Tityus scorpion stings are an important public health problem in Brazil, where the incidence of such stings exceeds the incidence of the health problems caused by other venomous animals, including snakes. In this study, we have analysed specific enzymatic activities of the venom from the Brazilian scorpions of Tityus genus, i.e., Tityus serrulatus, Tityus bahiensis and Tityus stigmurus. The data presented here revealed that Tityus spp. venoms exhibited significant hyaluronidase activity but no phospholipase activity. All the venom samples exhibited the ability to hydrolyse Abz-FLRRV-EDDnp and dynorphin 1-13 substrates. These activities were inhibited by 1,10-phenanthroline but not by PMSF, indicating the presence of metalloproteinases in the Tityus spp. venoms. The venom peptidase activity on Abz-FLRRV-EDDnp and on dynorphin 1-13 was partially inhibited by therapeutic Brazilian anti-scorpion and anti-arachnidic antivenoms. Dynorphin 1-13 (YGGFLRRIRPKLK) contains two scissile bonds between the residues Leu-Arg and Arg-Arg that are susceptible to cleavage by the Tityus venom metallopeptidase(s). Their cleavage releases leu-enkephalin, an important bioactive peptide. The detection of metalloproteinase(s) with specificity for both dynorphin 1-13 degradation and leu-enkephalin releasing can be important for the mechanistic understanding of hypotension and bradycardia induction in cases of scorpion stings, whereas hyaluronidases might contribute to the diffusion of the toxins present in these venoms. Furthermore, the limited inhibition of the toxic enzymatic activities by commercial antivenoms illustrates the necessity of improvements in current antivenom preparation.

Although proline-rich oligopeptides are known molecules, we present here 32 new sequences that are inhibitors of the angiotensin-converting enzyme and consistent with the symptoms of the victims of Bitis spp, who display severe hypotension.

The number of cases of envenomation by scorpions has grown significantly in Brazil since 2007, with the most severe cases being caused by the Tityus serrulatus scorpion. Although envenomed patients mostly suffer neurotoxic manifestations, other symptoms, such as hypertension, cannot be exclusively attributed to neurotoxins. Omics analyses have detected plentiful amounts of metalloproteases in T. serrulatus venom. However, the roles played by these enzymes in envenomation are still unclear. Endeavoring to investigate the functions of scorpion venom proteases, we describe here for the first time an Angiotensin I-Converting Enzyme-like peptidase (ACE-like) purified from T. serrulatus venom. The crude venom cleaved natural and fluorescent substrates and these activities were inhibited by captopril. Regarding the serum neutralization, the scorpion antivenom was more effective at blocking the ACE-like activity than arachnid antivenom, although neither completely inhibited the venom cleavage action, even at higher doses. ACE-like was purified from the venom after three chromatographic steps and its identity was confirmed by mass spectrometric and transcriptomic analyses. Bioinformatics analysis showed homology between the ACE-like transcript sequences from Tityus spp. and human testis ACE. These findings advance our understanding of T. serrulatus venom components and may improve treatment of envenomation victims, as ACE-like may contribute to envenomation symptoms, especially the resulting hypertension.

Snakebite envenomation is considered a highly relevant public health hazard in South America, having an impact in terms of mortality and morbidity. In Brazil, Bothrops (sensu latu) poisoning is responsible for 90% of the snakebites and in patients treated at the Vital Brazil Hospital (Butantan Institute) this index reaches 97.5%. The objective of the present study was to analyze more specifically the ability of the antibothropic antivenom, produced by the Butantan Institute, São Paulo, Brazil, to neutralize metallo-and serine peptidases, known as the major toxins present in Bothrops jararaca venom. A set of Fret peptides (Free Ressonance Energy Transfer) was studied using the BjV (B. jararaca venom) and site-directed inhibitors PMSF, EDTA and 1,10-phenanthroline. Two substrates were reached to be used as specific tools for studies with metallo peptidases, Abz-FASSAQ-EDDnp, and the serine peptidases, Abz-RPPGFSPFRQ-EDDnp. In disagreement with the literature, the use of both substrates and the antibothropic serum showed a weak neutralization of the serine peptidases present in this venom and a strong neutralization of the metallo peptidases. In order to investigate possible mechanisms of action that have not yet been described for the serine peptidases from the BjV, the present study shows for the first time a new tyrosine-specific chymotrypsin-like and angiotensin-degrading serine peptidase activity, that was partially blocked by the antibothropic serum. In conclusion, the antivenom presented a good neutralization of metallo peptidases but not of serine peptidases, indicating that further studies about serine peptidases immunogenicity are necessary to improve the antibothropic serum.

Envenoming by viperid snakes results in a complex pattern of tissue damage, including hemorrhage, which in severe cases may lead to permanent sequelae. Snake venom metalloproteinases (SVMPs) are main players in this pathogenesis, acting synergistically upon different mammalian proteomes. Hemorrhagic Factor 3 (HF3), a P-III class SVMP from Bothrops jararaca , induces severe local hemorrhage at pmol doses in a murine model. Our hypothesis is that in a complex scenario of tissue damage, HF3 triggers proteolytic cascades by acting on a partially known substrate repertoire. Here, we focused on the hypothesis that different proteoglycans, plasma proteins, and the platelet derived growth factor receptor (PDGFR) could be involved in the HF3-induced hemorrhagic process. In surface plasmon resonance assays, various proteoglycans were demonstrated to interact with HF3, and their incubation with HF3 showed degradation or limited proteolysis. Likewise, Western blot analysis showed in vivo degradation of biglycan, decorin, glypican, lumican and syndecan in the HF3-induced hemorrhagic process. Moreover, antithrombin III, complement components C3 and C4, factor II and plasminogen were cleaved in vitro by HF3. Notably, HF3 cleaved PDGFR (alpha and beta) and PDGF in vitro, while both receptor forms were detected as cleaved in vivo in the hemorrhagic process induced by HF3. These findings outline the multifactorial character of SVMP-induced tissue damage, including the transient activation of tissue proteinases, and underscore for the first time that endothelial glycocalyx proteoglycans and PDGFR are targets of SVMPs in the disruption of microvasculature integrity and generation of hemorrhage.

Scorpion venoms are composed of several substances with different pharmacological activities. Neurotoxins exert their effects by targeting ion channels resulting in toxic effects to mammals, insects and crustaceans. Tb II-I, a fraction isolated from Tityus bahiensis scorpion venom, was investigated for its ability to induce neurological and immune-inflammatory effects. Two putative β-sodium channel toxins were identified in this fraction, Tb2 II and Tb 4, the latter having been completely sequenced by mass spectrometry. Male Wistar rats, stereotaxically implanted with intrahippocampal cannulas and electrodes, were injected with Tb II-I (2 µg/2 µL) via the intrahippocampal route. The behavior, electrographic activity and cellular integrity of the animals were analyzed and the intracerebral level of cytokines determined. Tb II-I injection induced seizures and damage in the hippocampus. These alterations were correlated with the changes in the level of the cytokines tumoral necrosis factor-α (TNF-α) and interleukin-6 (IL-6). Therefore, the binding of Tb II-I to its target in the central nervous system may induce inflammation resulting in neuropathological and behavioral alterations.

Accidents caused by scorpions represent a relevant public health issue in Brazil, being more recurring than incidents with snakes and spiders. The main species responsible for this situation is the yellow scorpion, Tityus serrulatus, due especially to the great frequency with which accidents occur and the potential of its venom to induce severe clinical manifestations, even death, mainly among children. Although neurotoxins are well characterized, little information is known about other components of scorpion venoms, such as peptidases, and their effect on envenomation. Previous results from our group showed that the metallopeptidases present in this venom are capable of hydrolyzing the neuropeptide dynorphin 1-13 in vitro, releasing Leu-enkephalin, which may interact with ion channels and promote indirect neurotoxicity. Thus, this study aims to get more information about the effect of toxic peptidase activity present in the venom on biologically active peptides, and to evaluate the in vitro neutralizing potential of commercial antivenoms produced by the Butantan Institute. A set of human bioactive peptides were studied as substrates for the peptidases, and the members of the neuropeptide Y family were found to be the most susceptible ones. All new substrate hydrolyses were totally inhibited by ethylenediaminetetracetic and not blocked by phenylmethanesulfonylfluoride, indicating that metallopeptidases were responsible for the peptidase activity. Also, peptidase activities were only partially inhibited by therapeutic Brazilian scorpion antivenom (SAV) and arachnid antivenom (AAV). The dose-response inhibition by both antivenoms indicates that AAV neutralizes better than SAV at the used doses. These characterizations, unpublished until now, can contribute to the improvement of our knowledge about the venom and envenomation processes by T. serrulatus.

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