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.

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.

[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.

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.

Molecular diversity of toxic components from the scorpion Heterometrus petersii venom revealed by proteomic and transcriptome analysis.

Scorpion venoms contain a vast untapped reservoir of natural products, which have the potential for medicinal value in drug discovery. In this study, toxin components from the scorpion Heterometrus petersii venom were evaluated by transcriptome and proteome analysis.Ten known families of venom peptides and proteins were identified, which include: two families of potassium channel toxins, four families of antimicrobial and cytolytic peptides,and one family from each of the calcium channel toxins, La1-like peptides, phospholipase A2,and the serine proteases. In addition, we also identified 12 atypical families, which include the acid phosphatases, diuretic peptides, and ten orphan families. From the data presented here, the extreme diversity and convergence of toxic components in scorpion venom was uncovered. Our work demonstrates the power of combining transcriptomic and proteomic approaches in the study of animal venoms.

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.

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.

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.

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.

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".

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.

Mass spectrometry strategies for venom mapping and peptide sequencing from crude venoms: case applications with single arthropod specimen.

Due to their complexity and diversity, animal venoms represent an extensive source of bioactive compounds such as peptides and proteins. Conventional approaches for their characterization often require large quantities of biological material. Current mass spectrometry (MS) techniques now give access to a wealth of information in a short working time frame with minute amounts of sample. Such MS approaches may now be used for the discovery of novel compounds, and once their structure has been determined they may be synthesized and tested for functional activity. Molecular mass fingerprints of venoms allow the rapid identification of known toxins as well as preliminary structural characterization of new compounds. De novo peptide sequencing by tandem mass spectrometry (MS/MS) offers rapid access to partial or total primary peptide structures. This article, written as a tutorial, also contains new material: molecular mass fingerprint analysis of Orthochirus innesi scorpion venom, and identification of components from bumblebee Bombus lapidarius venom, both collected from one single specimen. The structure of the three major peptides detected in the Bombus venom was fully characterized in one working day by de novo sequencing using an electrospray ionization hybrid quadrupole time-of-flight instrument (ESI-QqTOF) and a matrix-assisted laser desorption ionization time-of-flight instrument (MALDI-LIFT-TOF-TOF). After presenting the MS-based sequence elucidation, perspectives in using MS and MS/MS techniques in toxinology are discussed.

Moving pieces in a taxonomic puzzle: venom 2D-LC/MS and data clustering analyses to infer phylogenetic relationships in some scorpions from the Buthidae family (Scorpiones).

The Buthidae is the most clinically important scorpion family, with over 500 species distributed worldwide. Taxonomical positions and phylogenetic relationships concerning the representative genera and species of this family have been mostly inferred based upon comparisons between morphological characters. Yet, some authors have performed such inferences by comparing some structural properties of a few selected molecules found in the venoms from these scorpions. Here, we propose a novel methodology pipeline designed to address these issues. We have analyzed the whole venoms from some species that exemplify peculiar cases in the Buthidae family (Tityus stigmurus, Tityus serrulatus, Tityus bahiensis, Leiurus quinquestriatus quinquestriatus and Leiurus quinquestriatus hebraeus), by means of a proteomic approach using a 2D-LC/MS technique. The molecules found in these venoms were clustered according to their physicochemical properties (molecular mass and hydrophobicity), by using the machine learning-based Weka software. The clusters assessment, along with the number of molecules found in a given cluster for each scorpion, which assigns for the venom and structural family complexities, respectively, was used to generate a phenetic correlation tree for positioning these species. Our results were in accordance with the classical taxonomy viewpoint, which places T. serrulatus and T. stigmurus as very close species, T. bahiensis as a less related species in the Tityus genus and L. q. quinquestriatus and L. q. hebraeus with small differences within the same species (L. quinquestriatus). Therefore, we believe that this is a well-suited method to determine venom complexities that reflect the scorpions' evolutionary history, which can be crucial to reconstruct their phylogeny through the molecular evolution of their venoms.

Effect of acid secretion blockade on acute gastric mucosal lesions induced by Tityus serrulatus scorpion toxin in anaesthetized rats.

Scorpion venom (TX) promotes gastric acid and pepsin secretion leading to acute gastric mucosal lesions (AGML), when injected in animals. The goal of the present study was to observe the effects of acid gastric secretion blockers over the incidence of TX-induced AGML in vivo. To verify this model, we used male albino rats, fasted 18-20 h (n=122) and anaesthetized with urethane (1.4 g/kg, i.p.). Their trachea and left femoral vein were both cannulated; the first to avoid airway obstructions during scorpion intoxication and the second for administration of saline, TX and acid blockers. Following the surgical procedure, the animals were divided in 10 groups of at least 10 animals each. Control groups were injected with NaCl 0.9% 1 ml/kg (n=10) or TX 375 microg/kg (n=32). Test groups (n=10, each) received atropine 5 mg/kg, cimetidine 10mg/kg, ranitidine 2.5mg/kg, ranitidine 5mg/kg, omeprazol 1 mg/kg, omeprazol 4 mg/kg, octreotide 80 and octreotide 100 microg/kg 10 min before the TX was injected. After 1h of intoxication, the stomach was resected for macroscopic study and the gastric secretion was collected for volume, pH and acid output assessment. We observed that all blockers were able to completely or partially prevent the TX-induced acid secretion as well as the AGML (p<0.05). Our data suggest the TX-induced AGML can be prevented by different class of acid blockers injected before the intoxication.

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.

Identification of second lipolysis activating protein from scorpion Buthus occitanus tunetanus.

Besides the previously described LVP1, a second protein, LVP2, inducing a lipolytic response in adipose cells, was purified from scorpion Buthus occitanus tunetanus venom. It represented 2% of crude venom proteins, with pHi = 6 and molecular mass of 16889 Da. The reduction and the alkylation of LVP2 revealed an heterodimeric structure. Isolated alpha and beta chains of LVP2 have a molecular weight (MW) of 8822 Da and 8902, respectively. This protein was not toxic to mice and stimulated lipolysis on freshly dissociated rat adipocytes in a dose-dependent manner with EC50 = 2 +/- 0.75 microg/ml. LVP2 subunits did not display any lipolytic activity. As previously described for venom and LVP1, beta adrenergic receptor (beta AR) antagonists interfere with LVP2 activity. Furthermore, it is shown that LVP2 competes with [3H] CGP 12177 (beta1/beta2 AR antagonist) for binding to adipocyte plasma membrane with an IC50 of about 10(-7)M. Thus, these results bring original information on the existence of proteins that are present in scorpion venoms and can exert a distinct biological activity on adipocyte lipolysis through a beta-type adreno-receptor pathway.

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.

Characterization of two different peptides from the venom of the scorpion Buthus sindicus.

Two disulfide-rich, low-molecular mass peptides (approximately 3 kDa and approximately 4 kDa) have been isolated from Buthus sindicus venom using ion-exchange and reverse-phase HPLC. Peptide I has 35 residues with 8 half-cystine residues and is clearly related to four-disulfide core proteins of the neurophysin type and to toxins of other scorpion species (55-63% residue identity). Peptide II, present in low yield, has 28 residues with 6 half-cystine residues and a structure largely dissimilar from that of peptide I and other characterized toxins, although probably still a member of the disulfide core peptide type. Consequently, scorpion venom contains, in addition to toxins characterized before, toxin-like compounds with distant relationships.

Primary structure of scorpion anti-insect toxins isolated from the venom of Leiurus quinquestriatus quinquestriatus.

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.