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Structural variability is a feature of snake venom proteins, and glycosylation is a post-translational modification that contributes to the diversification of venom proteomes. Studies by our group have shown that Bothrops venoms are distinctly defined by their glycoprotein content, and that most hybrid/complex N-glycans identified in these venoms contain sialic acid. Considering that metalloproteases and serine proteases are abundant components of Bothrops venoms and essential in the envenomation process, and that these enzymes contain several glycosylation sites, the role of sialic acid in venom proteolytic activity was evaluated. Here we show that removal of sialic acid by treatment of nine Bothrops venoms with neuraminidase (i) altered the pattern of gelatinolysis in zymography of most venoms and reduced the gelatinolytic activity of all venoms, (ii) decreased the proteolytic activity of some venoms on fibrinogen and the clotting activity of human plasma of all venoms, and (iii) altered the proteolysis profile of plasma proteins by B. jararaca venom, suggesting that sialic acid may play a role in the interaction of proteases with their protein substrates. In contrast, the profile of venom amidolytic activity on Bz-Arg-pNA did not change after removal of sialic acid, indicating that this monosaccharide is not essential in N-glycans of serine proteases acting on small substrates. In summary, these results expand the knowledge about the variability of the subproteomes of Bothrops venom proteases, and for the first time point to the importance of carbohydrate chains containing sialic acid in the enzymatic activities of venom proteases relevant in human envenomation.

Most characterized angiogenic modulators are proteolytic fragments of structural plasma and/or matrix components. Herein, we have identified a novel anti-angiogenic peptide generated by the in vitro hydrolysis of the C-terminal moiety of the fibrinogen alpha chain, produced by the snake venom metalloprotease bothropasin (SVMP), a hemorrhagic proteinase in Bothrops jararaca venom. The 14-amino acids peptide (alphastatin-C) is a potent antagonist of basic fibroblast growth factor, induced endothelial cell (HUVEC-CS) proliferation, migration and capillary tube formation in matrigel. It also inhibits cell adhesion to fibronectin. The basis of the antagonism between bFGF and alphastatin-C is elucidated by the inhibition of various bFGF induced signaling pathways and their molecular components modification, whenever the combination of the stimuli is provided, in comparison to the treatment with bFGF only. To corroborate to the potential therapeutic use of alphastatin-C, we have chosen to perform in vivo assays in two distinct angiogenic settings. In chick model, alphastatin-C inhibits chorioallantoic membrane angiogenesis. In mouse, it efficiently reduces tumor number and volume in a melanoma model, due to the impairment of tumor neovascularization in treated mice. In contrast, we show that the alphastatin-C peptide induces arteriogenesis, increasing pial collateral density in neonate mice. alphastatin-C is an efficient new antiangiogenic FGF-associated agent in vitro, it is an inhibitor of embryonic and tumor vascularization in vivo while, it is an arteriogenic agent. The results also suggest that SVMPs can be used as in vitro biochemical tools to process plasma and/or matrix macromolecular components unraveling new angiostatic peptides.

Snake venom proteomes have long been investigated to explore a multitude of biologically active components that are used for prey capture and defense, and are involved in the pathological effects observed upon mammalian envenomation. Glycosylation is a major protein post-translational modification in venoms and contributes to the diversification of proteomes. We have shown that Bothrops venoms are markedly defined by their content of glycoproteins, and that most N-glycan structures of eight Bothrops venoms contain sialic acid, while bisected N-acetylglucosamine was identified in Bothrops cotiara venom. To further investigate the mechanisms involved in the generation of different venoms by related snakes, here the glycoproteomes of nine Bothrops venoms (Bothrops atrox, B. cotiara, Bothrops erythromelas, Bothrops fonsecai, B. insularis, Bothrops jararaca, Bothrops jararacussu, Bothrops moojeni and Bothrops neuwiedi) were comparatively analyzed by enrichment with three lectins of different specificities, recognizing bisecting N-acetylglucosamine- and sialic acid-containing glycoproteins, and mass spectrometry. The lectin capture strategy generated venom fractions enriched with several glycoproteins, including metalloprotease, serine protease, and L- amino acid oxidase, in addition to various types of low abundant enzymes. The different contents of lectin-enriched proteins underscore novel aspects of the variability of the glycoprotein subproteomes of Bothrops venoms and point to the role of distinct types of glycan chains in generating different venoms by closely related snake species.

In Brazil, snakes from the Bothrops genus are responsible for thousands of accidents, and their venoms are mainly made up of proteolytic enzymes. Although the antibothropic serum produced by the Butantan Institute is remarkable in saving lives, studies show that some symptoms observed in cases of envenoming are not efficiently neutralized. Moreover, our group has shown that the commercial antivenom does not fully neutralize in vitro some serine proteases present in the Bothrops jararaca venom. Therefore, this study focuses on a new method in the production of specific immunoglobulins capable of neutralizing the activities of these enzymes in vitro. For this, a pool of serine proteases that was not inhibited by the commercial antivenom, made up of four enzymes (KN-BJ2, BjSP, HS112 and BPA) from the B. jararaca venom was obtained through two chromatographic steps (DEAE-HPLC and C8-RP-HPLC). The identities of these proteases were confirmed by SDS-PAGE, followed by tryptic digestion and mass spectrometry analysis. This pool was inoculated into BALB/c and C57BL/6 mice, using SBA-15 as adjuvant, and the produced IgGs were purified by affinity chromatography. The sera were characterized by ELISA, avidity and proteolytic neutralization assays. Both animal models responded to the immunization, producing higher IgGs titers when compared to the commercial antivenom. The experimental serum from BALB/c mice presented a better hydrolysis inhibition of the selective fluorescent substrate for serine proteases (~80%) when compared to C57BL/6 (~25%) and the commercial antivenom (

Cancer is characterized by the development of abnormal cells that divide in an uncontrolled way and may spread into other tissues where they may infiltrate and destroy normal body tissue. Several previous reports have described biochemical anti-tumorigenic properties of crude snake venom or its components, including their capability of inhibiting cell proliferation and promoting cell death. However, to the best of our knowledge, there is no work describing cancer cell proteomic changes following treatment with snake venoms. In this work we describe the quantitative changes in proteomics of MCF7 and MDA-MB-231 breast tumor cell lines following treatment with Bothrops jararaca snake venom, as well as the functional implications of the proteomic changes. Cell lines were treated with sub-toxic doses at either 0.63 μg/mL (low) or 2.5 μg/mL (high) of B. jararaca venom for 24 h, conditions that cause no cell death per se. Proteomics analysis was conducted on a nano-scale liquid chromatography coupled on-line with mass spectrometry (nLC-MS/MS). More than 1000 proteins were identified and evaluated from each cell line treated with either the low or high dose of the snake venom. Protein profiling upon venom treatment showed differential expression of several proteins related to cancer cell metabolism, immune response, and inflammation. Among the identified proteins we highlight histone H3, SNX3, HEL-S-156an, MTCH2, RPS, MCC2, IGF2BP1, and GSTM3. These data suggest that sub-toxic doses of B. jararaca venom have potential to modulate cancer-development related protein targets in cancer cells. This work illustrates a novel biochemical strategy to identify therapeutic targets against cancer cell growth and survival.

Made available in DSpace on 2021-06-25T10:44:33Z (GMT). No. of bitstreams: 0 Previous issue date: 2020-10-07. Added 1 bitstream(s) on 2021-07-15T15:22:57Z : No. of bitstreams: 1 S1678-91992020000100332.pdf: 2226438 bytes, checksum: fa2d02231c1d500b9f79508b01012af1 (MD5) Research Triangle Institute Background: Proteases play an important role for the proper physiological functions of the most diverse organisms. When unregulated, they are associated with several pathologies. Therefore, proteases have become potential therapeutic targets regarding the search for inhibitors. Snake venoms are complex mixtures of molecules that can feature a variety of functions, including peptidase inhibition. Considering this, the present study reports the purification and characterization of a Kunitz-type peptide present in the Dendroaspis polylepis venom as a simultaneous inhibitor of elastase-1 and cathepsin L. Methods: The low molecular weight pool from D. polylepis venom was fractionated in reverse phase HPLC and all peaks were tested in fluorimetric assays. The selected fraction that presented inhibitory activity over both proteases was submitted to mass spectrometry analysis, and the obtained sequence was determined as a Kunitz-type serine protease inhibitor homolog dendrotoxin I. The molecular docking of the Kunitz peptide on the elastase was carried out in the program Z-DOCK, and the program RosettaDock was used to add hydrogens to the models, which were re-ranked using ZRANK program. Results: The fraction containing the Kunitz molecule presented similar inhibition of both elastase-1 and cathepsin L. This Kunitz-type peptide was characterized as an uncompetitive inhibitor for elastase-1, presenting an inhibition constant (Ki) of 8 μM. The docking analysis led us to synthesize two peptides: PEP1, which was substrate for both elastase-1 and cathepsin L, and PEP2, a 30-mer cyclic peptide, which showed to be a cathepsin L competitive inhibitor, with a Ki of 1.96 µM, and an elastase-1 substrate. Conclusion: This work describes a Kunitz-type peptide toxin presenting inhibitory potential over serine and cysteine proteases, and this could contribute to further understand the envenomation process by D. polylepis. In addition, the PEP2 inhibits the cathepsin L activity with a low inhibition constant. Laboratory of Immunochemistry Butantan Institute Laboratory of Biochemistry and Biophysics Butantan Institute Department of Physics and Biophysics Botucatu Biosciences Institute (IBB) São Paulo State University (UNESP) Department of Physics and Biophysics Botucatu Biosciences Institute (IBB) São Paulo State University (UNESP)

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.

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 μM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice’s naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I

Although omics studies have indicated presence of proteases on the Tityus serrulatus venom (TsV), little is known about the function of these molecules. The TsV contains metalloproteases that cleave a series of human neuropeptides, including the dynorphin A (1-13) and the members of neuropeptide Y family. Aiming to isolate the proteases responsible for this activity, the metalloserrulase 3 and 4 (TsMS 3 and TsMS 4) were purified after two chromatographic steps and identified by mass spectrometry analysis. The biochemical parameters (pH, temperature and cation effects) were determined for both proteases, and the catalytic parameters (Km, kcat, cleavage sites) of TsMS 4 over fluorescent substrate were obtained. The metalloserrulases have a high preference for cleaving neuropeptides but presented different primary specificities. For example, the Leu-enkephalin released from dynorphin A (1-13) hydrolysis was exclusively performed by TsMS 3. Neutralization assays using Butantan Institute antivenoms show that both metalloserrulases were well blocked. Although TsMS 3 and TsMS 4 were previously described through cDNA library studies using the venom gland, this is the first time that both these toxins were purified. Thus, this study represents a step further in understanding the mechanism of scorpion venom metalloproteases, which may act as possible neuropeptidases in the envenomation process.

The scorpion Tityus serrulatus venom comprises a complex mixture of molecules that paralyzes and kills preys, especially insects. However, venom components also interact with molecules in humans, causing clinic envenomation. This cross-interaction may result from homologous molecular targets in mammalians and insects, such as (NEP)-like enzymes. In face of these similarities, we searched for peptides in Tityus serrulatus venom using human NEP as a screening tool. We found a NEP-inhibiting peptide with the primary sequence YLPT, which is very similar to that of the insect neuropeptide proctolin (RYLPT). Thus, we named the new peptide [des-Arg(1)]-proctolin. Comparative NEP activity assays using natural substrates demonstrated that [des-Arg(1)]-proctolin has high specificity for NEP and better inhibitory activity than proctolin. To test the initial hypothesis that molecular homologies allow Tityus serrulatus venom to act on both mammal and insect targets, we investigated the presence of a NEP-like in cockroaches, the main scorpion prey, that could be likewise inhibited by [des-Arg(1)]-proctolin. Indeed, we detected a possible NEP-like in a homogenate of cockroach heads whose activity was blocked by thiorphan and also by [des-Arg(1)]-proctolin. Western blot analysis using a human NEP monoclonal antibody suggested a NEP-like enzyme in the homogenate of cockroach heads. Our study describes for the first time a proctolin-like peptide, named [des-Arg(1)]-proctolin, isolated from Tityus serrulatus venom. The tetrapeptide inhibits human NEP activity and a NEP-like activity in a cockroach head homogenate, thus it may play a role in human envenomation as well as in the paralysis and death of scorpion preys.

TsTX-I, isolated from Tityus serrulatus scorpion venom, causes epileptic-like discharges when injected into the central nervous system. The involvement of excitatory amino acids and cytokines in this activity was investigated. Our results have demonstrated that TsTX-I increases the release of IFN-γ but does not alter the intracerebral concentration of the excitatory amino acids in rats. Thus, this cytokine seems to be more important in the convulsive process than glutamate.

Scorpion stings are the main cause of human envenomation in Brazil and, for the treatment of victims, the World Health Organization (WHO) recommends the use of antivenoms. The first step to achieve effective antivenom is to use a good quality venom pool and to evaluate it, with LD50 determination as the most accepted procedure. It is, however, time-consuming and requires advanced technical training. Further, there are significant ethical concerns regarding the number of animals required for testing. Hence, we investigated the correspondence between LD50 results, in vitro assays, and a strong correlation with proteolytic activity levels was observed, showing, remarkably, that proteases are potential toxicity markers for Tityus serrulatus venom. The comparison of reversed-phase chromatographic profiles also has a potential application in venoms’ quality control, as there were fewer neurotoxins detected in the venom with high LD50 value. These results were confirmed by mass spectrometry analysis. Therefore, these methods could precede the LD50 assay to evaluate the venom excellence by discriminating—and discarding—poor-quality batches, and, consequently, with a positive impact on the number of animals used. Notably, proposed assays are fast and inexpensive, being technically and economically feasible in Tityus serrulatus venom quality control to produce effective antivenoms.

In Brazil, envenomation by Bothrops pitvipers is responsible for over 73% of snakebites, and their venom is a rich source of proteolytic enzymes. Most studies have demonstrated that Bothrops jararaca venom acts on macromolecular substrates, causing an imbalance in the victim's hemostatic system. In contrast, fewer studies have examined the proteolytic activity on small molecules such as peptides. In this study, we used a set of bioactive peptides (insulin B chain, Met-enkephalin, Leu-enkephalin, neuropeptide Y, peptide YY, pancreatic polypeptide, substance P and somatostatin) to identify new peptide substrates for the metallopeptidases and serine peptidases from the B. jararaca venom. The majority of these peptides were substrates for the venom, but neuropeptide Y and pancreatic polypeptide presented higher hydrolyses rates. Although most of the peptides were simultaneously substrates for both classes of proteases, serine peptidases were the most active. Substance P was an exclusive substrate for metallopeptidases, while somatostatin was a selective substrate for serine peptidases. The neutralizing efficacy of the bothropic antivenom produced by the Butantan Institute was also assessed and found to totally prevent substance P hydrolysis, whereas somatostatin cleavage was not inhibited. Thus, the antivenom effectively inhibited metallopeptidase activity, but did not neutralize some of the serine peptidases. These results indicate that, in addition to cleaving proteins, the proteolytic enzymes from this venom also hydrolyze bioactive peptides, and this peptidase activity could effectively contribute to some of the many dire manifestations of envenomation.

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.

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.

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.

Most functions attributed to Tityus serrulatus venom (TsV) are related to active molecules on ion-channels; however, here we describe a new pentapeptide that was discovered through enzymatic assay selection using EP24.15. The primary structure analysis revealed the sequence KEXXG (X means Ile or Leu), similar to the sequence present in the β-KTX propeptide described from the venom of Tityus spp. We confirmed through HPLC analysis that KEILG is the peptide present in TsV, but that KELLG also inhibits EP24.15 although through different mechanisms.

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.