Understanding the complexity of Tityus serrulatus venom: A focus on high molecular weight components

Tityus serrulatus scorpion is responsible for a significant number of envenomings in Brazil, ranging from mild to severe, and in some cases, leading to fatalities. While supportive care is the primary treatment modality, moderate and severe cases require antivenom administration despite potential limitations and adverse effects. The remarkable proliferation of T. serrulatus scorpions, attributed to their biology and asexual reproduction, contributes to a high incidence of envenomation. T. serrulatus scorpion venom predominantly consists of short proteins acting as neurotoxins (α and ß), that primarily target ion channels. Nevertheless, high molecular weight compounds, including metalloproteases, serine proteases, phospholipases, and hyaluronidases, are also present in the venom. These compounds play a crucial role in envenomation, influencing the severity of symptoms and the spread of venom. This review endeavors to comprehensively understand the T. serrulatus scorpion venom by elucidating the primary high molecular weight compounds and exploring their potential contributions to envenomation. Understanding these compounds' mechanisms of action can aid in developing more effective treatments and prevention strategies, ultimately mitigating the impact of scorpion envenomation on public health in Brazil.

Terrestrial venomous animals, the envenomings they cause, and treatment perspectives in the Middle East and North Africa.

The Middle East and Northern Africa, collectively known as the MENA region, are inhabited by a plethora of venomous animals that cause up to 420,000 bites and stings each year. To understand the resultant health burden and the key variables affecting it, this review describes the epidemiology of snake, scorpion, and spider envenomings primarily based on heterogenous hospital data in the MENA region and the pathologies associated with their venoms. In addition, we discuss the venom composition and the key medically relevant toxins of these venomous animals, and, finally, the antivenoms that are currently in use to counteract them. Unlike Asia and sub-Saharan Africa, scorpion stings are significantly more common (approximately 350,000 cases/year) than snakebites (approximately 70,000 cases/year) and present the most significant contributor to the overall health burden of envenomings, with spider bites being negligible. However, this review also indicates that there is a substantial lack of high-quality envenoming data available for the MENA region, rendering many of these estimates speculative. Our understanding of the venoms and the toxins they contain is also incomplete, but already presents clear trends. For instance, the majority of snake venoms contain snake venom metalloproteinases, while sodium channel-binding toxins and potassium channel-binding toxins are the scorpion toxins that cause most health-related challenges. There also currently exist a plethora of antivenoms, yet only few are clinically validated, and their high cost and limited availability present a substantial health challenge. Yet, some of the insights presented in this review might help direct future research and policy efforts toward the appropriate prioritization of efforts and aid the development of future therapeutic solutions, such as next-generation antivenoms.

N-Terminal Tagging with GFP Enhances Selectivity of Agitoxin 2 to Kv1.3-Channel Binding Site.

Recently developed fluorescent protein-scorpion toxin chimeras (FP-Tx) show blocking activities for potassium voltage-gated channels of Kv1 family and retain almost fully pharmacological profiles of the parental peptide toxins (Kuzmenkov et al., Sci Rep. 2016, 6, 33314). Here we report on N-terminally green fluorescent protein (GFP)-tagged agitoxin 2 (GFP-L2-AgTx2) with high affinity and selectivity for the binding site of Kv1.3 channel involved in the pathogenesis of various (primarily of autoimmune origin) diseases. The basis for this selectivity relates to N-terminal location of GFP, since transposition of GFP to the C-terminus of AgTx2 recovered specific interactions with the Kv1.1 and Kv1.6 binding sites. Competitive binding experiments revealed that the binding site of GFP-L2-AgTx2 overlaps that of charybdotoxin, kaliotoxin 1, and agitoxin 2, the known Kv1.3-channel pore blockers. GFP-L2-AgTx2 was demonstrated to be applicable as a fluorescent probe to search for Kv1.3 pore blockers among individual compounds and in complex mixtures, to measure blocker affinities, and to visualize Kv1.3 distribution at the plasma membrane of Kv1.3-expressing HEK293 cells. Our studies show that definite combinations of fluorescent proteins and peptide blockers can result in considerable modulation of the natural blocker-channel binding profile yielding selective fluorescent ligands of certain channels.

Risk Assessment and the Effects of Refuge Availability on the Defensive Behaviors of the Southern Unstriped Scorpion (Vaejovis carolinianus).

Selection should favor individuals that acquire, process, and act on relevant environmental signals to avoid predation. Studies have found that scorpions control their use of venom: both when it is released and the total volume expelled. However, this research has not included how a scorpion's awareness of environmental features influences these decisions. The current study tested 18 Vaejovis carolinianus scorpions (nine females and nine males) by placing them in circular arenas supplied with varying numbers (zero, two, or four) of square refuges and by tracking their movements overnight. The following morning, defensive behaviors were elicited by prodding scorpions on the chelae, prosoma, and metasoma once per second over 90 s. We recorded stings, venom use, chelae pinches, and flee duration. We found strong evidence that, across all behaviors measured, V. carolinianus perceived prods to the prosoma as more threatening than prods to the other locations. We found that stinging was a common behavior and became more dominant as the threat persisted. Though tenuous, we found evidence that scorpions' defensive behaviors changed based on the number of refuges and that these differences may be sex specific. Our findings suggest that V. carolinianus can assess risk and features of the local environment and, therefore, alter their defensive strategies accordingly.

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.

Thermostable potassium channel-inhibiting neurotoxins in processed scorpion medicinal material revealed by proteomic analysis: Implications of its pharmaceutical basis in traditional Chinese medicine.

The neurotoxins of venomous scorpion act on ion channels. Whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used in traditional Chinese medicine materials is unknown. Comprehensive mass spectrometry-based proteomic characterization of functionally active toxins in the processed medicinal scorpion material revealed 22 full-length and 44 truncated thermostable potassium channel-modulatory toxins that preserved six conserved cysteine residues capable of forming the three disulfide bonds necessary for toxicity. Additionally, a broad spectrum of degraded toxin fragments was found, indicating their relative thermal instability which enabled toxicity reduction. Furthermore, the suppression of interleukin-2 (IL-2) production in Jurkat cells and the reduced delayed-type hypersensitivity (DTH) response demonstrated that the extracts have immunoregulatory activity both in vitro and in vivo. Our work describes the first "map" of functionally active scorpion toxins in processed scorpion medicinal material, which is helpful to unveil the pharmaceutical basis of the processed scorpion medicinal material in traditional Chinese medicine. BIOLOGICAL SIGNIFICANCE: Scorpions have been used as medicinal materials in China for more than one thousand years. This is an example of the well-known "Combat poison with poison" strategy common to traditional Chinese medicine. In the past 30 years, extensive investigations of Chinese scorpions have indicated that the neurotoxins in the scorpion venom are the main toxic components and they target various ion channels in cell membranes. However, whether these neurotoxins are retained in processed Buthus martensii Karsch scorpions used for traditional Chinese medicine remains unknown. Our study described the thermal stability and instability of potassium channel-modulatory neurotoxins in processed scorpions and helps to understand the pharmaceutical basis underling the strategy of "combat poison with poison to cure diseases".

Molecular Characterization of a Three-disulfide Bridges Beta-like Neurotoxin from Androctonus crassicauda Scorpion Venom.

Scorpion venom is the richest source of peptide toxins with high levels of specific interactions with different ion-channel membrane proteins. The present study involved the amplification and sequencing of a 310-bp cDNA fragment encoding a beta-like neurotoxin active on sodium ion-channel from the venom glands of scorpion Androctonus crassicauda belonging to the Buthidae family using reverse transcription polymerase chain reaction (RT-PCR) technique. The amplified complementary DNA (cDNA) fragment had a coding sequence of 240 bp. The deduced precursor open-reading frame was composed of 80 amino acid residues contain a signal peptide of 22 amino acid residues, followed by a mature toxin of 58 amino acids. It had a molecular mass of 6.84 kDa and isoelectric point of 4.58. The sequence similarity search revealed several matches with the scorpion toxin-like domain of toxin-3 superfamily with a homology range of 35- 75%. Multiple alignments and secondary structure prediction demonstrated that the toxin peptide deduced from the amplified cDNA was related to the long-chain neurotoxins in size but stabilized by three disulfide bridges instead of four. The level of difference implies that the corresponding genes have originated from a common ancestor. This level of difference may also confirm an evolutionary link between the ‘short-chain’ and ‘long-chain’ toxins. The analysis showed one major segment within this neurotoxin with maximal hydrophilicity which was predicted to be antigenic by inducing an antibody response.

Mass fingerprinting and electrophysiological analysis of the venom from the scorpion Centruroides hirsutipalpus (Scorpiones: Buthidae)

Background Centruroides hirsutipalpus, of the family Buthidae, is a scorpion endemic to the Western Pacific region of Mexico. Although medically important, its venom has not yet been studied. Therefore, this communication aims to identify their venom components and possible functions. Methods Fingerprinting mass analysis of the soluble venom from this scorpion was achieved by high-performance liquid chromatography and electrospray mass spectrometry. Furthermore, the soluble venom and its toxic effects were evaluated extensively via electrophysiological assays in HEK cells expressing human voltage-gated Na+ channels (hNav 1.1 to Nav1.6), CHO cells expressing hNav 1.7, potassium channel hERG 1 (Ether-à-go-go-related-gene) and the human K+-channel hKv1.1. Results The separation of soluble venom produced 60 fractions from which 83 distinct components were identified. The molecular mass distribution of these components varies from 340 to 21,120 Da. Most of the peptides have a molecular weight between 7001 and 8000 Da (46% components), a range that usually corresponds to peptides known to affect Na+ channels. Peptides with molecular masses from 3000 to 5000 Da (28% of the components) were identified within the range corresponding to K+-channel blocking toxins. Two peptides were obtained in pure format and completely sequenced: one with 29 amino acids, showing sequence similarity to an "orphan peptide" of C. limpidus, and the other with 65 amino acid residues shown to be an arthropod toxin (lethal to crustaceans and toxic to crickets). The electrophysiological results of the whole soluble venom show a beta type modification of the currents of channels Nav1.1, Nav1.2 and Nav1.6. The main effect observed in channels hERG and hKv 1.1 was a reduction of the currents. ..

Transcriptomic analysis of the venom glands from the scorpion Hadogenes troglodytes revealed unique and extremely high diversity of the venom peptides.

Hadogenes is a genus of large African scorpions with 18 described species. However, little is known about the venom peptide composition of any species from Hadogenes so far. Here, we fully explored the composition of venom gland peptides from Hadogenes troglodytes using transcriptomic approach. We discovered 121 novel peptides from the scorpion, including 20 new-type peptides cross-linked with one, two, three, four or seven disulfide bridges, respectively, 11 novel K+-channel toxin-like peptides, 2 novel ryanodine receptors-specific toxin-like peptides, a unique peptide containing the cysteine knots of spider toxins, 15 novel La1-like toxins, 3 novel TIL domain-containing peptides, 5 novel peptides with atypical cysteine patterns, 19 novel antimicrobial peptides, 6 novel cysteine-free peptides and 39 new-type cysteine-free peptides. Among them, the new-type peptides are largely dominant; this highlights the unique diversity of the venom gland peptides from H. troglodytes. Some of the new peptides would serve as new molecular probes for the investigations of cellular ion channels and other receptors, or offer new templates for the development of therapeutic drugs for the treatment of ion channel-associated diseases, and infections caused by antibiotics-resistant pathogens. BIOLOGICAL SIGNIFICANCE: In this study, we fully explored the composition of venom gland peptides from the scorpion Hadogenes troglodytes using transcriptomic approach. We discovered a total of 121 novel peptides from the venom glands of the scorpion, of which new-type peptides are largely dominant. These data highlight the unique diversity of the venom gland peptides from the scorpion H. troglodytes, gain insights into new mechanisms for the scorpion to subdue its prey and predators, and enlarge the protein database of scorpion venom glands. The discovery of a lot of novel peptides provides new templates for the development of therapeutic drugs, and offers new molecular materials for the basic researches of various cellular receptors, and for the evolutionary investigations of scorpion toxins.

Enhanced and selective permeability of gold nanoparticles functionalized with cell penetrating peptide derived from maurocalcine animal toxin.

Functionalization of gold nanoparticles (GNPs) is suitable for many applications such as biomedical imaging, clinical diagnosis, and targeted delivery by conjugating cell-penetrating peptides (CPPs). Here, we investigated intracellular uptake of GNP conjugated to MCaUF1-9(Ala) , a CPP derived from maurocalcine (MCa) animal toxin, and compared it with TAT functionalized GNP. Peptide conjugated GNP was characterized using UV-Visible spectroscopy, dynamic light scattering, zeta potential, and transmission electron microscopy. Uptake of MCaUF1-9(Ala) and TAT functionalized GNPs was evaluated in three cell lines, HeLa, MDA-MB-231, and A431, using dark field imaging and atomic absorption spectroscopy. According to peptide sequences and type of cells different cell penetrating activity was observed. Peptide functionalized GNP had little effect on cell viability and respect to net charge difference between peptide, showed interesting selectivity against three cell types. Peptide conjugated to GNPs displayed higher uptake than bare GNPs in the all cell lines except HeLa cell with lowest internalization. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2693-2700, 2016.

Peptide de novo sequencing of mixture tandem mass spectra.

The impact of mixture spectra deconvolution on the performance of four popular de novo sequencing programs was tested using artificially constructed mixture spectra as well as experimental proteomics data. Mixture fragmentation spectra are recognized as a limitation in proteomics because they decrease the identification performance using database search engines. De novo sequencing approaches are expected to be even more sensitive to the reduction in mass spectrum quality resulting from peptide precursor co-isolation and thus prone to false identifications. The deconvolution approach matched complementary b-, y-ions to each precursor peptide mass, which allowed the creation of virtual spectra containing sequence specific fragment ions of each co-isolated peptide. Deconvolution processing resulted in equally efficient identification rates but increased the absolute number of correctly sequenced peptides. The improvement was in the range of 20-35% additional peptide identifications for a HeLa lysate sample. Some correct sequences were identified only using unprocessed spectra; however, the number of these was lower than those where improvement was obtained by mass spectral deconvolution. Tight candidate peptide score distribution and high sensitivity to small changes in the mass spectrum introduced by the employed deconvolution method could explain some of the missing peptide identifications.

Variability in venom volume, flow rate and duration in defensive stings of five scorpion species.

Scorpions have been shown to control their venom usage in defensive encounters, depending on the perceived threat. Potentially, the venom amount that is injected could be controlled by reducing the flow speed, the flow duration, or both. We here investigated these variables by allowing scorpions to sting into an oil-filled chamber, and recording the accreting venom droplets with high-speed video. The size of the spherical droplets on the video can then be used to calculate their volume. We recorded defensive stings of 20 specimens representing 5 species. Significant differences in the flow rate and total expelled volume were found between species. These differences are likely due to differences in overall size between the species. Large variation in both venom flow speed and duration are described between stinging events of single individuals. Both venom flow rate and flow duration correlate highly with the total expelled volume, indicating that scorpions may control both variables in order to achieve a desired end volume of venom during a sting.

Antimicrobial peptides from scorpion venoms.

The need for new antimicrobial agents is becoming one of the most urgent requirements in modern medicine. The venoms of many different species are rich sources of biologically active components and various therapeutic agents have been characterized including antimicrobial peptides (AMPs). Due to their potent activity, low resistance rates and unique mode of action, AMPs have recently received much attention. This review focuses on AMPs from the venoms of scorpions and examines all classes of AMPs found to date. It gives details of their biological activities with reference to peptide structure. The review examines the mechanism of action of AMPs and with this information, suggests possible mechanisms of action of less well characterised peptides. Finally, the review examines current and future trends of scorpion AMP research, by discussing recent successes obtained through proteomic and transcriptomic approaches.

Ability of horse anti-Tityus discrepans F(ab')2 ELISA assay to recognize Tityus discrepans venom toxins.

Anti-Tityus discrepans F(ab')2 ELISA recognition of T. discrepans toxins was measured with regression analysis and its slope called ELISA recognition value (ERv). Fractions containing toxins affecting mammal macrophages or Na(+)-channels have Ervs >19. Toxins affecting potassium channels or insect NaV channels have ERvs <10. Fractions including curarizing or antineoplasic peptides had ERvs <1. Erv increases in proportion to mammalian toxin toxicity rather than to toxin molecular mass.

Mild reproductive effects of the Tityus bahiensis scorpion venom in rats

Scorpion envenoming is a public health problem in Brazil, where Tityus serrulatus and T. bahiensis are considered the most dangerous scorpions. They are well adapted to urbanized environments, and there is an increasing probability of human exposure to these venoms, including during pregnancy. Not much is known about the effects of prenatal exposure to the venom, and no information is available to aid in the rational treatment of victims stung during pregnancy. Thus, this study aimed to investigate whether venom from the scorpion T. bahiensis administered once to pregnant female rats at a dose that causes a moderate envenomation may lead to deleterious effects on the reproductive performance of the dams and on the development of their offspring. This is the first work demonstrating that T. bahiensis venom, when administered experimentally to rats, alters maternal reproductive performance and the morphological development of fetuses. The venom was given to dams on the 5th (GD5) or on the 10th (GD10) gestational day. After laparotomy, on GD21, fetuses and placentas were counted, weighed and externally analyzed. The corpora lutea were counted. The sex and vitality of fetuses were evaluated, and each litter was then randomly divided for visceral or skeletal analyses. Data were analyzed by ANOVA followed by the Tukey-Kramer test and Fisher's exact test. The significance level for all tests was set at p < 0.05.

Mild reproductive effects of the Tityus bahiensis scorpion venom in rats

Scorpion envenoming is a public health problem in Brazil, where Tityus serrulatus and T. bahiensis are considered the most dangerous scorpions. They are well adapted to urbanized environments, and there is an increasing probability of human exposure to these venoms, including during pregnancy. Not much is known about the effects of prenatal exposure to the venom, and no information is available to aid in the rational treatment of victims stung during pregnancy. Thus, this study aimed to investigate whether venom from the scorpion T. bahiensis administered once to pregnant female rats at a dose that causes a moderate envenomation may lead to deleterious effects on the reproductive performance of the dams and on the development of their offspring. This is the first work demonstrating that T. bahiensis venom, when administered experimentally to rats, alters maternal reproductive performance and the morphological development of fetuses. The venom was given to dams on the 5th (GD5) or on the 10th (GD10) gestational day. After laparotomy, on GD21, fetuses and placentas were counted, weighed and externally analyzed. The corpora lutea were counted. The sex and vitality of fetuses were evaluated, and each litter was then randomly divided for visceral or skeletal analyses. Data were analyzed by ANOVA followed by the Tukey-Kramer test and Fisher's exact test. The significance level for all tests was set at p < 0.05.

Subtype-selective activation of K(v)7 channels by AaTXKß2₋64, a novel toxin variant from the Androctonus australis scorpion venom.

K(v)7.4 channel subunits are expressed in central auditory pathways and in inner ear sensory hair cells and skeletal and smooth muscle cells. Openers of K(v)7.4 channels have been suggested to improve hearing loss, systemic or pulmonary arterial hypertension, urinary incontinence, gastrointestinal and neuropsychiatric diseases, and skeletal muscle disorders. Scorpion venoms are a large source of peptides active on K⁺ channels. Therefore, we have optimized a combined purification/screening procedure to identify specific modulator(s) of K(v)7.4 channels from the venom of the North African scorpion Androctonus australis (Aa). We report the isolation and functional characterization of AaTXKß2₋64, a novel variant of AaTXKß1₋64, in a high-performance liquid chromatography fraction from Aa venom (named P8), which acts as the first peptide activator of K(v)7.4 channels. In particular, in both Xenopus oocytes and mammalian Chinese hamster ovary cells, AaTXKß2₋64, but not AaTXKß1₋64, hyperpolarized the threshold voltage of current activation and increased the maximal currents of heterologously expressed K(v)7.4 channels. AaTXKß2₋64 also activated K(v)7.3, K(v)7.2/3, and K(v)7.5/3 channels, whereas homomeric K(v)1.1, K(v)7.1, and K(v)7.2 channels were unaffected. We anticipate that these results may prove useful in unraveling the novel biologic roles of AaTXKß2₋64-sensitive K(v)7 channels and developing novel pharmacologic tools that allow subtype-selective targeting of K(v)7 channels.

Anticoagulant and factor Xa-like activities of Tityus discrepans scorpion venom

Tityus discrepans venom (TdV) produces a variety of haemostatic manifestations including alveoli fbrin deposition and/ or prothrombin and partial thromboplastin time (PT, PTT) alterations in mammals. In vitro studies have demonstrated that TdV contains tissue plasminogen activator-like (t-PA), fbrinolytic and plasmin inhibitory compounds and produces platelets activation through GPVI and a novel Src-dependent signalling pathway. The aim of this study is to describe the initial characterization of procoagulant and anticoagulant components from TdV. This venom was fractionated by exclusion molecular chromatography on a Sephadex G-50 column. The eluted material was collected as fve fractions called S1 to S5. These fractions and the whole venom were used to evaluate factor Xa- and thrombin-like activities, fbrinogen degradation, furthermore thrombin- and factor Xa-inhibitory activities. The results demonstrated that TdV contain components with factor Xa-like activity (procoagulants) as well fbrinogenolytic compounds present in the fraction S1 and components with factor Xa inhibitory activity in the fractions S4 and S5 (anticoagulants).

El veneno de Tityus discrepans (TdV) produce en mamíferos una variedad de manifestaciones hemostáticas tales como depósitos de fbrina en alveolos y/o alteración en los tiempos de protrombina y tromboplastina parcial (PT, PTT). Estudios in vitro han demostrado que el TdV contiene componentes semejantes al activador del plasminógeno tipo tisular (t-PA), fbrino-líticos, compuestos que inhiben la actividad de plasmina y además componentes que promueven la activación de plaquetas a través del receptor GPVI y por una nueva vía de señalización dependiente de las Src kinasas. El objetivo de este estudio es describir la caracterización inicial de componentes procoagulantes y anticoagulantes a partir del TdV. Este veneno fue fraccionado por cromatografía de exclusión molecular sobre una columna Sephadex G-50. El material eluido fue colectado en cinco fracciones denominadas S1 a S5. Estas fracciones y el veneno completo fueron usados para evaluar actividades semejantes a factor Xa y trombina, degradación de fbrinógeno, como también la inhibición de la actividad del factor Xa y de la trombina. Los resultados demostraron que TdV contiene componentes con actividad semejante al factor Xa (procoagulantes) y compuestos fbrinogenolíticos presentes en la fracción S1, además de componentes con actividad inhibitoria del factor Xa presentes en la fracción S4 y S5 (anticoagulantes).

Effects of atropine and propranolol on lung inflammation in experimental envenomation: comparison of two buthidae venoms

Previous works had shown that scorpion venom induced neurotransmitter elevation and an inflammatory response associated with various anatomo-pathological modifications. The most dangerous scorpions species in Algeria responsible for these effects are Androctonus australis hector(Aah) and Androctonus amoreuxi (Aam). Results Comparison of the physiopathological effects induced by the two venoms showed differences in the kinetic of cytokine release and in lung injury. The lung edema was only observed in response to Aah venom and it was correlated with cell infiltration. In order to better understand the involved mechanism in inflammatory response, we used two antagonists, atropine (non-selective muscarinic antagonist) and propranolol (β adrenergic antagonist), which lead to a decrease of cell infiltration but has no effect on edema forming.Conclusion These results suggest another pathway in the development of lung injury following envenomation with Aam or Aah venom.

[Quod medicina aliis, aliis est acre venenum**--venoms as a source of anticancer agents]. / Quod medicina aliis, aliis est acre venenum*--jady jako zródlo substancji o dzialaniu przeciwnowotworowym.

Natural product derived from plants and animals were used in folk medicine for centuries. The venoms produced by animals for hunting of self-defence are rich in bioactive compounds with broad spectrum of biological activity. The papers presents the most promising compounds isolated from venoms of snakes, scorpions and toads. For these compounds both: mechanism of anticancer activity as well as possibilities of clinical use are presented.