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.
Scorpions belonging to the Buthidae family have traditionally drawn much of the biochemist's attention due to the strong toxicity of their venoms. Scorpions not toxic to mammals, however, also have ...complex venoms. They have been shown to be an important source of bioactive peptides, some of them identified as potential drug candidates for the treatment of several emerging diseases and conditions. It is therefore important to characterize the large diversity of components found in the non-Buthidae venoms. As a contribution to this goal, this manuscript reports the construction and characterization of cDNA libraries from four scorpion species belonging to the Vaejovis genus of the Vaejovidae family: Vaejovis mexicanus, V. intrepidus, V. subcristatus and V. punctatus. Some sequences coding for channel-acting toxins were found, as expected, but the main transcribed genes in the glands actively producing venom were those coding for non disulfide-bridged peptides. The ESTs coding for putative channel-acting toxins, corresponded to sodium channel β toxins, to members of the potassium channel-acting α or κ families, and to calcium channel-acting toxins of the calcin family. Transcripts for scorpine-like peptides of two different lengths were found, with some of the species coding for the two kinds. One sequence coding for La1-like peptides, of yet unknown function, was found for each species. Finally, the most abundant transcripts corresponded to peptides belonging to the long chain multifunctional NDBP-2 family and to the short antimicrobials of the NDBP-4 family. This apparent venom composition is in correspondence with the data obtained to date for other non-Buthidae species. Our study constitutes the first approach to the characterization of the venom gland transcriptome for scorpion species belonging to the Vaejovidae family.
The current trend of using recombinant antibody fragments in research to develop novel antidotes against scorpion stings has achieved excellent results. The polyclonal character of commercial ...antivenoms, obtained through the immunization of animals and which contain several neutralizing antibodies that recognize different epitopes on the toxins, guarantees the neutralization of the venoms. To avoid the use of animals, we aimed to develop an equivalent recombinant antivenom composed of a few neutralizing single chain antibody fragments (scFvs) that bind to two different epitopes on the scorpion toxins. In this study, we obtained scFv RU1 derived from scFv C1. RU1 showed a good capacity to neutralize the Cn2 toxin and whole venom of the scorpion Centruroides noxius. Previously, we had produced scFv LR, obtained from a different parental fragment (scFv 3F). LR also showed a similar neutralizing capacity. The simultaneous administration of both scFvs resulted in improved protection, which was translated as a rapid recovery of previously poisoned animals. The crystallographic structure of the ternary complex scFv LR-Cn2-scFv RU1 allowed us to identify the areas of interaction of both scFvs with the toxin, which correspond to non-overlapping sites. The epitope recognized by scFv RU1 seems to be related to a greater efficiency in the neutralization of the whole venom. In addition, the structural analysis of the complex helped us to explain the cross-reactivity of these scFvs and how they neutralize the venom.
Alpha-neurotoxins target voltage-gated sodium channels (Na(v)s) and constitute an important component in the venom of Buthidae scorpions. These toxins are short polypeptides highly conserved in ...sequence and three-dimensional structure, and yet they differ greatly in activity and preference for insect and various mammalian Na(v)s. Despite extensive studies of the structure-function relationship of these toxins, only little is known about their evolution and phylogeny. Using a broad data set based on published sequences and rigorous cloning, we reconstructed a reliable phylogenetic tree of scorpion alpha-toxins and estimated the evolutionary forces involved in the diversification of their genes using maximum likelihood-based methods. Although the toxins are largely conserved, four positions were found to evolve under positive selection, of which two (10 and 18; numbered according to LqhalphaIT and Lqh2 from the Israeli yellow scorpion Leiurus quinquestriatus hebraeus) have been previously shown to affect toxin activity. The putative role of the other two positions (39 and 41) was analyzed by mutagenesis of Lqh2 and LqhalphaIT. Whereas substitution P41K in Lqh2 did not alter its activity, substitution K41P in LqhalphaIT significantly decreased the activity at insect and mammalian Na(v)s. Surprisingly, not only that substitution A39L in both toxins increased their activity by 10-fold but also LqhalphaIT(A39L) was active at the mammalian brain channel rNa(v)1.2a, which otherwise is hardly affected by LqhalphaIT, and Lqh2(A39L) was active at the insect channel, DmNa(v)1, which is almost insensitive to Lqh2. Thus, position 39 is involved not only in activity but also in toxin selectivity. Overall, this study describes evolutionary forces involved in the diversification of scorpion alpha-toxins, highlights the key role of positions under positive selection for selectivity and potency, and raises new questions as to the toxin-channel face of interaction.
•The long-chain scorpion toxin BmKM9 was expressed without additional amino acid residues.•Scorpion toxin rBmKM9 inhibited the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels with ...different activities.•Scorpion toxin rBmKM9 displayed different effects on the gating of rNav1.4 and hNav1.5 channels.•Scorpion toxin rBmKM9 affected the recovery of hNav1.5 channels from inactivation more significantly than rNav1.4 channels.
Scorpion toxins are invaluable pharmacological tools for studying ion channels and potential drugs for channelopathies. The long-chain toxins from scorpion venom with four disulfide bridges exhibit their unusual bioactivity or biotoxicity by acting on the sodium channels. However, the functional properties of most toxins are still unclear due to their tiny amounts in crude venom and their challenging production by chemical and gene engineering techniques. Here, we expressed one of the long-chain α-toxins, BmKM9, found in the venom of the scorpion Buthus martensii Karsch and characterized its pharmacological properties on sodium channels. Unlike previous toxin production, the recombinant BmKM9 (rBmKM9) possessed no additional amino acid residues such as the His-tag and thrombin cleavage site. The refolded toxin could inhibit the inactivation of rNav1.4, hNav1.5 and hNav1.7 sodium channels. Dose-response experiments were further conducted on these channels. The calculated EC50 values were 131.7±6.6nM for rNav1.4, 454.2±50.1nM for hNav1.5 and 30.9±10.3μM for hNav1.7. The channel activation experiments indicated that the rBmKM9 toxin could shift the activation curves of rNav1.4 and hNav1.5 channels toward a more negative direction and present the typical features of a β-toxin. However, instead of the same activation property on sodium channels, the rBmKM9 toxin could result in different inactivation shift capabilities on rNav1.4 and hNav1.5 channels. The V1/2 values of the steady-state inactivation were altered to be more positive for rNav1.4 and more negative for hNav1.5. Moreover, the recovery of the hNav1.5 channel from inactivation was more significantly delayed than that of the rNav1.4 channel by exposure to rBmKM9. Together, these findings highlighted that the rBmKM9 toxin presents the pharmacological properties of both α- and β-toxins, which would increase the challenge to the classical classification of scorpion toxins. Furthermore, the expression method and functional information on sodium channels would promote the potential application of toxins and contribute to further channel structural and functional studies.
Australian scorpion venoms have been poorly studied, probably because they do not pose an evident threat to humans. In addition, the continent has other medically important venomous animals capable ...of causing serious health problems. Urodacus yaschenkoi belongs to the most widely distributed family of Australian scorpions (Urodacidae) and it is found all over the continent, making it a useful model system for studying venom composition and evolution. This communication reports the whole set of mRNA transcripts produced by the venom gland. U. yaschenkoi venom is as complex as its overseas counterparts. These transcripts certainly code for several components similar to known scorpion venom components, such as: alpha-KTxs, beta-KTxs, calcins, protease inhibitors, antimicrobial peptides, sodium-channel toxins, toxin-like peptides, allergens, La1-like, hyaluronidases, ribosomal proteins, proteasome components and proteins related to cellular processes. A comparison with the venom gland transcriptome of Centruroides noxius (Buthidae) showed that these two scorpions have similar components related to biological processes, although important differences occur among the venom toxins. In contrast, a comparison with sequences reported for Urodacus manicatus revealed that these two Urodacidae species possess the same subfamily of scorpion toxins. A comparison with sequences of an U. yaschenkoi cDNA library previously reported by our group showed that both techniques are reliable for the description of the venom components, but the whole transcriptome generated with Next Generation Sequencing platform provides sequences of all transcripts expressed. Several of which were identified in the proteome, but many more transcripts were identified including uncommon transcripts. The information reported here constitutes a reference for non-Buthidae scorpion venoms, providing a comprehensive view of genes that are involved in venom production. Further, this work identifies new putative bioactive compounds that could be used to seed research into new pharmacological compounds and increase our understanding of the function of different ion channels.
We report isolation, sequencing, and electrophysiological characterization of OSK3 (α-KTx 8.8 in Kalium and Uniprot databases), a potassium channel blocker from the scorpion Orthochirus scrobiculosus ...venom. Using the voltage clamp technique, OSK3 was tested on a wide panel of 11 voltage-gated potassium channels expressed in Xenopus oocytes, and was found to potently inhibit Kv1.2 and Kv1.3 with IC50 values of ~331nM and ~503nM, respectively. OdK1 produced by the scorpion Odontobuthus doriae differs by just two C-terminal residues from OSK3, but shows marked preference to Kv1.2. Based on the charybdotoxin-potassium channel complex crystal structure, a model was built to explain the role of the variable residues in OdK1 and OSK3 selectivity.
•We purified novel potassium channel blocker (OSK3) from the venom of Orthochirus scrobiculosus.•We determined OSK3 activity on Kv1.2, Kv1.3 and Kv1.6 from the wide panel of tested potassium channels.•C-Terminal residues of OSK3 and OdK1 are responsible for toxin selectivity.
•We cloned three toxin peptides, Ctri18, Ctry68 and Ctry2908, from non-Buthidae scorpions.•Recombinant Ctri18, Ctry68 and Ctry2908 peptides were obtained.•Functionally, they can inhibit Kv1.3 ...channels and T cell IL-2 secretions.
Non-Buthidae venomous scorpions are huge natural sources of toxin peptides; however, only a few studies have been done to understand their toxin peptides. Herein, we describe three new potential immunomodulating toxin peptides, Ctri18, Ctry68 and Ctry2908, from two non-Buthidae scorpions, Chaerilus tricostatus and Chaerilus tryznai. Sequence alignment analyses showed that Ctri18, Ctry68 and Ctry2908 are three new members of the scorpion toxin α-KTx15 subfamily. Electrophysiological experiments showed that Ctri18, Ctry68 and Ctry2908 blocked the Kv1.3 channel at micromole to nanomole levels, but had weak effects on potassium channel KCNQ1 and sodium channel Nav1.4, which indicated that Ctri18, Ctry68 and Ctry2908 might have specific inhibiting effects on the Kv1.3 channel. ELISA experiments showed that Ctri18, Ctry68 and Ctry2908 inhibited IL-2 cytokine secretions of activated T lymphocyte in human PBMCs. Excitingly, consistent with the good Kv1.3 channel inhibitory activity, Ctry2908 inhibited cytokine IL-2 secretion in nanomole level, which indicated that Ctry2908 might be a new lead drug template toward Kv1.3 channels. Together, these studies discovered three new toxin peptides, Ctri18, Ctry68 and Ctry2908, with Kv1.3 channel and IL-2 cytokine-inhibiting activities from two scorpions, C. tricostatus and C. tryznai, and highlighted that non-Buthidae venomous scorpions are new natural toxin peptide sources.
Scorpion envenomation poses a global public health issue, with an estimated 1,500,000 cases worldwide annually resulting in 2600 deaths. North Africa, particularly Morocco, experiences severe ...envenomations, mainly attributed to
and
in Morocco, and
and
in Algeria and Tunisia, with case numbers often underestimated. Current treatment relies mainly on symptomatic approaches, except in Morocco, where management is limited to symptomatic treatment due to controversies regarding specific treatment. In Morocco, between 30,000 and 50,000 scorpion envenomation cases are reported annually, leading to hundreds of deaths, mainly among children. Controversies among clinicians persist regarding the appropriate course of action, often limiting treatments to symptomatic measures. The absence of a specific antivenom for the venoms of the most lethal scorpions further exacerbates the situation. This study aims to address this gap by developing a monovalent antivenom against the endemic and most dangerous scorpion,
. The antivenom was produced by immunizing albino rabbits with a mixture of
venom collected from high-risk areas in Morocco. Immunizations were performed by subcutaneous injections at multiple sites near the lymphatic system, following an immunization schedule. Production control of neutralizing antibody titers was conducted through immunodiffusion. Once a sufficient antibody titer was achieved, blood collection was performed, and the recovered plasma underwent affinity chromatography. The efficacy of purified IgG was evaluated by determining the ED
in mice, complemented by histological and immunohistochemical studies on its ability to neutralize venom-induced tissue alterations and the neutralization of toxins bound to receptors in the studied organs. The monovalent antivenom demonstrated specificity against
venom and effective cross-protection against the venom of the scorpions
and
, highly implicated in lethal envenomations in the Maghreb. This study shows that the developed monovalent antivenom exhibits notable efficacy against local scorpions and a surprising ability to neutralize the most lethal envenomations in North Africa. These results pave the way for a new, more specific, and promising therapeutic approach to countering severe scorpion envenomations, especially in Morocco, where specific treatment is lacking.
Many efforts aim at solving the serious problems encountered with immunotherapy against scorpion envenoming. The most attractive approach consists in generating single-chain antibody fragments (scFv) ...as their pharmaco-kinetic properties should match closely those of the scorpion toxins. Although high affinity scFv reagents have been generated in the past, their production level, stability, and toxin neutralizing capacity remain disappointingly poor. In the current study, we identified one Nanobody (Nb), a single-domain antigen-binding fragment of a dromedary Heavy-chain antibody (HCAb) that recognizes specifically the
Androctonus australis hector AahI′ toxin. This Nb has excellent production, stability and solubility characteristics. With this Nb we further manufactured a tandem linked bivalent construct and assembled a HCAb with improved antigen binding due to avidity effects. All these constructs were shown in mouse models to possess a scorpion toxin neutralization capacity that exceeds by far all previous attempts with scFv-based materials, even when used at lower doses. It is therefore clear that in the near future Nanobodies will be at the core of novel serotherapeutics as they combine multiple benefits over other reagents to treat scorpion envenomed patients.
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