Characterization of Sodium Channel Peptides Obtained from the Venom of the Scorpion Centruroides bonito.

Five peptides were isolated from the venom of the Mexican scorpion Centruroides bonito by chromatographic procedures (molecular weight sieving, ion exchange columns, and HPLC) and were denoted Cbo1 to Cbo5. The first four peptides contain 66 amino acid residues and the last one contains 65 amino acids, stabilized by four disulfide bonds, with a molecular weight spanning from about 7.5 to 7.8 kDa. Four of them are toxic to mice, and their function on human Na+ channels expressed in HEK and CHO cells was verified. One of them (Cbo5) did not show any physiological effects. The ones toxic to mice showed that they are modifiers of the gating mechanism of the channels and belong to the beta type scorpion toxin (ß-ScTx), affecting mainly the Nav1.6 channels. A phylogenetic tree analysis of their sequences confirmed the high degree of amino acid similarities with other known bona fide ß-ScTx. The envenomation caused by this venom in mice is treated by using commercially horse antivenom available in Mexico. The potential neutralization of the toxic components was evaluated by means of surface plasmon resonance using four antibody fragments (10FG2, HV, LR, and 11F) which have been developed by our group. These antitoxins are antibody fragments of single-chain antibody type, expressed in E. coli and capable of recognizing Cbo1 to Cbo4 toxins to various degrees.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus.

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

An overview of some enzymes from buthid scorpion venoms from Colombia: Centruroides margaritatus, Tityus pachyurus, and Tityus n. sp. aff. metuendus

Background: In Colombia, several species of Buthidae scorpions belonging to the genera Centruroides and Tityus coexist, and their stings are considered life-threatening to humans because of their venom neurotoxins. Despite previous studies focusing on neurotoxins from these scorpion genera, little is known about the enzymes present in their venoms and their relationship with whole venom toxicity. Methods: Here, using proteomic and biochemical protocols the enzymatic activities of the venoms of three Colombian scorpion species, C. margaritatus, T. pachyurus, and T. n. sp. aff. metuendus, were compared to establish the presence and absence of enzymes such as phospholipases, hyaluronidases, and proteases that could be related to venom toxicity. Results: C. margaritatus was positive for hyaluronidases, T. n. sp. aff. metuendus for proteases, and T. pachyurus exhibited activity for all three mentioned enzymes. Conclusion: This information provides valuable insights into the specific enzyme diversity of each species' venom and their potential role in venom toxicity, which could contribute to the development of better treatments and prevention strategies for scorpion envenomation.

Unveiling the Protein Components of the Secretory-Venom Gland and Venom of the Scorpion Centruroides possanii (Buthidae) through Omic Technologies.

Centruroides possanii is a recently discovered species of "striped scorpion" found in Mexico. Certain species of Centruroides are known to be toxic to mammals, leading to numerous cases of human intoxications in the country. Venom components are thought to possess therapeutic potential and/or biotechnological applications. Hence, obtaining and analyzing the secretory gland transcriptome and venom proteome of C. possanii is relevant, and that is what is described in this communication. Since this is a newly described species, first, its LD50 to mice was determined and estimated to be 659 ng/g mouse weight. Using RNA extracted from this species and preparing their corresponding cDNA fragments, a transcriptome analysis was obtained on a Genome Analyzer (Illumina) using the 76-base pair-end sequencing protocol. Via high-throughput sequencing, 19,158,736 reads were obtained and ensembled in 835,204 sequences. Of them, 28,399 transcripts were annotated with Pfam. A total of 244 complete transcripts were identified in the transcriptome of C. possanii. Of these, 109 sequences showed identity to toxins that act on ion channels, 47 enzymes, 17 protease inhibitors (PINs), 11 defense peptides (HDPs), and 60 in other components. In addition, a sample of the soluble venom obtained from this scorpion was analyzed using an Orbitrap Velos apparatus, which allowed for identification by liquid chromatography followed by mass spectrometry (LC-MS/MS) of 70 peptides and proteins: 23 toxins, 27 enzymes, 6 PINs, 3 HDPs, and 11 other components. Until now, this work has the highest number of scorpion venom components identified through omics technologies. The main novel findings described here were analyzed in comparison with the known data from the literature, and this process permitted some new insights in this field.

Of Seven New K+ Channel Inhibitor Peptides of Centruroides bonito, α-KTx 2.24 Has a Picomolar Affinity for Kv1.2.

Seven new peptides denominated CboK1 to CboK7 were isolated from the venom of the Mexican scorpion Centruroides bonito and their primary structures were determined. The molecular weights ranged between 3760.4 Da and 4357.9 Da, containing 32 to 39 amino acid residues with three putative disulfide bridges. The comparison of amino acid sequences with known potassium scorpion toxins (KTx) and phylogenetic analysis revealed that CboK1 (α-KTx 10.5) and CboK2 (α-KTx 10.6) belong to the α-KTx 10.x subfamily, whereas CboK3 (α-KTx 2.22), CboK4 (α-KTx 2.23), CboK6 (α-KTx 2.21), and CboK7 (α-KTx 2.24) bear > 95% amino acid similarity with members of the α-KTx 2.x subfamily, and CboK5 is identical to Ce3 toxin (α-KTx 2.10). Electrophysiological assays demonstrated that except CboK1, all six other peptides blocked the Kv1.2 channel with Kd values in the picomolar range (24-763 pM) and inhibited the Kv1.3 channel with comparatively less potency (Kd values between 20-171 nM). CboK3 and CboK4 inhibited less than 10% and CboK7 inhibited about 42% of Kv1.1 currents at 100 nM concentration. Among all, CboK7 showed out-standing affinity for Kv1.2 (Kd = 24 pM), as well as high selectivity over Kv1.3 (850-fold) and Kv1.1 (~6000-fold). These characteristics of CboK7 may provide a framework for developing tools to treat Kv1.2-related channelopathies.

Medicinal plants from Mexico used in the treatment of scorpion sting.

Scorpion sting envenomation is a major public health in Mexico. Rural communities rarely have antivenoms in the health centers, therefore, the people commonly resort to using medicinal plants to treat the symptoms of envenoming caused by scorpion venom, but this knowledge has not yet been reported in detail. In this review, we carry out a review of the medicinal plants used in Mexico against scorpion stings. PubMed, Google, Science Direct, and the Digital Library of Mexican Traditional Medicine (DLMTM) were used to collect data. The results showed the use of at least 48 medicinal plants distributed in 26 families, where Fabaceae (14.6%), Lamiaceae (10.4%), and Asteraceae (10.4%) have the maximum representation. The application of leaves (32%) was preferred followed by roots (20%), stem (17.3%), flowers (16%), and bark (8%). In addition, the most common method of use to treat scorpion stings is decoction (32.5%). The oral and topical routes of administration have similar percentages of use. In vitro and in vivo studies of Aristolochia elegans, Bouvardia ternifolia, and Mimosa tenuiflora were found, which showed an antagonistic effect on the contraction of the ileum caused by the venom of C. limpidus, likewise, they increased the LD50 of said venom and even B. ternofila showed reduced albumin extravasation. The results of these studies demonstrate the promising use of medicinal plants for future pharmacological applications; nevertheless, validation, bioactive compound isolation and toxicity studies are necessary to support and improve therapeutics.

Characterization and Chemical Synthesis of Cm39 (α-KTx 4.8): A Scorpion Toxin That Inhibits Voltage-Gated K+ Channel KV1.2 and Small- and Intermediate-Conductance Ca2+-Activated K+ Channels KCa2.2 and KCa3.1.

A novel peptide, Cm39, was identified in the venom of the scorpion Centruroides margaritatus. Its primary structure was determined. It consists of 37 amino acid residues with a MW of 3980.2 Da. The full chemical synthesis and proper folding of Cm39 was obtained. Based on amino acid sequence alignment with different K+ channel inhibitor scorpion toxin (KTx) families and phylogenetic analysis, Cm39 belongs to the α-KTx 4 family and was registered with the systematic number of α-KTx 4.8. Synthetic Cm39 inhibits the voltage-gated K+ channel hKV1.2 with high affinity (Kd = 65 nM). The conductance-voltage relationship of KV1.2 was not altered in the presence of Cm39, and the analysis of the toxin binding kinetics was consistent with a bimolecular interaction between the peptide and the channel; therefore, the pore blocking mechanism is proposed for the toxin-channel interaction. Cm39 also inhibits the Ca2+-activated KCa2.2 and KCa3.1 channels, with Kd = 502 nM, and Kd = 58 nM, respectively. However, the peptide does not inhibit hKV1.1, hKV1.3, hKV1.4, hKV1.5, hKV1.6, hKV11.1, mKCa1.1 K+ channels or the hNaV1.5 and hNaV1.4 Na+ channels at 1 µM concentrations. Understanding the unusual selectivity profile of Cm39 motivates further experiments to reveal novel interactions with the vestibule of toxin-sensitive channels.

Heterologous expression of four recombinant toxins from Panamanian scorpions of the genus Tityus and Centruroides for production of antivenom.

BACKGROUND: The development of more effective antivenoms remains a necessity for countries where scorpionism is a public health problem. Also, the regionalization of antivenoms may be important for some countries with special scorpionism characteristics. OBJECTIVE: Production of antibodies capable of neutralizing the lethal effect of the venom of three scorpion species from Panama. METHODS: The primary structures of two neurotoxins from T. pachyurus, one from T. cerroazul and another from C. bicolor were elucidated using N-terminal amino acid degradation and Sanger gene cloned sequencing. The obtained mRNA transcripts were cloned and expressed using E. coli vectors. Different bacterial expression conditions were tested and the best culture conditions for each expressed protein is reported. The expressed scorpion toxins were purified by chromatographic methods and used as immunogens in rabbits. RESULTS: The antibodies produced under the reported immunization scheme show better neutralization (ED50) than other reported commercial antivenoms used to neutralize similar species scorpion venoms under similar LD50 conditions. CONCLUSION: The information reported here shows the proof of concept for selecting recombinant immunogens with the ability to produce antibodies for neutralizing the lethal effects of the most important medical species of scorpions in Panama.

Reduced Toxicity of Centruroides vittatus (Say, 1821) May Result from Lowered Sodium ß Toxin Gene Expression and Toxin Protein Production.

Body tissue and venom glands from an eastern population of the scorpion Centruroides vittatus (Say, 1821) were homogenized and molecular constituents removed to characterize putative sodium ß toxin gene diversity, RT-qPCR, transcriptomic, and proteomic variation. We cloned sodium ß toxins from genomic DNA, conducted RT-qPCR experiments with seven sodium ß toxin variants, performed venom gland tissue RNA-seq, and isolated venom proteins for mass spectrophotometry. We identified >70 putative novel sodium ß toxin genes, 111 toxin gene transcripts, 24 different toxin proteins, and quantified sodium ß toxin gene expression variation among individuals and between sexes. Our analyses contribute to the growing evidence that venom toxicity among scorpion taxa and their populations may be associated with toxin gene diversity, specific toxin transcripts variation, and subsequent protein production. Here, slight transcript variation among toxin gene variants may contribute to the major toxin protein variation in individual scorpion venom composition.

Comparison of the Scorpionism Caused by Centruroidesmargaritatus, Tityuspachyurus and Tityus n. sp. aff. metuendus Scorpion Venoms in Colombia.

Among other scorpion species, Colombia has two genera of the Buthidae family Centruroides and Tityus, considered to be dangerous to humans. This research shares scientific knowledge aiming to a better understanding about the pathophysiological effects of such venoms. The venom of the three species: Centruroides margaritarus, Tityus pachyurus, and T. n. sp. aff. metuendus with biomedical interest were studied. An initial pre-glycemic sample was taken from ICR mice. They were later intraperitoneally inoculated with doses of 35% and 70% of LD50 of total venom. Poisoning signs were observed during a 6-h period to determine the level of scorpionism. After observation, a second glycemic sample was taken, and a histopathological evaluation of different organs was performed. This work revealed that all three venoms showed considerably notorious histopathological alterations in main organs such as heart and lungs; and inducing multiple organ failure, in relation to the glycemia values, only C. margaritatus and T. n. sp. aff. metuendus showed significant changes through manifestation of hyperglycemia. According to the Colombian scorpionism level; signs were mild to severe affecting the autonomous nervous system.

Full Neutralization of Centruroidessculpturatus Scorpion Venom by Combining Two Human Antibody Fragments.

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

A novel lineage of polyomaviruses identified in bark scorpions.

Polyomaviruses are non-enveloped viruses with circular double-stranded DNA genomes (~4-7 kb). Initially identified in mammals, polyomaviruses have now been identified in birds and a few fish species. Although fragmentary polyomavirus-like sequences have been detected as apparent 'hitchhikers' in shotgun genomics datasets of various arthropods, the possible diversity of these viruses in invertebrates remains unclear. Scorpions are predatory arachnids that are among the oldest terrestrial animals. Using high-throughput sequencing and traditional molecular techniques we determine the genome sequences of eight novel polyomaviruses in scorpions (Centruroides sculpturatus) from the greater Phoenix area, Arizona, USA. Analysis of Centruroides transcriptomic datasets elucidated the splicing of the viral late gene array, which is more complex than that of vertebrate polyomaviruses. Phylogenetic analysis provides further evidence of co-divergence of polyomaviruses with their hosts, suggesting that at least one ancestral species of polyomaviruses was circulating amongst the primitive common ancestors of arthropods and chordates.

Identification and Characterization of Novel Proteins from Arizona Bark Scorpion Venom That Inhibit Nav1.8, a Voltage-Gated Sodium Channel Regulator of Pain Signaling.

The voltage-gated sodium channel Nav1.8 is linked to neuropathic and inflammatory pain, highlighting the potential to serve as a drug target. However, the biophysical mechanisms that regulate Nav1.8 activation and inactivation gating are not completely understood. Progress has been hindered by a lack of biochemical tools for examining Nav1.8 gating mechanisms. Arizona bark scorpion (Centruroides sculpturatus) venom proteins inhibit Nav1.8 and block pain in grasshopper mice (Onychomys torridus). These proteins provide tools for examining Nav1.8 structure-activity relationships. To identify proteins that inhibit Nav1.8 activity, venom samples were fractioned using liquid chromatography (reversed-phase and ion exchange). A recombinant Nav1.8 clone expressed in ND7/23 cells was used to identify subfractions that inhibited Nav1.8 Na+ current. Mass-spectrometry-based bottom-up proteomic analyses identified unique peptides from inhibitory subfractions. A search of the peptides against the AZ bark scorpion venom gland transcriptome revealed four novel proteins between 40 and 60% conserved with venom proteins from scorpions in four genera (Centruroides, Parabuthus, Androctonus, and Tityus). Ranging from 63 to 82 amino acids, each primary structure includes eight cysteines and a "CXCE" motif, where X = an aromatic residue (tryptophan, tyrosine, or phenylalanine). Electrophysiology data demonstrated that the inhibitory effects of bioactive subfractions can be removed by hyperpolarizing the channels, suggesting that proteins may function as gating modifiers as opposed to pore blockers.

The venom of the scorpion Centruroides limpidus, which causes the highest number of stings in Mexico, is neutralized by two recombinant antibody fragments.

Phage display and directed evolution have made it possible to generate recombinant antibodies in the format of single chain variable fragments (scFvs) capable of neutralizing different toxins and venoms of Mexican scorpions. Despite having managed to neutralize a significant number of venoms, some others have not yet been completely neutralized, due to the diversity of the toxic components present in them. An example is the venom of the scorpion Centruroides limpidus, which contains three toxins of medical importance, called Cll1, Cll2 and Cl13. The first two are neutralized by scFv 10FG2, while Cl13, due to its sequence divergence, was not even recognized. For this reason, the aim of the present work was the generation of a new scFv capable of neutralizing Cl13 toxin and thereby helping to neutralize the whole venom of this scorpion. By hybridoma technology, a monoclonal antibody (mAb B7) was generated, which was able to recognize and partially neutralize Cl13 toxin. From mAb B7, its scFv format was obtained, named scFv B7 and subjected to three cycles of directed evolution. At the end of these processes, scFv 11F which neutralized Cl13 toxin was obtained. This scFv, administered in conjunction with scFv 10FG2, allowed to fully neutralize the whole venom of Centruroides limpidus scorpion.

Colombian Scorpion Centruroides margaritatus: Purification and Characterization of a Gamma Potassium Toxin with Full-Block Activity on the hERG1 Channel.

The Colombian scorpion Centruroides margaritatus produces a venom considered of low toxicity. Nevertheless, there are known cases of envenomation resulting in cardiovascular disorders, probably due to venom components that target ion channels. Among them, the humanether-à-go-go-Related gene (hERG1) potassium channels are critical for cardiac action potential repolarization and alteration in its functionality are associated with cardiac disorders. This work describes the purification and electrophysiological characterization of a Centruroides margaritatus venom component acting on hERG1 channels, the CmERG1 toxin. This novel peptide is composed of 42 amino acids with a MW of 4792.88 Da, folded by four disulfide bonds and it is classified as member number 10 of the γ-KTx1 toxin family. CmERG1 inhibits hERG1 currents with an IC50 of 3.4 ± 0.2 nM. Despite its 90.5% identity with toxin É£-KTx1.1, isolated from Centruroides noxius, CmERG1 completely blocks hERG1 current, suggesting a more stable plug of the hERG channel, compared to that formed by other É£-KTx.

Scorpion Stings and Antivenom Use in Arizona.

BACKGROUND: Arizona's rugged desert landscape harbors many venomous animals, including a small nocturnal scorpion, Centruroides sculpturatus, whose venom can cause severe neuromotor disturbance. An effective antivenom is available at selected health care facilities in the state. METHODS: We analyzed 4398 calls of scorpion stings to the Arizona Poison and Drug Information Center (APDIC) in Tucson over a period of 3 years, from January 2017 to December 2019. RESULTS: We followed 1952 (44.4%) of the victims to resolution. We excluded 2253 callers with minimal effects of the sting and 193 victims with possible toxic effects who were lost to follow-up. The most common complaints among callers were pain at the sting site in 88.9% and local numbness in 62.2%. Detailed clinical information was obtained from 593 calls from a health care facility. Neuromotor signs consistent with C. sculpuratus envenomation included nystagmus in 163 (27.5%), hypersalivation in 91 (15.3%), and fasciculations in 88 (14.8%). Antivenom (Anascorp; Rare Disease Therapeutics, Inc., Franklin, Tenn) was administered to 145 patients. Most were children <5 years old (n = 76, or 54.4%); 27 (18.6%) were 5-9 years old and 42 (30.0%) were ≥10 years of age. About half, 79 of 145 (54.5%) victims who received antivenom, met the APDIC recommended use criteria. CONCLUSIONS: Patients treated with antivenom exhibited a rapid resolution of symptoms without immediate or delayed hypersensitivity reactions. We recommend broadened availability of antivenom at sites where it is most needed.

Envenomation by Centruroides pococki scorpion with neuromuscular toxicity.

Cases of human envenomation by Centruroides pococki are exceptionally reported in human. We report a Class III human envenomation by C. pococki in Guadeloupe, French West Indies, with neuromuscular toxicity that has never been described before. Symptoms resolved within a few hours, without the need for intravenous scorpion-specific antivenom.

Centruroides margaritatus scorpion complete venom exerts cardiovascular effects through alpha-1 adrenergic receptors.

Centruroides margaritatus scorpion stings are common in Colombia. However, the cardiovascular toxicity of the venom has not been clarified. AIM: To study the effect and mechanisms of action of the complete venom of C. margaritatus (CmV) on the murine cardiovascular system. METHODS: We evaluated the in vivo effect of CmV LD50 on the mean arterial pressure (MABP), heart rate, and surface electrocardiogram in male adult normotensive Wistar rats. Ex vivo, we evaluated the vascular reactivity of rat aortic rings to increasing concentrations (1 to 60 µg/mL) of CmV using the blockers L-NAME, indomethacin, seratrodast, and prazosin. RESULTS: In the first hour of poisoning, CmV increased the MABP. In the second hour after poisoning, the heart rate decreased as the normalized PR interval and QT corrected increased. After that, cardiovascular shock was demonstrated by a drastic fall in the MABP and signs of cardiac conduction system block. In aortic rings, CmV caused a direct vasoconstrictor effect mediated by alpha-1 adrenergic receptors and counteracted by nitric oxide. CONCLUSION: The direct vascular and probably the cardiac alpha-1 effects likely explain the transient hypertension and the maintenance of cardiac function, while interval lengthening may be due to K+ channel blockage. Afterwards, the effects of both the alpha-1 pathway and the K+ channel pathway converged, resulting in fatal cardiovascular shock. This knowledge could aid in understanding the dynamics of the effects of the venom and in designing treatments to address its cardiovascular effects.

Biochemical, electrophysiological and immunological characterization of the venom from Centruroides baergi, a new scorpion species of medical importance in Mexico.

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