Thus, many efforts have been made to introduce new replacement therapeutics that cause less adverse reactions. One of the most attractive approaches to replacing the available therapy is offered by single-domain antibody fragments, or nanobodies (Nbs). We immunized dromedaries with H. lepturus toxin and identified a functional recombinant Nb (referred to as F7Nb) against heminecrolysin (HNc), the major known hemolytic and dermonecrotic fraction of H. lepturus venom. This Nb was retrieved from the immune library by phage display selection. The in vitro neutralization tests indicated that 17.5 nmol of the F7Nb can inhibit 45% of the hemolytic activity of 1 EC100 (7.5 μg/ml) of HNc. The in vivo neutralization tests demonstrated that F7Nb had good antihemolytic and antidermonecrotic effects against HNc in all tested mice. Surprisingly, F7Nb (8.75 nmol) neutralized 1 LD100 of HNc (10 μg) via an intracerebroventricular route or 1 LD100 (80 μg) via a subcutaneous route. All of the control mice died. Hence, this Nb is a potential leading novel candidate for treating H. lepturus scorpionism in the near future.-Yardehnavi, N., Behdani, M., Pooshang Bagheri, K., Mahmoodzadeh, A., Khanahmad, H., Shahbazzadeh, D., Habibi-Anbouhi, M., Hassanzadeh Ghassabeh, G., Muyldermans, S. A camelid antibody candidate for development of a therapeutic agent against Hemiscorpius lepturus envenomation.
... Previous studies have shown that this type of expression system is particularly well adapted to disulfide rich proteins. It has been used to express erabutoxin and BotXIV insect specific toxin of the scorpion Buthus occitanus tunetanus (14). Hence, it seems that the entire recombinant product expectedly goes to the inclusion bodies. ...
Scorpion venom is important and rich source of peptides, most of which have been widely used as pharmacological tools for unraveling structure-function relationship of various ion channels. Naturally occurring toxins can be also considered as lead compounds in the development of novel drugs.
In this context, the scorpion-derived peptide neurotoxins specific to sodium channels have shown promise as potential therapeutic targets for the treatment of various human diseases.
A cDNA library from the extracted RNA was constructed using RT-PCR and semi-nested RT-PCR. DNA sequencing followed by phylogenetic analysis was applied to screen the cDNA library clones. For molecular characterization of the BMK gene we used cloning and recombinant protein expression techniques based on E.coli systems. Then we performed mice immunization and Western blot and Immunodot analyses.
A novel BMK neurotoxin has been cloned, expressed and characterized from the Iranian scorpion M. eupeus venom. We analyzed the recombinant BMK by immunoblotting with treated antiserum. The result showed that mice antiserum can react also with scorpion crude venom, so is able to recognize native BMK toxin.
The newly produced recombinant protein BMK revealed to be immunogenic. Moreover, anti-BMK antibodies produced in mice were able to recognize both the recombinant BMK neurotoxin and the one in M. eupeus crude venome. Taken together, the molecular characterization and recombinant production of the Iranian scorpion M. eupeus venom component can serve as a new probe for further studies of sodium channels function and physiology. This provides a promising perspective for the future design of selective drugs, as well as for research of antivenom production.
... Finally, we observed no cross-reaction with serum raised against β-type toxins, thereby distancing the Buthus occitanus Amoreux scorpion from its New World brethren. Typically, the immunological classification of scorpion toxins is superimposed on the structural and pharmacological characteristics, primarily because particular epitopes are implicated in both immunological cross reactivity and mode of action (Bouhaouala-Zahar et al. 1996). As such, we propose that the toxins from the Buthus occitanus venoms of French origin studied here are closely related in sequence to those present in Buthus species from North Africa and Asia, taking in mind that identical MS data may also result from a few amino acid changes or extensive amino acid replacements. ...
... Non toxic recombinant BotXIV fused with synthetic ZZ domain of protein A displayed antigenic cross-reactivity with BotI and homologous toxins. When used as immunogen, it induced protection in mice against BotI and the Bot G-50 toxic fraction [4]. ...
Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins.
In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion α-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.
... Although higher dosages could not be tested, data from the previous studies appear to corroborate this result as m-agatoxin IV showed no toxicity when injected intracerebroventricularly in mice [46] and was shown to be selective for insect sodium channels [47,48]. As the toxicity of mammalian toxins is more likely to be in the submicrogram range, d-palutoxins can arguably be considered insect-specific toxins [49]. However, injection of d-paluIT2 at the same concentration in mouse brain resulted in leg paralysis of a short duration, which suggests that this group of toxins may recognize a receptor site common to both insects and mammals, with different relative affinities according to their primary sequence. ...
Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named δ-palutoxins IT1 to IT4. The four toxins are homologous 36–37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The δ-palutoxins are highly homologous with the previously described µ-agatoxins and curtatoxins (77–97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9–50 µg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the δ-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that δ-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion α and α-like toxins and is thus likely to bind to channel receptor site 3. However, δ-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. δ-Palutoxins are thus proposed as new insecticidal toxins related to the α and α-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.
... Different studies show that some epitopes are probably both implicated in toxins immunological cross-reactivity and/or common mode of action (Bouhaouala-Zahar et al., 1996;Fourquet et al., 1988). So far, the immunological classification of the toxins might be superimposed on the pharmacological one (Table 1). ...
In this study, we have characterized the immunological and pharmacological properties of the three major alpha-type toxins from the scorpion Androctonus amoreuxi, AamH1, AamH2 and AamH3, which were previously described as putative toxins from cDNAs [Chen, T. et al., 2003. Regul. Pept. 115, 115-121]. The immunological tests (ELISA, RIA) have demonstrated that AamH1, AamH2 and AamH3 belong to the immunological groups 3 and 4 of alpha-type toxins. Analysis of the three toxin effects on currents through rat brain (rNav1.2), rat muscle (rNav1.4) and Drosophila (DmNav1) sodium channels expressed in Xenopus oocytes revealed that AamH1 and AamH2, but not AamH3, have anti-insect and anti-mammal activities and can be classified as alpha-like toxins. While AamH1 removes fast inactivation only in neuronal rNav1.2 channel and has no effect on muscular rNav1.4 channel, AamH2 affects both neuronal rNav1.2 and muscular rNav1.4 channels. AamH3 was lethal to mice by intracerebroventricular injection despite its lack of activity on the neuronal rNav1.2 channel. Finally, we have shown that the A. amoreuxi venom was better neutralized by the antiserum raised against the venom of Buthus occitanus tunetanus than by the antisera raised against scorpion venoms from the same genus Androctonus.
... Non toxic recombinant BotXIV fused with synthetic ZZ domain of protein A displayed antigenic cross-reactivity with BotI and homologous toxins. When used as immunogen, it induced protection in mice against BotI and the Bot G-50 toxic fraction [4]. ...
Two toxin-like proteins (AahTL1 and AahTL3) were purified from the venom of the scorpion Androctonus australis Hector (Aah). AahTL1 and AahTL3 are the first non toxic proteins cross-reacting with AahI toxins group which indicates that these proteins can be used as a model of vaccins. In order to study structure-function relationships, their complete amino-acid sequences (66 residues) were determined, by automated Edman degradation. They show more than 50% of similarity with both AahI and AahIII antimammal toxins. Three-dimensional structural models of AahTL1 and AahTL3 constructed by homology suggest that the two proteins are structurally similar to antimammal scorpion alpha-toxins specific to voltage dependent Na+ channels. The models showed also that amino-acid changes between potent Aah toxins and both AahTL1 and AahTL3 disrupt the electrostatic potential gradient at their surface preventing their interaction with the receptor, which may explain their non toxicity.
... Although production yields can be up to 10-20 mg/L (Li et al. 2000), the refolding step is usually the limiting factor in obtaining sufficient amounts of active toxin, as the refolding yield can be less than 10% of the original product. Other strategies based on fusion proteins have permitted the production of soluble, folded forms of the toxins in E. coli using a thioredoxin reductase-deficient strain (Johnson et al. 2000) or two protein A IgGbinding domains (ZZ) leading to periplasmic expression (Bouhaouala-Zahar et al. 1996;Korolkova et al. 2001). A yeast expression system has also been used successfully with yields in the 8-10 mg/L range . ...
Acid-sensing ion channels (ASICs) are thought to be important ion channels, particularly for the perception of pain. Some of them may also contribute to synaptic plasticity, learning, and memory. Psalmotoxin 1 (PcTx1), the first potent and specific blocker of the ASIC1a proton-sensing channel, has been successfully expressed in the Drosophila melanogaster S2 cell recombinant expression system used here for the first time to produce a spider toxin. The recombinant toxin was identical in all respects to the native peptide, and its three-dimensional structure in solution was determined by means of (1)H 2D NMR spectroscopy. Surface characteristics of PcTx1 provide insights on key structural elements involved in the binding of PcTx1 to ASIC1a channels. They appear to be localized in the beta-sheet and the beta-turn linking the strands, as indicated by electrostatic anisotropy calculations, surface charge distribution, and the presence of residues known to be implicated in channel recognition by other inhibitor cystine knot (ICK) toxins.
... Thus, considering the difficulties in expressing functional small polypeptides in the cytosol of E. coli due to their degradation [18,32], our strategy was to produce rBotIII-OH and its mutants as fused proteins to ZZ domains [38]. The use of such system would allow the export of the fusion proteins to the periplasmic area where the machinery needed for the disulfide bond formation is located [6,15]. Approximately 1 mg/l of homogeneous soluble active rBotIII-OH and its mutants were obtained after factor Xa cleavage of ZZ-BotIII. ...
Alpha scorpion toxins bind to receptor site 3 on voltage-dependent sodium channels and inhibit their inactivation. The alpha-scorpion toxin BotIII is the most toxic protein of Buthus occitanus tunetanus. Its sequence differs only by three amino acid residues from that of AahII, the most active alpha-toxin. Due to their high affinity and selectivity for mammalian sodium channels, BotIII and AahII represent powerful tools for studying the molecular determinants of specificity for voltage-dependent sodium channels. Sequence analysis of BotIII gene has revealed two exons separated by a 381-bp intron and a signal peptide of 19 amino acids. We succeeded in expressing BotIII in significantly higher amounts than AahII the only expressed strict alpha anti-mammalian scorpion toxin reported in the literature. We have also modified specific amino acid residues of BotIII. The recombinant and the natural toxins differ by the amidation of the C-terminal residue. Toxicity and binding experiments indicated: (a) the affinity of rBotIII-OH and rAahII-OH (rBotIII-OH with the 3 mutations R10V, V51L, N64H) for the voltage-dependent sodium channels is reduced compared to the natural toxins. This data revealed the important role of the C-terminal amidation for the biological activity of BotIII and AahII; (b) the single mutation N64H is responsible for the difference of toxicity and affinity between rBotIII-OH and rAahII-OH; (c) the addition of the sequence GR to rBotIII-OH leads to the loss of biological activity. This study is in agreement with the important role attributed to the C-terminal sequence of alpha-toxins in their interaction with sodium channels receptors.
... Expression assays were explored for BotIT2. Recombinant BotIT2 was expressed in HB101/ E. coli strain, as a fusion protein with two IgG-Binding (ZZ) domains derived from protein A of Staphylococcus aureus using the expression vector, pEZZ18 26,27,28,29 . ...
Numerous toxins from scorpion venoms are much more toxic to insects than to other animal classes, and possess high affinity to Na+ channels. Many of them active on insects were purified from the venom of Buthus occitanus tunetanus. Using amino acid sequences of BotIT2 and RACE-PCR amplification (Rapid amplification of cDNA ends) technique, we isolated, identified and sequenced the nucleotide sequence from the venom glands of the scorpion Buthus occitanus tunetanus. The cDNA encodes a precursor of an insect toxin of 60 amino acid residues. The deduced nucleotide sequence toxin was identical to the determined amino acid sequence of BotIT2. BotIT2 is more similar to the excitatory toxins in its mode of action and to the depressant toxins in its primary structure.
Background:
Scorpion neurotoxins such as those that modify the mammalian voltage-gated sodium ion channels (Nav) are the main responsible for scorpion envenomation. Their neutralization is crucial in the production of antivenoms against scorpion stings.
Methods:
In the present study, two in silico designed genes - one that codes for a native neurotoxin from the venom of the Anatolian scorpion Androctonus crassicauda, named Acra 4 - and another non-native toxin - named consensus scorpion toxin (SccTx) obtained from the alignment of the primary structures of the most toxic neurotoxins from the Middle Eastern and North African scorpions - were recombinantly expressed in E. coli Origami.
Results:
Following bacterial expression, the two expressed neurotoxins, hereafter named HisrAcra4 and HisrSccTx, were obtained from inclusion bodies. Both recombinant neurotoxins were obtained in multiple Cys-Cys isoforms. After refolding, the active protein fractions were identified with molecular masses of 8,947.6 and 9,989.1 Da for HisrAcra4 and HisrSccTx, respectively, which agreed with their expected theoretical masses. HisrAcra4 and HisrSccTx were used as antigens to immunize two groups of rabbits, to produce either anti-HisrAcra4 or anti-HisrSccTx serum antibodies, which in turn could recognize and neutralize neurotoxins from venoms of scorpion species from the Middle East and North Africa. The antibodies obtained from rabbits neutralized the 3LD50 of Androctonus australis, Leiurus quinquestriatus hebraeus and Buthus occitanus venoms, but they did not neutralize A. crassicauda and A. mauritanicus venoms. In addition, the anti-HisrAcra4 antibodies did not neutralize any of the five scorpion venoms tested. However, an antibody blend of anti-HisrAcra4 and anti-HisrSccTx was able to neutralize A. crassicauda and A. mauritanicus venoms.
Conclusions:
Two recombinant Nav neurotoxins, from different peptide families, were used as antigens to generate IgGs for neutralizing scorpion venoms of species from the Middle East and North Africa.
Snakes, scorpions, and spiders are venomous animals that pose a threat to human health, and severe envenomings from the bites or stings of these animals must be treated with antivenom. Current antivenoms are based on plasma-derived immunoglobulins or immunoglobulin fragments from hyper-immunized animals. Although these medicines have been life-saving for more than 120 years, opportunities to improve envenoming therapy exist. In the later decades, new biotechnological tools have been applied with the aim of improving the efficacy, safety, and affordability of antivenoms. Within the avenues explored, novel immunization strategies using synthetic peptide epitopes, recombinant toxins (or toxoids), or DNA strings as immunogens have demonstrated potential for generating antivenoms with high therapeutic antibody titers and broad neutralizing capacity. Furthermore, these approaches circumvent the need for venom in the production process of antivenoms, thereby limiting some of the complications associated with animal captivity and venom collection. Finally, an important benefit of innovative immunization approaches is that they are often compatible with existing antivenom manufacturing setups. In this review, we compile all reported studies examining venom-independent innovative immunization strategies for antivenom development. In addition, a brief description of toxin families of medical relevance found in snake, scorpion, and spider venoms is presented, as well as how biochemical, bioinformatic, and omics tools could aid the development of next-generation antivenoms.
Key Contribution: An exhaustive compilation of the studies exploring innovative immunization strategies for antivenom development, mainly focusing on venom-independent approaches.
Scorpion venom are composed mainly of bioactive proteins and peptides that may serve as lead compounds for the design of biotechnological tools and therapeutic drugs. However, exploring the therapeutic potential of scorpion venom components is mainly impaired by the low yield of purified toxins from milked venom. Therefore, production of toxin-derived peptides and proteins by heterologous expression is the strategy of choice for research groups and pharmaceutical industry to overcome this limitation. Recombinant expression in microorganisms is often the first choice, since bacteria and yeast systems combine high level of recombinant protein expression, fast cell growth and multiplication and simple media requirement. Herein, we present a comprehensive revision, which describes the scorpion venom components that were produced in their recombinant forms using microbial systems. In addition, we highlight the pros and cons of performing the heterologous expression of these compounds, regarding the particularities of each microorganism and how these processes can affect the application of these venom components. The most used microbial system in the heterologous expression of scorpion venom components is Escherichia coli (85%), and among all the recombinant venom components produced, 69% were neurotoxins. This review may light up future researchers in the choice of the best expression system to produce scorpion venom components of interest.
Spiders and scorpions are notorious for their fearful dispositions and their ability to inject venom into prey and predators, causing symptoms such as necrosis, paralysis, and excruciating pain. Information on venom composition and the toxins present in these species is growing due to an interest in using bioactive toxins from spiders and scorpions for drug discovery purposes and for solving crystal structures of membrane-embedded receptors. Additionally, the identification and isolation of a myriad of spider and scorpion toxins has allowed research within next generation antivenoms to progress at an increasingly faster pace. In this review, the current knowledge of spider and scorpion venoms is presented, followed by a discussion of all published biotechnological efforts within development of spider and scorpion antitoxins based on small molecules, antibodies and fragments thereof, and next generation immunization strategies. The increasing number of discovery and development efforts within this field may point towards an upcoming transition from serum-based antivenoms towards therapeutic solutions based on modern biotechnology.
Toxic proteins from animal venoms usually act on molecular targets that are critically involved in the function of a physiological system of a prey. With the ultimate view of designing new pharmacological tools as well as original drugs acting on these targets, animal toxins are the subject of extensive molecular analyses. In particular, the topographies by which toxins bind to their targets and hence affect the function of the associated physiological system clearly need to be identified.
Scorpions use a cocktail of toxins to immobilize their prey. Their venoms constitute a complex mixure 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. 1)The a long << chain toxins >> (60-70 amino acids residues cross-linked by 3 disulfide-bridges) affect exclusively voltage-dependent Na; channels of excitable cells from mammals and insects; 2) The << short chain toxins >> (30-40 amino acids residues cross-linked by 3 or 4 disulfide-bridges) block several types of K+ channels in different cells. At the structural level, scorpion toxins show a dense core of secondary elements, 2 1/2 turns of an alpha-helix, and a short segment of anti-parallel beta-sheet, already found in all known structures of scorpion toxins, irrespective of their size. sequence and function. From cDNA libraries, full-lengh cDNAs encoding precursors of these toxins have been isolated and could be used in heterologous expression systems, in order to produce recombinant toxins. They will provide a template for the design of new biopesticide agents, able to mimic the interactive surface of the toxins.
The following studies on molecular biology of scorpion toxin genes were reviewed: (1) Strategies of cloning cDNAs or genomic genes encoding scorpion toxins; (2) scorpion toxin cDNAs and postprotein processing of precursor toxins; (3) Structural organization and pre-mRNA processing of genomic genes encoding scorpion toxins; (4) Expression of recombinant scorpion toxin genes in cells or organisms.
To study the structural variability of the Os3 neurotoxin from the venom of scorpion Orthochirus scrobiculosus inhabiting Uzbekistan and Turkmenia, a cloning method for the family of cDNAs that encode the highly homologous Os3-like polypeptides was developed. The Turkmenian scorpion venom was shown to contain a family of closely related Os3-like polypeptides. Mass spectrometry indicated that crude venoms from scorpions inhabiting these two regions of Central Asia differ in the amino acid composition of their toxic components depending on the habitat.
Les toxines de venins de scorpion sont des mini-protéines basiques, de 60–70 résidus d'acides aminés, possédant quatre ponts disulfure dans le cas des toxines modulant les canaux Na+ voltage-dépendants des cellules excitables, et de 30–40 résidus, avec trois ou quatre ponts disulfure, pour les toxines bloquant divers canaux K+. Leurs structures tridimensionnelles montrent l'existence d'une hélice α et d'un feuillet β hautement conservés. Une approche génétique a isolé les ADNc codant pour leurs précurseurs, puis décrypté les séquences promotrices et structurales de leurs gènes. Des activités diverses sur les canaux Ca2+ et Cl−1 coexistent aussi dans les venins de scorpion.
Long chain scorpion toxins (made of 60 to 70 amino acids) acting on voltage-gated sodium channels in excitable cells are responsible for human envenomation, and comprise a-toxins that inhibit sodium current inactivation and p-toxins, that modify the activation process. These toxins may be further divided according to their pharmacological activities. Thus, a-toxins highly active on mammals or insects, as well as α like toxins may be distinguished within the α-toxin class. The β-toxin class includes toxins active on mammals and, as a separate group, the excitatory and depressant toxins active exclusively on insects. All these toxins possess 4 disulfide bridges and share 15 similar non cystine residues. Accordingly, their 3D structure is highly conserved, comprising an α-helix and a triple stranded β-sheet. The most solvent exposed turns of this structure are prone to insertions or deletions, and accordingly correspond to the most structurally variable regions of the toxins. They have been tested by chemical modification (on several toxins) and site-directed mutagenesis analysis (of LqhαIT) for their possible involvement in the interactions with sodium channels.
Significant progress has been made in immunological studies of scorpion toxins and several formats of antibodies directed against scorpion toxins have been reported. Some of these are commonly used in a specific treatment against envenoming; others are primarily used for immuno-biochemical characterizations. The preparation protocol of the antibody or its fragments can be substantially different from one laboratory to another, which complicates a direct comparison of the potency of the antivenom. The use of immune sera, the total immunoglobulin fraction or Fab and Fab'2 fragments as the therapeutic agent is widespread. A number of monoclonal antibodies have also been reported and used for engineering of Fv, ScFv or Fab fragments. Recently, a novel antibody format - known as nanobodies - derived from HCAbs of camelids and selected after phage display shows great potential to provide a more efficient therapy against scorpion envenoming. Subsequent bispecific derivatives have been designed and their pharmacokinetics have been studied. Distinct advantages and disadvantages have been attributed to these equine, murine or camelid antibodies and their derived fragments. Some fragments are easily amenable into next generation therapeutics after proper manufacturing and provide an ensured availability of the product to the medical community. Through examples, we will show how the comparison of the serotherapeutic effectiveness is compromised due to the absence of standardization, on the preparation of immunogens, production processes and / or nature of the products. We will report on recent advances in the field.
Peptidyl scorpion toxins are known to block diverse types of K+ channels with high affinity and, thus, can be used as powerful tools to study the physiological role of the ionic selectivity, and the architecture of the pore-region of this class of channels. Yet, diversity among K+ channels is large and there has been a profusion of research for new selective ligands in order to elucidate their mechanisms of action and pharmacology significance. Scorpion toxins active on K+ channels are short polypeptides of about 30 to 40 amino acid residues, cross-linked by three or four disulfide bridges. They display a high degree of primary sequence homologies. 1H Nuclear Magnetic Resonance (NMR) analysis has demonstrated that these toxins are composed of an -helix and a two-stranded antiparallel -sheet, linked by two disulfide bridges. This structural motif is also found in the insect defensins. A 370 bp cDNA encoding the Kaliotoxin 2 (KTX2) precursor (a 37 amino acid residues peptide purified from the North African scorpion Androctonus australis and acting as a high affinity blocker of K+ channels) was obtained by PCR amplification and the organization of the KTX2 precursor depicted. This precursor is composed of a signal peptide followed by the mature toxin. The transcriptional unit and the promotor region of the gene encoding KTX2 was then amplified from the genomic DNA of Androctonus australis and its sequence determined. A single intron of 87 bp, located close to the region encoding the C-terminal part of the signal peptide, was found. Its A+T content was particularly high (up to 86%). The transcription unit of the gene was 390 bp long. Regulatory consensus sequences were identified. The genes of scorpion short toxins active on K+ channels are organized similarly to those of the scorpion long toxins active on Na+ channels and not like those of structurally related insect defensins, which are intronless.
During scorpion envenoming, highly toxic small polypeptides of the venom diffuse rapidly within the victim, causing serious
medical problems. Nanobodies (Nbs), the recombinant single-domain antigen-binding fragments of camel-specific heavy-chain
only antibodies, offer special advantages in therapy over classic antibody fragments due to their robustness and smaller size,
matching the size of the scorpion toxins. Recently, a potent AahII scorpion toxin-neutralizing Nb was identified. However,
this NbAahII10 contains a single Cys in its first antigen-binding loop, leading to Nb dimerization upon prolonged storage.
In this work, we first investigate the efficacy of NbAahII10 variants in which this Cys was substituted by Ala, Ser or Thr.
Second, the NbAahII10 Cys/Ser mutant displaying the best functional properties is subsequently humanized. It is demonstrated
that the maximally humanized version of NbAahII10 Cys/Ser maintains its high affinity for the antigen without conceding much
on expression yield and stability. More importantly, its neutralizing capacity is preserved as all mice survive injections
of seven LD50 and 50% of mice survived nine LD50 of the scorpion toxin. Thus, this humanized Nb is the best candidate to develop a therapy in human against the most toxic
venom compound of one of the most dangerous scorpions.
Scorpion venom, containing highly toxic, small polypeptides that diffuse rapidly within the patient, causes serious medical problems. Nanobodies, single-domain antigen-binding fragments derived from dromedary heavy-chain antibodies, have a size that closely matches that of scorpion toxins. Therefore these nanobodies might be developed into potent immunotherapeutics to treat scorpion envenoming. Multiple nanobodies of sub-nanomolar affinity to AahII, the most toxic polypeptide within the Androctonus australis hector venom, were isolated from a dromedary immunized with AahII. These nanobodies neutralize the lethal effect of AahII to various extents without clear correlation with the kinetic rate constants kon or koff, or the equilibrium dissociation constant, KD. One particular nanobody, referred to as NbAahII10, which targets a unique epitope on AahII, neutralizes 7 LD50 of this toxin in mice, corresponding to a neutralizing capacity of approx. 37000 LD50 of AahII/mg of nanobody. Such high neutralizing potency has never been reached before by any other monoclonal antibody fragment.
A novel peptide named Pg8 was purified from the venom of the South African scorpion Parabuthus granulatus and its primary structure was determined. It contains 63 amino acid residues tightly folded by 4 disulfide bridges. The gene coding for this peptide was cloned from a cDNA library. By recursive PCR strategy a hybrid gene was constructed having a factor X recognition site for proteolysis and a modified sequence for preferential codon usage of E. coli. A pQE30 molecular vector already contained a His-tag was used for expression. This construction was expressed in BL21 and Origami strains. The fusion protein from inclusion bodies was separated by HPLC (yield approximately 5mg/L) and properly folded in vitro. Lethality tests showed that the recombinant peptide was toxic and was used to immunize mice. A volume of 0.25ml of the anti-serum produced was capable of protecting up to 3 LD(50) doses of pure toxin Pg8 but also, and more importantly, the entire soluble venom.
Scorpion stings are a public health problem in Brazil, with most incidents involving the species Tityus serrulatus. Some T. serrulatus toxins may act as immunogens for the production of a specific anti-venom, but many of the component toxins remain poorly characterized. Here, we describe the immunological characteristics of the toxin Ts1 (also known as TsVII and Ts-gamma) and evaluate production of neutralizing antibodies against the crude venom of T. serrulatus. Recombinant Ts1 with one copy (Ts1(1)) or two copies in tandem (Ts1(2)) was expressed in BL21 (DE3) cells. Rabbits and mice were immunized with the recombinant proteins (inclusion bodies) and then tested for production of neutralizing antibodies. Neutralization assays showed that anti-Ts1(1) and anti-Ts1(2) protected animals challenged with T. serrulatus crude venom and native Ts1. Thus, Ts1 could be used in a mixed "cocktail" of immunogens for T. serrulatus anti-venom production.
The selective toxicity of depressant scorpion neurotoxins to insects is useful in studying insect sodium channel gating and has an applied potential. In order to establish a genetic system enabling a structure-activity approach, the functional expression of such polypeptides is required. By engineering the cDNA encoding the depressant scorpion neurotoxin, LahIT2, behind the T7 promoter, large amounts of recombinant insoluble and nonactive toxin were obtained in Escherichia coli. Following denaturation and reduction, the recombinant protein, constructed with an additional N-terminal methionine residue, was subjected to renaturation. Optimal conditions for reconstitution of a functional toxin, having a dominant fold over many other possible isoforms, were established. The recombinant active toxin was purified by RP-HPLC and characterized. Toxicity (ED50) to insects, binding affinity (IC50) to an insect receptor site, and electrophysiological effect on an insect axonal preparation were found to be similar to those of the native toxin. Substitution of the C-terminal glycine by a Gly-Lys-Lys triplet did not abolish folding but affected toxicity (3.5-fold decrease) of LqhIT2. Apparently, this efficient bacterial expression system (500 micrograms HPLC-purified toxin/1 liter E. coli culture) provides the means for studying structure/ activity relationship and the molecular basis for the phylogenetic selectivity of scorpion depressant neurotoxins.
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.
An immunogenic nontoxic protein (TsNTxP) was purified from the venom of the scorpion Tityus serrulatus (Ts). This peptide is composed of 63 amino acid residues with a high degree of structural homology with the toxins isolated from Ts. The nucleotide sequence of the gene that encodes TsNTxP was obtained and also showed a high degree of similarity with genes encoding Tityus toxins [Guatimosim, S.C.F., Prado, V.F., Diniz, C.R., Chávez-Olórtegui, C.. Kalapothakis, E., 1999. Molecular cloning and genomic analysis of TsNTxP; an immunogenic protein from Tityus serrulatus scorpion venom. Toxicon 37, 507-517]. In the present study the TsNTxP gene was expressed in E. coli BL21DE3 cells as a fusion protein with maltose-binding protein. The recombinant protein (TsNTxPrec) was purified by affinity chromatography and used as an immunogen in rabbits. The antigenic specificity of anti-TsNTxPrec antibodies was compared by an enzyme-linked immunosorbent assay using TsNTxP, TstFG50 (the fraction of Ts venom that represents most of the toxicity of the crude venom) and the crude venom, to coat microtitration plates. Anti-TsNTxPrec antibodies had a comparable high cross-reactivity for all antigens tested. Concentrations of Ts venom equivalent to 20 LD50 were effectively neutralized by 1 ml of the anti-TsNTxPrec serum. This result provides basic data for the use of such recombinant scorpion protein as an immunogen in the development of antivenoms for clinical use.
Three-finger proteins form a structurally related family of compounds that exhibit a great variety of biological properties. To address the question of the prediction of functional areas on their surfaces, we tentatively conferred the acetylcholinesterase inhibitory activity of fasciculins on a short-chain curaremimetic toxin. For this purpose, we assimilated the three-dimensional structure of fasciculin 2 with the one of toxin alpha. This comparison revealed that the tips of the first and second loops, together with the C terminus residue, deviated most. A first recombinant fasciculin/toxin alpha chimera was designed by transferring loop 1 in its entirety together with the tip of loop 2 of fasciculin 2 into the toxin alpha scaffold. A second chimera (rChII) was obtained by adding the point Asn-61 --> Tyr substitution. Comparison of functional and structural properties of both chimeras show that rChII can accommodate the imposed modifications and displays nearly all the acetylcholinesterase-blocking activities of fasciculins. The three-dimensional structure of rChII demonstrates that rChII adopts a typical three-fingered fold with structural features of both parent toxins. Taken together, these results emphasize the great structural flexibility and functional adaptability of that fold and confirm that structural deviations between fasciculins and short-chain neurotoxins do indeed reflect functional diversity.
Four novel insecticidal toxins were isolated from the venom of the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) and named delta-palutoxins IT1 to IT4. The four toxins are homologous 36-37 amino acid peptides reticulated by four disulfide bridges and three have amidated C-terminal residues. The delta-palutoxins are highly homologous with the previously described mu-agatoxins and curtatoxins (77-97%). The four peptides demonstrated significant toxicity against larvae of the crop pest Spodoptera litura (Lepidoptera: Noctuidae) in a microinjection bioassay, with LD50 values in the 9-50 microg per g of insect range. This level of toxicity is equivalent to that of several of the most active scorpion toxins used in the development of recombinant baculoviruses, and the delta-palutoxins appear to be insect specific. Electrophysiological experiments demonstrated that delta-palutoxin IT1, the most active toxin acts by affecting insect sodium channel inactivation, resulting in the appearance of a late-maintained sodium current, in a similar fashion to insecticidal scorpion alpha and alpha-like toxins and is thus likely to bind to channel receptor site 3. However, delta-palutoxin IT1 was distinguished by its lack of effect on peak sodium conductance, on the early phase of sodium current inactivation and the absence of a shift in the activation voltage of the sodium channels. delta-Palutoxins are thus proposed as new insecticidal toxins related to the alpha and alpha-like scorpion toxins. They will be useful both in the development of recombinant baculoviruses in agrochemical applications and also as molecular probes for the investigation of molecular mechanisms of insect selectivity and structure and function of sodium channels.
The isolation of the peptide inhibitor of M-type K+ current, BeKm-1, from the venom of the Central Asian scorpion Buthus eupeus has been described previously (Fillipov A. K., Kozlov, S. A., Pluzhnikov, K. A., Grishin, E. V., and Brown, D. A. (1996)
FEBS Lett. 384, 277–280). Here we report the cloning, expression, and selectivity of BeKm-1. A full-length cDNA of 365 nucleotides
encoding the precursor of BeKm-1 was isolated using the rapid amplification of cDNA ends polymerase chain reaction technique
from mRNA obtained from scorpion telsons. Sequence analysis of the cDNA revealed that the precursor contains a signal peptide
of 21 amino acid residues. The mature toxin consists of 36 amino acid residues. BeKm-1 belongs to the family of scorpion venom
potassium channel blockers and represents a new subgroup of these toxins. The recombinant BeKm-1 was produced as a Protein
A fusion product in the periplasm of Escherichia coli. After cleavage and high performance liquid chromatography purification, recombinant BeKm-1 displayed the same properties
as the native toxin. Three BeKm-1 mutants (R27K, F32K, and R27K/F32K) were generated, purified, and characterized. Recombinant
wild-type BeKm-1 and the three mutants partly inhibited the native M-like current in NG108-15 at 100 nm. The effect of the recombinant BeKm-1 on different K+ channels was also studied. BeKm-1 inhibited hERG1 channels with an IC50 of 3.3 nm, but had no effect at 100 nm on hEAG, hSK1, rSK2, hIK, hBK, KCNQ1/KCNE1, KCNQ2/KCNQ3, KCNQ4 channels, and minimal effect on rELK1. Thus, BeKm-1 was shown
to be a novel specific blocker of hERG1 potassium channels.
We report the use of a recombinant Loxosceles intermedia spider protein in the form of a fusion protein as an antigen for immunization in rabbits and mice. The aim is to produce model protective antisera in these animals against dermonecrotic and lethal activities of the venom from the Brazilian spider responsible for 3000 cases, reported annually, of spider bites in South Brazil. A protein homologous to the dermonecrotic toxin was cloned from a cDNA expression library made with L. intermedia venom glands, expressed in E. coli cells as a fusion protein with beta-galactosidase and the recombinant protein (Li-rec protein) was purified by molecular filtration and affinity chromatography [Kalapothakis et al., Toxicon (2002) in press]. The Li-rec protein was characterized and used as an antigen to generate antibodies in rabbits and mice. These specifically raised antibodies recognized the native venom. In vitro neutralization assay of lethal effects indicated that 1 ml of rabbit serum raised against Li-rec protein was able to neutralize 25 LD(50) of the whole venom. In vivo protection experiments, the fusion proteins induced a long-term protection in rabbits against the dermonecrotic activity of the native venom. Immunized mice were challenged with various doses of the Loxosceles venom. Mice were fully protected against 2.5 LD(50) of venom. This result provides basic data for the use of such recombinant spider proteins as immunogens in the development of anti-venoms for clinical use or can be used as a vaccine providing efficient immune protection against L. intermedia venom.
We have purified a new toxin (BmK 17[4]) from Asian scorption (Buthus martensii Karsch) venom that possesses a distinctive structural motif in its N-terminal (positions 8–12) that is similarly found in two other previously described α-like toxins. BmK 17[4] prolongs action potentials (APs) in frog nerve and was purified using gel filtration, ion exchange, fast protein liquid chromatography (FPLC), and high-performance liquid chromatography (HPLC). BmK 17[4] significantly prolonged frog APs but it did not alter APs from an insect ventral nerve cord at similar doses. When applied to voltage-clamped frog muscle single fibers, BmK 17[4] prolonged fast inactivation. Because the polypeptide prolongs APs when both K+ and Ca2+ channels were blocked, BMK 17[4] acts to selectively alter Na+ channel inactivation. The N-terminal sequence of BmK 17[4] was found to be VRDAYIAKPENCVYXC—. The molar mass of BmK 17[4] was determined by LC/MS/MS to be 7097 Daltons. The N-terminal motif (KPENC), which introduces a reverse turn in residues 8–12, does not appear in previously characterized BmK α-toxins and may be characteristic of α-like toxins. Sequence similarity database searches were used to test whether the N-terminal sequences of α-like polypeptide toxins from B. martensii Karsch possess a distinctive structural motif in its 5-residue reverse turn (α-turn) that is conserved. Sequence similarities with putative polypetides encoded by cDNAs obtained from a cDNA library [Zhu, S. Y., Li, W. X., Zenq, X. C., et al. (2000) Nine novel precursors of Buthus martensii scorpiox alpha-toxin homologues. Toxicon
38, 1653–1661] from BmK venom glands showed that an active polypeptide toxin cleaved from the putative propolypeptide toxin BmK M9 is likely identical to BmK 17[4]. Sequence comparisons with toxins and putative toxins from B. martensii Karsch and other species revealed that a group of these toxins possess a common structural motif in their α-turn. A neighbor-joining phylogenetic analysis suggests that there are two phylogenetic sister groups of related BmK polypeptides; one possesses the KPENC motif and the other possesses a modifed version (KPHNC) of it.
Three different immunogens from the venom of the Mexican scorpion Centruroides noxius Hoffmann were used to study protective antibody response in mice and rabbits, challenged with toxin Cn2, one of the most abundant toxic peptide of this venom. The immunogens were: Cn5, a crustacean specific toxin; a recombinant protein containing the peptide Cn5 linked to the maltose transporter and a sub-fraction (F.II.5) containing 25 distinct peptides, among which is Cn5. Mice immunized with these three preparations, when directly challenged with Cn2 presented no apparent protection, whereas anti-sera produced in rabbits with these three immunogens were capable of partially neutralizing the effect of Cn2, when injected into naive mice. Cn5 rabbit anti-serum showed a better protective effect on mice, than the rabbit sera obtained against the two other antigens. The subcutaneous route of challenging mice was shown to be better than intraperitoneal injections. Comparative structural analysis of Cn5 with other toxins of this venom showed that our results are important to be taken into consideration, when choosing appropriate immunogens aimed at the production of better anti-venoms or for the rational design of possible vaccines.
Gene duplication followed by positive Darwinian selection is an important evolutionary event at the molecular level, by which a gene can gain new functions. Such an event might have occurred in the evolution of scorpion sodium channel toxin genes (alpha- and beta-groups). To test this hypothesis, a robust statistical method from Yang and co-workers based on the estimation of the nonsynonymous-to-synonymous rate ratio (omega = d(N)/ d(S)) was performed. The results provide clear statistical evidence for adaptive molecular evolution of scorpion alpha- and beta-toxin genes. A good match between the positively selected sites (evolutionary epitopes) and the putative bioactive surface (functional epitopes) indicates that these sites are most likely involved in functional recognition of sodium channels. Our results also shed light on the importance of the B-loop in the functional diversification of scorpion alpha- and beta-toxins.
From a cDNA library made from telsons of scorpions of the species Androctonus australis, full-length cDNAs of about 370 nucleotides encoding precursors of toxins active on mammals or on insects have been isolated using oligonucleotide probes. Sequence analysis of the cDNAs revealed that the precursors contain signal peptides of about 20 amino acid residues. In addition, precursors of toxins active on mammals have extensions at their COOH-terminal ends: Arg or Gly-Arg. The processing steps required to generate toxins from their respective precursors are thus not identical for all of them. Southern blot analysis performed at the genomic level with a cDNA encoding toxin II suggested a single copy gene having a minimum size of 2800 base pairs. Finally, in an attempt to successfully express an animal toxin, monkey kidney COS-7 cells transfected with a plasmid harboring a cDNA encoding toxin II transiently expressed a recombinant toxin having the immunological and biological properties of toxin II.
Using site-directed mutagenesis, we previously identified some residues that probably belong to the site by which Erabutoxin
a (Ea), a sea snake toxin, recognizes the nicotinic acetylcholine receptor (AcChoR) (Pillet, L., Trémeau, O., Ducancel, F.
Drevet, P., Zinn-Justin, S., Pinkasfeld, S., Boulain, J.-C., and Ménez, A. (1993) J. Biol. Chem. 268, 909-916). We have now studied the effect of mutating 26 new positions on the affinity of Ea for AcChoR. The mutations
are F4A, N5V, H6A, Q7L, S9G, Q10A, P11N, Q12A, T13V, T14A, K15A, T16A, ΔS18, E21A, Y25F, Q28A, S30A, T35A, I36R, P44V, T45A,
V46A, K47A, P48Q, I50Q, and S53A. Binding affinity decreases upon mutation at Gln-7, Gln-10 and to a lesser extent at His-6,
Ser-9 and Tyr-25 whereas it increases upon mutation at Ile-36. Other mutations have no effect on Ea affinity. In addition,
new mutations of the previously explored Ser-8, Asp-31, Arg-33, and Glu-38 better explain the functional role of these residues
in Ea. The previous and present mutational analysis suggest that the “functional” site of Ea covers a homogeneous surface
of at least 680 A2, encompassing the three toxin loops, and includes both conserved and variant residues. The variable residues might contribute
to the selectivity of Ea for some AcChoRs, including those from fish, the prey of sea snakes.
The binding of the radioiodinated insect selective neurotoxin from the venom of the scorpion Androctonus australis (AaIT), to synaptic plasma membrane vesicles derived from osmotically shocked insect synaptosomes was studied under kinetic and equilibrium conditions. The integrity of these vesicles and the existence of membrane potential and its modifiability were demonstrated by assays of the uptake of the lipophilic cation tetraphenylphosphonium. It has been shown that 125I-labeled AaIT binds specifically and reversibly to a single class of noninteracting binding sites of high affinity () and low capacity (1.2–2.0 pmol/mg protein). The values of the rate association and dissociation constants k1 and k−1 are, respectively, 1.36 · 106 M−1 · s−1 and 1.9 · 10−3 s−1, and are in a good accordance with the equilibrium constant. The use of various ionophores and changes in external potassium concentration shown to modify the membrane potential of the present neuronal preparation, did not affect the binding of 125I-AaIT, thus indicating its voltage-independence. Veratridine, tetrodotoxin, sea anemone toxin and the α and β scorpion toxins specific for vertebrates did not affect the binding of 125I-AaIT. Furthermore, the above scorpion toxins were devoid of specific binding to the present insect neuronal preparation. Two additional insect toxins derived from the venom of the scorpion Buthotus judaicus, BjIT1 (spastic-excitatory toxin, homologus to the AaIT) and BjIT2 (flaccidity inducing-depressory toxin), were both shown to displace the 125I-AaIT with a high affinity (Kd = 2.2 and 1.3 nM, respectively). These data are compared and discussed in light of the information concerning the interaction of scorpion venom toxins affecting vertebrates with mammalian neuronal tissues.
A novel method to obtain specific antibodies against short peptides is described, involving synthesis of the corresponding oligodeoxynucleotides followed by cloning into a new set of fusion vectors, pEZZS and pEZZ 18, based on two synthetic IgG-binding domains (ZZ) of Staphylococcus aureus protein A. The soluble gene fusion product thus obtained, can be collected from the culture medium of Escherichia coli and rapidly recovered in a one-step procedure by IgG affinity chromatography. The system was used to express a fusion protein consisting of the two Z fragments and the C-terminal part [amino acids (aa) 57–70] of human insulin-like growth factor I (IGF-I). This 16-kDa protein was purified by affinity chromatography on IgG Sepharose and antibodies were raised in rabbits. The fusion protein elicited peptide-specific antibodies, as measured by solid-phase radioimmuno assay and Western blotting, reactive with both synthetic C-terminal peptide and the native human IGF-I protein. The result suggests that the gene fusion system can be used for efficient antibody production against short peptides encoded by synthetic oligodeoxynucleotides.
A synthetic IgG-binding domain based on staphylococcal protein A was designed with the aid of sequence comparisons and computer
graphic analysis. A strategy, utilizing non-palindromic restriction sites, was used to overcome the difficulties of introducing
she-specific changes into the repetitive gene. A single mutagenized gene fragment was polymerized to different multiplicities,
and the different gene products were expressed in Escherichia coli. Using this scheme, protein A-like proteins composed of different numbers of IgG-binding domains were produced. These domains
were changed to lack asparagine-glycine dipeptide sequences as well as methionine residues and are thus, in contrast to native
protein A, resistant to treatment with hydroxylamine and cyanogen bromide.
Two insect selective toxins were purified by gel-permeation and ion-exchange chromatographies from the venom of the scorpion, Leiurus quinquestriatus quinquestriatus, and their chemical and pharmacological properties were studied. The first toxin (LqqIT1) induces a fast excitatory contraction paralysis of fly larvae and is about 40 times more toxic than the crude venom. It is a polypeptide composed of 71 amino acids, including 8 half-cystines and devoid of methionine and tryptophan, with an estimated molecular weight of 8189 and a pI value of 8.5. The second toxin (LqqIT2) induces a slow depressant, flaccid paralysis of fly larvae. It is composed of 72 amino acids, including 8 half-cystines, is devoid of proline methionine and histidine, and has an estimated molecular weight of 7990 and a pI value of 8.3. The contrasting symptomatology of these toxins is interpreted in terms of their effects on an isolated axonal preparation of the cockroach in current and voltage clamp conditions. LqqIT1 (0.5-4 microM) induced repetitive firing of the axon which was attributable to two changes in the sodium conductance, a small increase in the peak conductance and a slowing of its turning off. LqqIT2 (1-8 microM) caused a blockage of the evoked action potentials, attributable to both a strong depolarization of the axonal membrane and a progressive suppression of the sodium current. Neither toxin affected potassium conductance. The two toxins differ mainly in their opposite effects on the activatable sodium permeability. In binding assays to a preparation of insect synaptosomal membrane vesicles, the two toxins were shown to competitively displace the radioiodinated excitatory insect toxin derived from the venom of the scorpion, Androctonus australis [( 125I]AaIT), which strongly resembles, in its chemistry and action, the LqqIT1 toxin. The present two toxins have demonstrated a strong affinity closely resembling the AaIT, with KD values of 0.4, 1.9, and 1.0 nM for LqqIT1, LqqIT2, and AaIT, respectively. These data suggest the possibility that the excitatory and depressant insect toxins share a common binding site associated with sodium channels in insect neuronal membranes.
Factors that affect the probability of genetic transformation of Escherichia coli by plasmids have been evaluated. A set of conditions is described under which about one in every 400 plasmid molecules produces a transformed cell. These conditions include cell growth in medium containing elevated levels of Mg2+, and incubation of the cells at 0 degrees C in a solution of Mn2+, Ca2+, Rb+ or K+, dimethyl sulfoxide, dithiothreitol, and hexamine cobalt (III). Transformation efficiency declines linearly with increasing plasmid size. Relaxed and supercoiled plasmids transform with similar probabilities. Non-transforming DNAs compete consistent with mass. No significant variation is observed between competing DNAs of different source, complexity, length or form. Competition with both transforming and non-transforming plasmids indicates that each cell is capable of taking up many DNA molecules, and that the establishment of a transformation event is neither helped nor hindered significantly by the presence of multiple plasmids.
The neuromuscular effects of four purified toxins and crude venom from the scorpion Androctonus australis were investigated in the extensor tibiae nerve-muscle preparation of the locust Locusta migratoria. Insect and crustacean toxin and the mammal toxins I and II which have previously been shown to act on fly larvae, isopods, and mice all paralyse locust larvae. The paralytic potencies decrease in the following order: insect toxin → mammal toxin I → crustacean toxin → mammal toxin II.The toxins and crude venom cause repetitive activity of the motor axons. This leads to long spontaneous trains of junction potentials in the case of crude venom and insect toxin. The other toxins chiefly cause short bursts of action and junction potentials following single stimuli.The ‘slow’ excitatory motor axon invariably is affected sooner than the inhibitory or the ‘fast’ excitatory one. The minimal doses of toxins required to affect the ‘slow’ motor axon decrease in an order somewhat different from that established for their paralytic potencies: insect toxin → crustacean toxin → mammal toxin I → mammal toxin II.Crude venom depolarises and destabilises the muscle membrane potential at low doses. At high doses it decreases the membrane resistance, whereas insect toxin leads to an increase.Crude venom and insect toxin enhance the frequency of mejps, whereas mammal toxin I leads to the occurrence of ‘giant’ mejps.The pattern of axonal activities indicates that the various peripheral branches of the motor nerve are the primary target of the toxins.The time course of nerve action potentials is affected by mammal toxin I and crustacean toxin which cause anomalous shapes and prolongations not caused by insect toxin.The results with other animals suggest that only the insect toxin is selective in its activity. The way it affects the axon might be quite different from that previously reported for scorpion venoms or toxins.
We constructed a synthetic gene encoding the published amino acid sequence of DTx from Dendroaspis angusticeps, a ligand of voltage-dependent postassium channels that facilitates neurotransmitter release. We expressed it in Escherichia coli as a fusion protein secreted in the culture medium. The recombinant DTx was generated in vitro by chemical treatment and recovered as two isoforms. One of them (rDTx), like the venom toxin, has an N-terminal pyroglutamate whereas the other (rQDTx) has a free N-terminal glutamine. Chromatographic differences between rDTx and natural DTx led us to re-examine the amino acid sequence of natural DTx. In contrast to what was previously published, position 12 was an Asp and not Asn. Despite this difference, rDTx and DTx had similar toxicity in mice and binding affinity to synaptosomes, suggesting that residue 12 is not important for DTx function. Nor is the N-terminal residue implicated in DTx function since rDTx and rQDTx also had similar biological activities. We also synthesized and expressed a mutant of the DTx gene in which the lysine triplet 28–30 was changed into Ala-Ala-Gly. The two resulting recombinant isoforms exhibited only small decreases in biological activity, excluding the possibility that the positively charged lysine triplet 28–30 of DTx is directly involved in the toxin functional site.
Two toxic proteins, insect toxins I and II, selectively paralytic and lethal to insects, were purified from the venom of the Black scorpion, Buthotus judaicus, using gel permeation and ion-exchange chromatography. Their chemical purity and characteristics were assessed by column chromatography, disc electrophoresis, isoelectrofocusing and amino acid analyses. Their biological specificity and toxicity were determined by a series of paralysis and lethality assays employing the larvae of the flesh fly, Sarcophaga falculata, and the locust, Locust migratoria. Their modes of action were investigated employing in vitro cockroach axonal preparations in current- and voltage-clamp conditions. Insect toxin I (IT-I) is approximately 40-times more toxic than the crude venom (according to its fly larvae paralysing activity), is composed of 67 amino acids including six half cystines, and has an estimated molecular weight of 7532 and a pI value of 8.20. It causes an immediate contraction paralysis of fly larvae and a quick excitatory ‘knock-down’ effect on locusts. Insect toxin II (IT-II) is about 36-times more toxic than the crude venom according to the paralytic potency in fly larvae. It is composed of 69 amino acids including six half cystines and has an estimated molecular weight of 7894, a unique amino acid composition and a pI value of 8.30. IT-II causes a flaccid paralysis of fly larvae and a slow progressive paralysis and eventually death of locusts. In in vitro current clamp experiments, IT-I induces repetitive activities, and IT-II, a block of the evoked action potentials. These two opposite effects may be explained by their different effects on sodium permeabilities. Both toxins increase the sodium resting permeability (IT-II markedly more) resulting in a progressive depolarization of the axonal membrane. Concerning the activable sodium permeability, both toxins slightly slow the sodium transient inward current turning off. The peak sodium current is increased by IT-I and decreased byIT-II. This essential difference may, at least partially, account for the contrasting symptoms they induce in the whole insect.
A new method for determining nucleotide sequences in DNA is described. It is similar to the "plus and minus" method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441-448] but makes use of the 2',3'-dideoxy and arabinonucleoside analogues of the normal deoxynucleoside triphosphates, which act as specific chain-terminating inhibitors of DNA polymerase. The technique has been applied to the DNA of bacteriophage varphiX174 and is more rapid and more accurate than either the plus or the minus method.
The sequence A-A-U-A-A-A is present in six different purified messenger RNA molecules (specifically the alpha-and beta-globulin mRNAs of rabbit and human, the immunoglobulin light chain mRNA of mouse (MOPC 21) and the ovalbumin mRNA of chicken) about 20 residues away from the 3'-terminal poly (A) sequence. In addition, a large selection of the 3' non-coding regions of rabbit and human globulin mRNAs (both the alpha and beta globin mRNAs) are 85% homologous, demonstrating that this region is significantly conserved in evolution.
Depolarization of neuroblastoma cells causes a 70-fold increase in the apparent dissociation constant KD for scorpion toxin enhancement of activation of the action potential Na+ ionophore by veratridine and a large increase in the rate of reversal of scorpion toxin action. Depolarization also inhibits binding of 125I-labeled scorpion toxin to a small number of saturable binding sites on electrically excitable neuroblastoma cells and increases the rate of dissociation of scorpion toxin from these sites. The results suggest that scorpion toxin binds to a regulatory component of the action potential Na+ ionophore whose conformation changes on depolarization.
We have expressed a synthetic gene encoding the insecticidal neurotoxin of scorpion Androctonus australis (AaIT) in NIH/3T3 mouse fibroblast cells under the transcriptional control of a murine retroviral long terminal repeat. The secretion of the toxin into the culture medium was directed by the signal peptide of human interleukin-2. The recombinant AaIT produced was selectively toxic to yellow-fever mosquito larvae and harmless to mice.
The nucleotide sequence encoding the scorpion insectotoxin I5A was chemically synthesized and expressed in yeast, bacteria and tobacco. The I5A peptides produced in these organisms were purified using an immunoaffinity chromatography procedure. I5A produced using the bacterial secretion system was efficiently secreted and released into the culture medium. In contrast, only a trace amount of I5A was detected in bacterial cytosols when expressed from a direct expression vector, suggesting that I5A was unstable in bacterial cells. I5A secreted from yeast using an alpha-factor signal sequence was shown to have an N-terminal (Glu-Ala)2 extension, indicating incomplete processing of the secreted peptide by dipeptidyl aminopeptidase A. In tobacco, a nonsecreted form of the protein was produced. No measurable insect toxicity was observed when insect larvae were assayed, regardless of whether I5A was produced in yeast, bacteria or tobacco. The lack of toxicity is almost certainly the result of improper folding due to incorrect disulfide bond formation. The inability to produce a biologically active peptide must be overcome before scorpion toxins might be used for the genetic engineering of plants for insect resistance. The yeast and bacterial expression systems described here may be useful for further studies on the problem of expressing a biologically active peptide.
The possibility of raising a humoral immune response able to induce protection from the lethal effects of scorpion toxins was evaluated in the mouse model. A toxic fraction from the venom of the scorpion Tityus serrulatus was entrapped in sphingomyelin-cholesterol liposomes which yielded a conveniently detoxified immunogen. After three injections of this immunogen, all but three of a group of 18 mice developed an IgG response which was shown to be both specific and of good affinity for the toxic antigen. In vitro neutralization assays indicated that pre-incubation of a lethal dose of the toxic fraction with immune sera strongly diminished its toxicity. In vivo protection assays showed that mice with the highest levels of circulating anti-toxin antibodies could resist the challenge by double the normal LD50 of the toxic fraction, which killed all control non-immune mice. The protection was, however, found to be limited both in its duration and its effectiveness against higher amounts of toxin.
Sequence-specific nuclear magnetic resonance assignments for the polypeptide backbone and for most of the amino acid side-chain protons, as well as the general folding of AaH IT, are described. AaH IT is a neurotoxin purified from the venom of the scorpion Androctonus australis Hector and is specifically active on the insect nervous system. The secondary structure and the hydrogen-bonding patterns in the regular secondary structure elements are deduced from nuclear Overhauser effects and the sequence locations of the slowly exchanging amide protons. The backbone folding is determined by distance geometry calculations with the DISMAN program. The regular secondary structure includes two and a half turns of alpha-helix running from residues 21 to 30 and a three-stranded antiparallel beta-sheet including peptides 3-5, 34-38, and 41-46. Two tight turns are present, one connecting the end of the alpha-helix to an external strand of the beta-sheet, i.e., turn 31-34, and another connecting this same strand to the central one, i.e., turn 38-41. These structure elements are very similar to the secondary structure reported in single crystals for either variant 3 from the scorpion Centruroides sculpturatus Ewing (CsE V3) or toxin II from the scorpion A. australis Hector (AaH II). The differences in the specificity of these related proteins, which are able to discriminate between mammalian and insect voltage-dependent sodium channels of excitable tissues, are most probably brought about by the position of the C-terminal peptide with regard to a hydrophobic surface common to all scorpion toxins examined thus far. This surface is made of an aromatic cluster that is surrounded by long hydrophobic side-chain residues, as well as the loops protruding out of it. Thus, the interaction of a given scorpion toxin with its receptor might well be governed by the presence of this solvent-exposed hydrophobic surface, whereas adjacent areas modulate the specificity of the interaction.
Depressant insect-selective neurotoxins derived from scorpion venoms (a) induce in blowfly larvae a short, transient phase of contraction similar to that induced by excitatory neurotoxins followed by a prolonged flaccid paralysis and (b) displace excitatory toxins from their binding sites on insect neuronal membranes. The present study was undertaken in order to examine the basis of these similarities by comparing the primary structures and neuromuscular effects of depressant and excitatory toxins. A new depressant toxin (LqhIT2) was purified from the venom of the Israeli yellow scorpion. The effects of this toxin on a prepupal housefly neuromuscular preparation mimic the effects on the intact animal; i.e., a brief period of repetitive bursts of junction potentials is followed by suppression of their amplitude and finally by a block of neuromuscular transmission. Loose patch clamp recordings indicate that the repetitive activity has a presynaptic origin in the motor nerve and closely resembles the effect of the excitatory toxin AaIT. The final synaptic block is attributed to neuronal membrane depolarization, which results in an increase in spontaneous transmitter release; this effect is not induced by excitatory toxin. The amino acid sequences of three depressant toxins were determined by automatic Edman degradation. The depressant toxins comprise a well-defined family of polypeptides with a high degree of sequence conservation. This group differs considerably in primary structure from the excitatory toxin, with which it shares identical or related binding sites, and from the two groups of scorpion toxins that affect sodium conductance in mammals. The two opposing pharmacological effects of depressant toxins are discussed in light of the above data.
The amino acid sequences of insect-selective scorpion toxins, purified from the venom of Leiurus quinquestriatus quinquestriatus, have been determined by automatic phenyl isothiocyanate degradation of the S-carboxymethylated proteins and derived proteolytic peptides. The excitatory toxin Lqq IT1 and Lqq IT1' (70 residues) show the shift of one half-cystine from an external position, which is characteristic of anti-mammal toxins, to an internal sequence position. Lqq IT2 (61 residues) displays the half-cystine residue in position 12, common to the sequence of all known anti-mammal toxins; it induces flaccid paralysis on insects but is non-toxic for the mouse. Lqq IT2 structurally defines a new type of anti-insect toxins from scorpion venoms. CD spectra and immunological data are in agreement with this finding.
We previously reported the production of a fused snake neurotoxin composed of protein A and erabutoxin a in E. coli. The hybrid had much lower toxicity and affinity for the acetylcholine nicotinic receptor than natural erabutoxin. By treating the hybrid with cyanogen bromide we generated a toxin which was purified in a single step by RP-HPLC. This compound, produced in a good yield, recovered all properties of native erabutoxin a, implying that the lower toxic activities of the hybrid were due to the bulky protein A and not to an incorrect folding of the toxin. This work serves as a basis for future studies of toxin-receptor interactions using engineered toxin mutants.
A new toxin, Lqh alpha IT, which caused a unique mode of paralysis of blowfly larvae, was purified from the venom of the scorpion Leiurus quinquestriatus hebraeus, and its structural and pharmacological properties were compared to those of three other groups of neurotoxins found in Buthinae scorpion venoms. Like the excitatory and depressant insect-selective neurotoxins, Lqh alpha IT was highly toxic to insects, but it differed from these toxins in two important characteristics: (a) Lqh alpha IT lacked strict selectivity for insects; it was highly toxic to crustaceans and had a measurable but low toxicity to mice. (b) It did not displace an excitatory insect toxin, 125I-AaIT, from its binding sites in the insect neuronal membrane; this indicates that the binding sites for Lqh alpha IT are different from those shared by the excitatory and depressant toxins. However, in its primary structure and its effect on excitable tissues, Lqh alpha IT strongly resembled the well-characterized alpha scorpion toxins, which affect mammals. The amino acid sequence was identical with alpha toxin sequences in 55%-75% of positions. This degree of similarity is comparable to that seen among the alpha toxins themselves. Voltage- and current-clamp studies showed that Lqh alpha IT caused an extreme prolongation of the action potential in both cockroach giant axon and rat skeletal muscle preparations as a result of the slowing and incomplete inactivation of the sodium currents. These observations indicate that Lqh alpha IT is an alpha toxin which acts on insect sodium channels.(ABSTRACT TRUNCATED AT 250 WORDS)
Electrical excitability is one of the most important and characteristic properties of neurons. Most vertebrate cells, including neurons, maintain large ionic gradients across their surface membranes such that the intracellular fluid contains a high concentration of potassium ions and low concentrations of sodium ions and calcium ions relative to the extracellular fluid. These large ion gradients are maintained by the action of energy-dependent ion pumps specific for Na+ and K+, or for Ca2+. In addition, essentially all vertebrate cells maintain an internally negative membrane potential of the order of -60 mV, since their surface membranes are specifically permeable to K+, and this allows K+ to leak out of cells faster than Na+ and Ca2+ can leak in. Nerve cells are electrically excitable because of the presence, in their surface membranes, of voltage-sensitive ion channels that are selective for Na+, K+, or Ca2+. One class of Na+ channels and many classes of Ca2+ and K+ channels have been described in neurons. The channels open and close as a function of membrane voltage, allowing rapid movement of the appropriate ions down their concentration gradient so that ionic current passes into or out of the cell, depolarizing or hyperpolarizing the membrane.
Some beta-toxins from the South American scorpion Tityus serrulatus (e.g. Ts VII) are highly toxic both for mouse and fly larva. Radioiodinated Ts VII and the insect toxin from the North African scorpion Androctonus australis Hector (AaH IT) bind to the same site on a house fly head synaptosomal fraction. These results reinforce the hypothesis about the existence of a correlated series of scorpion toxins as previously defined by amino acid compositions and sequences, and immunological and circular dichroism studies, in suggesting that Ts VII constitutes a link which may fill the pharmacological gap existing between beta-toxins and insect toxins such as AaH IT.
We constructed a recombinant expression plasmid encoding a protein A–neurotoxin fusion protein. The fused toxin is directly
expressed in the periplasmic space of Escherichia coli and can be purified in the milligram range by a single immuno-affinity step. The LD50 values of the fused toxin and native toxin are 130 and 20 nmol/kg mouse respectively. The Kd values characterizing their binding to the nicotinic acetylcholine receptor (AcChoR) are respectively 4.8 ± 0.8 and 0.07
± 0.03 nM. In contrast, the fused and native toxins are equally well recognized by a toxin-specific monoclonal antibody which
recognizes the AcChoR binding site. The lower toxicity of the fused toxin might result, therefore, from a steric hindrance,
due to the presence of the bulky protein A moiety (mol. wt = 31 kd) rather than to a direct alteration of the ‘toxic’ site.
The fused toxin is more immunogenic than native toxin, since 1 nmol of hybrid toxin and 14 nmol of native toxin give rise
to comparable titers of antitoxin antibodies which, furthermore, are equally potent at neutralizing neurotoxicity. The work
described in this paper shows that the use of fused toxins may be of paramount importance for future development of serotherapy
against envenomation by snake bites.
From a cDNA library made from telsons of scorpions of the species Androctonus australis, full-length cDNAs of about 370 nucleotides encoding precursors of toxins active on mammals or on insects have been isolated using oligonucleotide probes. Sequence analysis of the cDNAs revealed that the precursors contain signal peptides of about 20 amino acid residues. In addition, precursors of toxins active on mammals have extensions at their COOH-terminal ends: Arg or Gly-Arg. The processing steps required to generate toxins from their respective precursors are thus not identical for all of them. Southern blot analysis performed at the genomic level with a cDNA encoding toxin II suggested a single copy gene having a minimum size of 2800 base pairs. Finally, in an attempt to successfully express an animal toxin, monkey kidney COS-7 cells transfected with a plasmid harboring a cDNA encoding toxin II transiently expressed a recombinant toxin having the immunological and biological properties of toxin II.
When the venom of the Moroccan scorpion Buthus occitanus mardochei was submitted to a combination of several chromatographic steps (including gel-filtration and ion-exchange chromatographies), seven proteins were obtained, six being lethal to mice. These proteins have been characterized by their chemical, immunological and toxic properties. The amino acid sequence (66 residues) of Bom III, the most noteworthy toxin of the venom as for its amino acid composition, is proposed following automatic sequencing of the reduced and S-methylated protein and of chymotryptic peptides. It was obvious that this sequence is somewhat different from those of toxins belonging to the same structural and immunological group (Bom III was found to be immunologically related to Buthus occitanus tunetanus toxins I and II which both share with it 56% of homology. Furthermore, Bom III was found to be unable to compete (as does Bot I) with toxin II of Androctonus australis Hector (an alpha-type toxin) for neurotoxin binding site 3 on the sodium channel of rat brain synaptosomes. Bom III was also unable to compete with toxin II of Centruroides suffusus suffusus (a beta-type toxin) to neurotoxin binding site 4 of the same channel.
Orthorhombic crystals (space group P212121, a = 45.94 A, b = 40.68 A, c = 29.93 A) of the potent scorpion alpha-toxin II from Androctonus australis Hector were grown using sterile techniques. The structure was solved by a combination of heavy-atom and model phasing. Subsequently, it was refined at 1.8 A resolution by a fast-Fourier restrained least-squares procedure. The crystallographic R factor is 0.152 for data with 7.0 A greater than d greater than 1.8 A and F greater than 2.5 sigma (F) and 0.177 when all data are considered. Eighty-nine solvent molecules have been incorporated into the model. The dense core formed by the alpha-helical and antiparallel beta-sheet moieties and three of the four disulfide bridges is similar in variant 3, a toxin purified from the North American scorpion Centruroides sculpturatus, and in toxin II. However, the two molecules differ markedly in the orientation of loops protruding from the core. Toxin II seems to contain several highly ordered solvent molecules. Eight of them occupy a cavity consisting of the C-terminal region and a loop found only in scorpion alpha-toxins. The highly reactive and pharmacologically important Lys-58 is found at one of the extremes of this cavity, where it establishes a series of hydrogen bonds with protein and solvent atoms. The reactivities of the five lysine residues of toxin II are highly correlated with the formation of hydrogen bonds, hydrophobic interactions, and salt links.
We have explored the possibility of improving baculovirus pesticides by incorporating an insect-specific neurotoxin gene into a baculovirus genome. A 112-bp gene (BeIt) encoding insectotoxin-1 of the scorpion Buthus eupeus was synthesized and cloned in Escherichia coli. For expression, BeIt was transferred to the DNA genome of Autographa californica nuclear polyhedrosis virus (AcMNPV). Three different recombinant AcMNPVs, carrying BeIt under the control of the strong AcMNPV polyhedrin promoter, were constructed and expression of BeIt was monitored upon infection of Spodoptera frugiperda (Sf) cells. Toxin expression was low using a recombinant virus in which BeIt was inserted 6 nucleotides (nt) downstream from the intact polyhedrin mRNA leader. More expression was observed when a signal-peptide was attached in-frame to the N terminus of BeIt. The highest level of expression was observed with a fusion gene comprised of the 58 N-terminal codons of polyhedrin fused to BeIt; however, the level of expression was ten- to twenty-fold below that for polyhedrin. Polyhedrin promoter-directed transcripts of all three recombinants accumulated to levels similar to those of wild-type polyhedrin transcripts, indicating that the limitation to expression of unfused BeIt was not at the level of transcription but rather at the posttranscriptional level including translation or protein stability. Paralytic activity of the toxin products was not detected.
Specific chemical modifications of scorpion α and β toxins have been used to study the involvement of particular residues in both the pharmacological and the antigenic sites of these toxins. Modification by 1,2-cyclohexanedione of arginine-27 of a β toxin, Centruroides suffusus suffusus toxin II, drastically decrease the antigenic activity without any influence on the pharmacological activity. Conversely, modification by the same reagent of arginine-2 of an α toxin, Androctonus australis Hector toxin III, led to a 100-times less pharmacologically potent derivative and did not induce a significant loss of antigenic activity. Excision of the N-terminal pentapeptide of another α toxin, Buthus occitanus mardochei toxin III, by pepsin digestion led to a non-toxic derivative retaining full antigenic activity. Thus, the N-terminal part of the conserved hydrophobic surface of the toxin is highly implicated in the pharmacological activity, whereas the region of arginine-27, located in the α helix situated on the back surface, opposite the conserved hydrophobic region, is fully implicated in the antigenic activity and is far from the pharmacological site.
These results are good arguments in favor of the idea that in scorpion toxins the surfaces implicated in the pharmacological and the antigenic activities do not overlap. Since the antigenic sites are present in highly variable sequence the development of an efficient polyvalent serotherapy is questionable.
Using highly specific radioimmunoassays for toxins I, II and III of the scorpion Androctonus australis Hector, the concentrations of these neurotoxins have been determined in pooled as well as in individual samples. Variations were found that support polymorphism of scorpion toxins at an individual level. Radioimmunoassays were also used to detect toxin I of Buthus occitanus tunetanus and toxin II of Androctonus australis Hector and also antigenically homologous toxins in the venoms of several North African scorpions. These results are interpreted in terms of scorpion serotherapy.
A complementation analysis of host-controlled modification and restriction of DNA by Escherichia coli has been carried out by examining the restriction and modification phenotypes of partial, permanent diploids containing various arrangements of wild type and mutant restriction and modification alleles. Intercistronic complementation was observed between three classes of restriction and modification mutants of E. coli B, indicating that at least three cistrons (the ram cistrons) are involved in the genetic control of the [restriction and modification of DNA. Mutations in one cistron (ramA) result in a loss of restriction activity but not in modification activity (r−m+). Mutations in a second cistron (ramC) result in a loss of restriction and modification activities (r−m−). Mutations in a third cistron result in a loss of modification activity and appear to be lethal unless accompanied by a mutation in the ramA or ramC cistrons. A fourth class of mutations, which are linked to the other ram cistrons and are expressed phenotypically as r−m− mutants, are trans dominant to the wild-type ram alleles. It is not known if this latter class of mutants represents a fourth cistron of the ram locus. Complementation was observed between E. coli K12 and B ramA and ramC mutations and the host specificity of the restored restriction activity was dependent on an intact ramC cistron. However, complementation was not detected between the P1 and K12 or P1 and B ram alleles. A general model for the genetic control of the restriction and modification properties of E. coli strains and their episomes is presented.
Die Reinigung eines für Insekten toxischen Proteins aus dem Gift des Skorpions Androctonus australis Hector ist mittels Rezyklieren von Sephadex G50-Filtration und Chromatographie en DEAE-Sephadex A50 und Amberlit CG-50 durchgeführt worden. Das Endprodukt wurde 267 Mal gereinigt im Vergleich zu dem rohen Gift. Die Gift ausbeute war 95 %. Die Reinheit des Insekttoxins wurde durch Aminosäureanalyse, sequentiale N-End-Degradation, C-End-Aminosäure-Bestimmung und Zonen Elektrophorese geschätzt. Sein Molekulargewicht ist 7498. Das Insekttoxin unterscheidet sich von den für Säugetiere giftigen Toxinen, die in demselben Gift enthalten sind. Die Sequenz der ersten 15 Aminosäurereste aus den N-Enden der beiden Toxinarten wird verglichen. Die physiologische Bedeutung dieser neurotoxischen Proteine wird besprochen.
The purification of eleven neurotoxins from the venom of three scorpions (Androctonus australis Hector, Buthus occitanus tunetanus and Leiurus quinquestriatus quinquestriatus) has been performed by Sephadex G-50 filtration (with and without recycling) and equilibrium chromatography on Amberlite CG-50, CM-Sephadex C-50 and DEAE-Sephadex A-50. All the chromatographic steps were carried out in ammonium acetate buffers. Purity of the neurotoxins was assessed by amino acid analysis and zone electrophoresis. All have a molecular weight of about 7000 daltons and do not contain methionine. As shown by amino acid composition and chain length scorpion neurotoxins show a high degree of homology.
Using the same method of purification of small molecular weight proteins worked out to obtain pure scorpion neurotoxins, four neurotoxins have been isolated from the venom of the cobra Naja haje.
N. haje venom from the Miami Serpentarium contains two neurotoxins (I and II) whereas the venom of the same species obtained from the Institut Pasteur contains three neurotoxins (I, II′ and III). Toxins I, II and II′ consist of 61 amino acid residues and toxin III of 71. All are basic proteins containing four or five disulphide bridges. It is likely that the difference observed in neurotoxin composition of N. haje venoms from two different sources has a taxonomical counterpart, each venom originating from a different sub-species.
By use of starch gel zone electrophoresis and Sephadex G-50 gel filtration a toxic protein with contraction-paralysis as well as lethal activity to fly larvae has been isolated from the venom of the scorpion Androctonus australis Hector. This larva-toxic protein is different from the mice-lethal neurotoxins of the same venom. The significance of this finding is discussed.
1. An experimental method of recording and controlling the membrane potential of a small area of the membrane of the cockroach giant axon is described.
2. The recorded action potentials were essentially similar to those previously recorded by other methods.
3. The membrane currents resemble those reported for the squid axon, the node of Ranvier in frog nerve and the lobster giant axon.
4. Small cathodal polarizations gave only small outward currents; larger depolarizations (10-100 mV.) gave an initial inward current which changed into a delayed outward current.
5. The initial inward current attained a maximum with depolarizing pulses of 40-50 mV. and showed a reversed, outward, flow of about 100 mV.
6. Delayed outward currents increased continuously with increasing impulse voltage.
7. The initial inward current was larger when the pulse was preceded by an hyperpolarizing prepulse.
8. It is concluded that, although the early inward currents were in all probability related to Na+ ions and the delayed outward currents to K+ ions, the possible participation of Ca2+ and Cl- ions to the ionic currents cannot be excluded.
Scorpion neurotoxins active on mammals, i.e. alpha- and beta-toxins, have been divided into several groups according to structural and also immunological criteria. A study of the alpha-toxins has been performed in order to determine the number of antigenic sites; a minimum of four Fab fragments were found to bind simultaneously to toxins I and II of Androctonus australis Hector, and toxin I of Buthus occitanus tunetanus. Taking advantage of the loss of one common antigenic site between toxin II of Androctonus australis Hector and toxin III of Buthus occitanus tunetanus, a highly purified antibody population towards a single site of toxin II of Androctonus australis Hector was isolated and characterized. This result is discussed in terms of sequence homologies between the two proteins as the amino acid sequence of toxin III of Buthus occitanus tunetanus has been determined using standard procedures: the two proteins differ at three positions, two of which (positions 10 and 64) are in the vicinity of the disulfide bridge Cys 12-Cys 63, the third (position 51) is the only one to be conservative.
Three toxins, i.e. toxins II and III of Androctonus australis Hector and toxin I of Buthus occitanus tunetanus, were labeled with 125I. High specific radioactivities were obtained (490-1100 Ci/mmole) that allowed the setting up of three radioimmunoassays. We were able to quantify the amount of toxin in 0.1 ml of 10(-9)-10(-10) M solutions and to test antigenic homologies between toxins belonging to different structural groups. Three possibilities exist: (1) no cross-reactivity when sequence difference is greater than 25%; (2) full cross-reactivity when this difference is lower than 25%; (3) partial cross-reactivity, which is interpreted as a loss of some common antigenic sites.