ADDovenom: Thermostable Protein-Based ADDomer Nanoparticles as New Therapeutics for Snakebite Envenoming.

Snakebite envenoming can be a life-threatening medical emergency that requires prompt medical intervention to neutralise the effects of venom toxins. Each year up to 138,000 people die from snakebites and threefold more victims suffer life-altering disabilities. The current treatment of snakebite relies solely on antivenom-polyclonal antibodies isolated from the plasma of hyperimmunised animals-which is associated with numerous deficiencies. The ADDovenom project seeks to deliver a novel snakebite therapy, through the use of an innovative protein-based scaffold as a next-generation antivenom. The ADDomer is a megadalton-sized, thermostable synthetic nanoparticle derived from the adenovirus penton base protein; it has 60 high-avidity binding sites to neutralise venom toxins. Here, we outline our experimental strategies to achieve this goal using state-of-the-art protein engineering, expression technology and mass spectrometry, as well as in vitro and in vivo venom neutralisation assays. We anticipate that the approaches described here will produce antivenom with unparalleled efficacy, safety and affordability.

Unravelling the Gastroprotective Potential of Kefir: Exploring Antioxidant Effects in Preventing Gastric Ulcers.

The present study was conducted to evaluate the protective effect of milk kefir against NSAID-induced gastric ulcers. Male Swiss mice were divided into three groups: control (Vehicle; UHT milk at a dose of 0.3 mL/100 g), proton pump inhibitor (PPI; lansoprazole 30 mg/kg), and 4% milk kefir (Kefir; 0.3 mL/100 g). After 14 days of treatment, gastric ulcer was induced by oral administration of indomethacin (40 mg/kg). Reactive oxygen species (ROS), nitric oxide (NO), DNA content, cellular apoptosis, IL-10 and TNF-α levels, and myeloperoxidase (MPO) enzyme activity were determined. The interaction networks between NADPH oxidase 2 and kefir peptides 1-35 were determined using the Residue Interaction Network Generator (RING) webserver. Pretreatment with kefir for 14 days prevented gastric lesions. In addition, kefir administration reduced ROS production, DNA fragmentation, apoptosis, and TNF-α systemic levels. Simultaneously, kefir increased NO bioavailability in gastric cells and IL-10 systemic levels. A total of 35 kefir peptides showed affinity with NADPH oxidase 2. These findings suggest that the gastroprotective effect of kefir is due to its antioxidant and anti-inflammatory properties. Kefir could be a promising natural therapy for gastric ulcers, opening new perspectives for future research.

Next-Generation Sequencing for Venomics: Application of Multi-Enzymatic Limited Digestion for Inventorying the Snake Venom Arsenal.

To improve the characterization of snake venom protein profiles, we report the application of a new generation of proteomic methodology to deeply characterize complex protein mixtures. The new approach, combining a synergic multi-enzymatic and a time-limited digestion (MELD), is a versatile and straightforward protocol previously developed by our group. The higher number of overlapping peptides generated during MELD increases the quality of downstream peptide sequencing and of protein identification. In this context, this work aims at applying the MELD strategy to a venomics purpose for the first time, and especially for the characterization of snake venoms. We used four venoms as the test models for this proof of concept: two Elapidae (Dendroaspis polylepis and Naja naja) and two Viperidae (Bitis arietans and Echis ocellatus). Each venom was reduced and alkylated before being submitted to two different protocols: the classical bottom-up proteomics strategy including a digestion step with trypsin only, or MELD, which combines the activities of trypsin, Glu-C and chymotrypsin with a limited digestion approach. The resulting samples were then injected on an M-Class chromatographic system, and hyphenated to a Q-Exactive Mass Spectrometer. Toxins and protein identification were performed by Peaks Studio X+. The results show that MELD considerably improves the number of sequenced (de novo) peptides and identified peptides from protein databases, leading to the unambiguous identification of a greater number of toxins and proteins. For each venom, MELD was successful, not only in terms of the identification of the major toxins (increasing of sequence coverage), but also concerning the less abundant cellular components (identification of new groups of proteins). In light of these results, MELD represents a credible methodology to be applied as the next generation of proteomics approaches dedicated to venomic analysis. It may open new perspectives for the sequencing and inventorying of the venom arsenal and should expand global knowledge about venom composition.

Oxidative stress induced by Pollonein-LAAO, a new L-amino acid oxidase from Bothrops moojeni venom, prompts prostate tumor spheroid cell death and impairs the cellular invasion process in vitro.

Cancer cells produce abnormal levels of reactive oxygen species (ROS) that contribute to promote their malignant phenotype. In this framework, we hypothesized that the change in ROS concentration above threshold could impair key events of prostate cancer cells (PC-3) progression. Our results demonstrated that Pollonein-LAAO, a new L-amino acid oxidase obtained from Bothrops moojeni venom, was cytotoxic to PC-3 cells in two-dimensional and in tumor spheroid assays. Pollonein-LAAO was able to increase the intracellular ROS generation that culminates in cell death from apoptosis by both intrinsic and extrinsic pathways due to the up-regulation of TP53, BAX, BAD, TNFRSF10B and CASP8. Additionally, Pollonein-LAAO reduced mitochondrial membrane potential and caused G0/G1 phase to delay, due to the up-regulation of CDKN1A and the down-regulation of the expression of CDK2 and E2F. Interestingly, Pollonein-LAAO inhibited critical steps of the cellular invasion process (migration, invasion and adhesion), due to the down-regulation of SNAI1, VIM, MMP2, ITGA2, ITGAV and ITGB3. Furthermore, the Pollonein-LAAO effects were associated with the intracellular ROS production, since the presence of catalase restored the invasiveness of PC-3 cells. In this sense, this study contributes to the potential use of Pollonein-LAAO as ROS-based agent to enhance the current understanding of cancer treatment strategies.

Biochemical characterization and assessment of leishmanicidal effects of a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom (CollinLA AO-I).

This study reports the isolation of CollinLAAO-I, a new L-amino acid oxidase from Crotalus durissus collilineatus snake venom, its biochemical characterization and leishmanicidal potential in Leishmania spp. CollinLAAO-I (63.1 kDa) was successfully isolated with high purity using two chromatographic steps and represents 2.5% of total venom proteins. CollinLAAO-I displayed high enzymatic activity (4262.83 U/mg/min), significantly reducing after 28 days. The enzymatic activity of CollinLAAO-I revealed higher affinity for hydrophobic amino acids such as L-leucine, high enzymatic activity in a wide pH range (6.0-10.0), at temperatures from 0 to 25 °C, and showed complete inhibition in the presence of Na+ and K+. Cytotoxicity assays revealed IC50 of 18.49 and 11.66 µg/mL for Leishmania (L.) amazonensis and Leishmania (L.) infantum, respectively, and the cytotoxicity was completely suppressed by catalase. CollinLAAO-I significantly increased the intracellular concentration of reactive oxygen species (ROS) and reduced the mitochondrial potential of both Leishmania species. Furthermore, CollinLAAO-I decreased the parasite capacity to infect macrophages by around 70%, indicating that even subtoxic concentrations of CollinLAAO-I can interfere with Leishmania vital processes. Thus, the results obtained for CollinLAAO-I provide important support for developing therapeutic strategies against leishmaniasis.

Isolation and characterization of the first phosphodiesterase (Bj-PDE) from the venom of Bothrops jararacussu snake.

Phosphodiesterases are exonucleases that sequentially hydrolyse phosphodiester bonds of polynucleotides from the 3'-end and release 5-mononucleotides. After more than one decade without any advance in the study of Bothropic phosphodiesterases, we described here the isolation of the first phosphodiesterase from Bothrops jararacussu, which we named Bj-PDE. A five-step column chromatography procedure (size exclusion, hydrophobic interaction, cation exchange, lentil lectin affinity, and blue sepharose affinity) enabled isolation of Bj-PDE with preserved and stable enzymatic activity (bis(p-nitrophenyl) phosphate substrate), Km = 6.9 mM (± 0.7 mM), kcat/Km = 1.7 × 104 M-1 s-1 (± 0.2 × 104 M-1 s-1), MW = 116 kDa (SDS-PAGE), optimum activity around 45 °C at pH 8.0, and stability for 81 days at different storage temperatures (8, -20, and - 80 °C). Ca2+ and Mg2+ ions positively influenced Bj-PDE activity, while EDTA had the opposite action. Zn2+ restored >50 % of enzyme activity after its inhibition by EDTA. The Bj-PDE partial sequence identified by mass spectrometry was very similar to the sequence of BATXPDE1 from Bothrops atrox, which was evolutionarily close to this new PDE. Therefore, our study represents an important progress on the isolation of this minor toxin and sheds new lights on the properties and bioprospection of bothropic phosphodiesterases.

Heterologous expression of Ts8, a neurotoxin from Tityus serrulatus venom, evidences its antifungal activity.

In this study we expressed the Ts8, a neurotoxin from Tityus serrulatus scorpion venom, in Pichia pastoris yeast. We evaluated the peptide expression in different conditions, such as pH, temperature, and addition of casamino acids supplement. Analyses of expressed products by mass spectrometry and Edman degradation showed that rTs8 has sites that allow its cleavage by yeast proteases released into the culture medium. The casamino acids addition was favourable for toxin expression, however, was not sufficient to minimize proteolytic degradation. Functional assays with recombinant toxin fragments and native toxins have demonstrated the release of cytokines such as TNF-α and IL-1ß in some peptides tested. In addition, the toxins were shown to inhibit the Pichia pastoris growth in antifungal test and were not toxic to alveolar macrophages cells at the concentrations analyzed The electrophysiological screening, by voltage clamp technique, showed that the rTs8 fragment with the highest molecular weight inhibited the Kv1.3 channel, whereas the N-terminal fragment had no activity on the ion channels tested.

Insights into structure and function of CdcVEGFs, the vascular endothelial growth factor from Crotalus durissus collilineatus snake venom.

Vascular endothelial growth factors (VEGFs) are crucial molecules involved in the modulation of angiogenesis. Snake venom-derived VEGFs (svVEGFs) are known to contribute significantly to the envenoming due to their capacity of increasing vascular permeability. In our work, we isolated and analyzed the biochemical and functional properties of the VEGF from Crotalus durissus collilineatus venom (CdcVEGF). The venom was fractionated by reversed phase chromatography on FPLC system (Fast Protein Liquid Chromatography) and the eluted fractions were submitted to an ELISA assay using an anti-VEGF-F antibody, for identification of svVEGF. Positive fractions for svVEGF were submitted to SDS-PAGE and to an anion exchange chromatography to isolate the molecule. The subfractions were analyzed by ELISA and SDS-PAGE and six of them presented svVEGFs, named CdcVEGF1 (Q23-3), CdcVEGF2 (Q24-3), CdcVEGF3 (Q24-4), CdcVEGF4 (Q25-3), CdcVEGF5 (Q25-4), and CdcVEGF6 (Q25-5). Their structural characterization was accomplished by mass spectrometry analysis using MALDI-TOF to determine their molecular masses and UPLC-ESI-QTOF to determine their amino acid sequence. Interestingly, all isolated CdcVEGFs induced angiogenesis on HUVEC cells through tube formation on Matrigel when compared to culture medium (negative control). Moreover, CdcVEGF2 and CdcVEGF3 also induced a significant increase in tube formation when compared to the positive control (basic fibroblast growth factor - bFGF). Additionally, crotalid antivenom produced by the Instituto Butantan was able to recognize CdcVEGFs, demonstrating to be immunogenic. This study demonstrates that snake venom cocktail can reveal novel and important molecules, which are potential molecular tools to study diverse biological processes, such as angiogenesis.

Shedding Lights on Crude Venom from Solitary Foraging Predatory Ant Ectatomma opaciventre: Initial Toxinological Investigation.

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1-116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

Shedding lights on crude venom from solitary foraging predatory ant Ectatomma opaciventre: initial toxinological investigation

Some species of primitive predatory ants, despite living in a colony, exercise their hunting collection strategy individually; their venom is painful, paralyzing, digestive, and lethal for their prey, yet the toxins responsible for these effects are poorly known. Ectatomma opaciventre is a previously unrecorded solitary hunting ant from the Brazilian Cerrado. To overcome this hindrance, the present study performed the in vitro enzymatic, biochemical, and biological activities of E. opaciventre to better understand the properties of this venom. Its venom showed several proteins with masses ranging from 1–116 kDa, highlighting the complexity of this venom. Compounds with high enzymatic activity were described, elucidating different enzyme classes present in the venom, with the presence of the first L-amino acid oxidase in Hymenoptera venoms being reported. Its crude venom contributes to a state of blood incoagulability, acting on primary hemostasis, inhibiting collagen-induced platelet aggregation, and operating on the fibrinolysis of loose red clots. Furthermore, the E. opaciventre venom preferentially induced cytotoxic effects on lung cancer cell lines and three different species of Leishmania. These data shed a comprehensive portrait of enzymatic components, biochemical and biological effects in vitro, opening perspectives for bio-pharmacological application of E. opaciventre venom molecules.

De Novo Transcriptome Analysis of the Venom of Latrodectus geometricus with the Discovery of an Insect-Selective Na Channel Modulator.

The brown widow spider, Latrodectus geometricus, is a predator of a variety of agricultural insects and is also hazardous for humans. Its venom is a true pharmacopeia representing neurotoxic peptides targeting the ion channels and/or receptors of both vertebrates and invertebrates. The lack of transcriptomic information, however, limits our knowledge of the diversity of components present in its venom. The purpose of this study was two-fold: (1) carry out a transcriptomic analysis of the venom, and (2) investigate the bioactivity of the venom using an electrophysiological bioassay. From 32,505 assembled transcripts, 8 toxin families were classified, and the ankyrin repeats (ANK), agatoxin, centipede toxin, ctenitoxin, lycotoxin, scorpion toxin-like, and SCP families were reported in the L. geometricus venom gland. The diversity of L. geometricus venom was also uncovered by the transcriptomics approach with the presence of defensins, chitinases, translationally controlled tumor proteins (TCTPs), leucine-rich proteins, serine proteases, and other important venom components. The venom was also chromatographically purified, and the activity contained in the fractions was investigated using an electrophysiological bioassay with the use of a voltage clamp on ion channels in order to find if the neurotoxic effects of the spider venom could be linked to a particular molecular target. The findings show that U24-ctenitoxin-Pn1a involves the inhibition of the insect sodium (Nav) channels, BgNav and DmNav. This study provides an overview of the molecular diversity of L. geometricus venom, which can be used as a reference for the venom of other spider species. The venom composition profile also increases our knowledge for the development of novel insecticides targeting voltage-gated sodium channels.

A comparative study on the leishmanicidal activity of the L-amino acid oxidases BjussuLAAO-II and BmooLAAO-II isolated from Brazilian Bothrops snake venoms.

This study aims to examine whether two L-amino acid oxidases isolated from Bothrops snake venom (SV-LAAOs) were cytotoxic to Leishmania (Leishmania) amazonensis and Leishmania (Viannia) braziliensis, two causative agents of leishmaniasis, which is an endemic disease in tropical and subtropical countries. The SV-LAAOs BjussuLAAO-II and BmooLAAO-II were isolated from Bothrops jararacussu and Bothrops moojeni venom, respectively, through a three-step chromatography process that used molecular exclusion, hydrophobic interaction, and affinity columns. BmooLAAO-II is a new SV-LAAO isoform that we isolated in this study. The purified BjussuLAAO-II and BmooLAAO-II had high L-amino acid oxidase-specific activity: 3481.17 and 4924.77 U/mg/min, respectively. Both SV-LAAOs were strongly cytotoxic to the two Leishmania species, even at low concentrations. At the same concentration, BjussuLAAO-II and BmooLAAO-II exerted different cytotoxic effects on the parasites. We reported for the first time that the SV-LAAOs suppressed cell proliferation and altered the mitochondrial membrane potential of the two Leishmania species. Surprisingly, BjussuLAAO-II increased the intracellular reactive oxygen species production only in L. (L.) amazonensis, while BmooLAAO-II increased the intracellular reactive oxygen species production only in L. (V.) braziliensis, indicating that these SV-LAAOs had a certain specificity of action.

Decellularized Splenic Matrix as a Scaffold for Spleen Bioengineering.

The spleen is considered a non-essential organ. However, its importance is increasingly clear, given the serious disorders caused by its absence or dysfunction, e.g., greater susceptibility to infections, thromboembolism and cancer. Surgical techniques to preserve the spleen and maintain splenic function have become increasingly common. However, the morbidity and mortality associated with its absence and dysfunction are still high. We used the decellularization technique to obtain a viable splenic scaffold for recellularization in vitro and propose the idea of bioengineered spleen transplantation to the host. We observed the maintenance of important structural components such as white pulp, marginal zone and red pulp, in addition to the network of vascular ducts. The decellularized scaffold presents minimal residual DNA and SDS, which are essential to prevent immunogenic responses and transplantation failure. Also, the main components of the splenic matrix were preserved after decellularization, with retention of approximately 72% in the matrisomal protein content. The scaffold we developed was partially recellularized with stromal cells from the spleen of neonatal rats, demonstrating adhesion, proliferation and viability of cells. Therefore, the splenic scaffold is very promising for use in studies on spleen reconstruction and transplantation, with the aim of complete recovery of splenic function.

From Animal Poisons and Venoms to Medicines: Achievements, Challenges and Perspectives in Drug Discovery.

Animal poisons and venoms are comprised of different classes of molecules displaying wide-ranging pharmacological activities. This review aims to provide an in-depth view of toxin-based compounds from terrestrial and marine organisms used as diagnostic tools, experimental molecules to validate postulated therapeutic targets, drug libraries, prototypes for the design of drugs, cosmeceuticals, and therapeutic agents. However, making these molecules applicable requires extensive preclinical trials, with some applications also demanding clinical trials, in order to validate their molecular target, mechanism of action, effective dose, potential adverse effects, as well as other fundamental parameters. Here we go through the pitfalls for a toxin-based potential therapeutic drug to become eligible for clinical trials and marketing. The manuscript also presents an overview of the current picture for several molecules from different animal venoms and poisons (such as those from amphibians, cone snails, hymenopterans, scorpions, sea anemones, snakes, spiders, tetraodontiformes, bats, and shrews) that have been used in clinical trials. Advances and perspectives on the therapeutic potential of molecules from other underexploited animals, such as caterpillars and ticks, are also reported. The challenges faced during the lengthy and costly preclinical and clinical studies and how to overcome these hindrances are also discussed for that drug candidates going to the bedside. It covers most of the drugs developed using toxins, the molecules that have failed and those that are currently in clinical trials. The article presents a detailed overview of toxins that have been used as therapeutic agents, including their discovery, formulation, dosage, indications, main adverse effects, and pregnancy and breastfeeding prescription warnings. Toxins in diagnosis, as well as cosmeceuticals and atypical therapies (bee venom and leech therapies) are also reported. The level of cumulative and detailed information provided in this review may help pharmacists, physicians, biotechnologists, pharmacologists, and scientists interested in toxinology, drug discovery, and development of toxin-based products.

Decellularized splenic matrix as a scaffold for spleen bioengineering

The spleen is considered a non-essential organ. However, its importance is increasingly clear, given the serious disorders caused by its absence or dysfunction, e.g., greater susceptibility to infections, thromboembolism and cancer. Surgical techniques to preserve the spleen and maintain splenic function have become increasingly common. However, the morbidity and mortality associated with its absence and dysfunction are still high. We used the decellularization technique to obtain a viable splenic scaffold for recellularization in vitro and propose the idea of bioengineered spleen transplantation to the host. We observed the maintenance of important structural components such as white pulp, marginal zone and red pulp, in addition to the network of vascular ducts. The decellularized scaffold presents minimal residual DNA and SDS, which are essential to prevent immunogenic responses and transplantation failure. Also, the main components of the splenic matrix were preserved after decellularization, with retention of approximately 72% in the matrisomal protein content. The scaffold we developed was partially recellularized with stromal cells from the spleen of neonatal rats, demonstrating adhesion, proliferation and viability of cells. Therefore, the splenic scaffold is very promising for use in studies on spleen reconstruction and transplantation, with the aim of complete recovery of splenic function.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins.

BACKGROUND: Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. METHODS: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. RESULTS: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). CONCLUSIONS: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.

Identification of new bioactive peptides from Kefir milk through proteopeptidomics: Bioprospection of antihypertensive molecules.

Kefir, a probiotic beverage prepared from fermented milk, has been associated with antihypertensive activity. However, the bioactive molecules responsible for this activity still remain unclear. Therefore, in this study we aim to evaluate the mechanisms of the antihypertensive effects of Kefir in the two-kidney one-clip hypertension model, and to bioprospect for bioactive peptides identified by proteomic methodologies. Treatment with Kefir was performed via gavage, and resulted in a 37 mmHg reduction in systolic arterial pressure and 19% inhibition of angiotensin converting enzyme (ACE) activity. For the proteopeptidomic study, the protein extract of Kefir beverage and non-fermented bovine milk were analysed by MALDI-TOF mass spectrometry, and their tryptic digestion products sequenced via Shotgun proteomics (Q-Exactive mass spectrometer). A list of 35 peptides with potential hypertensive activity due to ACE inhibition were identified. These results demonstrate the benefits of Kefir products, and may guide the design of new antihypertensive drugs.

Proteome of fraction from Tityus serrulatus venom reveals new enzymes and toxins

Tityus serrulatus venom (Ts venom) is a complex mixture of several compounds with biotechnological and therapeutical potentials, which highlights the importance of the identification and characterization of these components. Although a considerable number of studies have been dedicated to the characterization of this complex cocktail, there is still a limitation of knowledge concerning its venom composition. Most of Ts venom studies aim to isolate and characterize their neurotoxins, which are small, basic proteins and are eluted with high buffer concentrations on cation exchange chromatography. The first and largest fraction from carboxymethyl cellulose-52 (CMC-52) chromatography of Ts venom, named fraction I (Fr I), is a mixture of proteins of high and low molecular masses, which do not interact with the cation exchange resin, being therefore a probable source of components still unknown of this venom. Thus, the present study aimed to perform the proteome study of Fraction I from Ts venom, by high resolution mass spectrometry, and its biochemical characterization, by the determination of several enzymatic activities. Methods: Fraction I was obtained by a cation exchange chromatography using 50 mg of crude venom. This fraction was subjected to a biochemical characterization, including determination of L-amino acid oxidase, phospholipase, hyaluronidase, proteases activities and inhibition of angiotensin converting enzyme (ACE) activity. Fraction I was submitted to reduction, alkylation and digestion processes, and the tryptic digested peptides obtained were analyzed in a Q-Exactive Orbitrap mass spectrometer. Data analysis was performed by PEAKS 8.5 software against NCBI database. Results: Fraction I exhibits proteolytic activity and it was able to inhibit ACE activity. Its proteome analysis identified 8 different classes of venom components, among them: neurotoxins (48%), metalloproteinases (21%), hypotensive peptides (11%), cysteine-rich venom protein (9%), antimicrobial peptides (AMP), phospholipases and other enzymes (chymotrypsin and lysozymes) (3%) and phosphodiesterases (2%). Conclusions: The combination of a proteomic and biochemical characterization strategies leads us to identify new components in the T. serrulatus scorpion venom. The proteome of venom´s fraction can provide valuable direction in the obtainment of components in their native forms in order to perform a preliminary characterization and, consequently, to promote advances in biological discoveries in toxinology.(AU)

Microbial production of toxins from the scorpion venom: properties and applications.

Scorpion venom are composed mainly of bioactive proteins and peptides that may serve as lead compounds for the design of biotechnological tools and therapeutic drugs. However, exploring the therapeutic potential of scorpion venom components is mainly impaired by the low yield of purified toxins from milked venom. Therefore, production of toxin-derived peptides and proteins by heterologous expression is the strategy of choice for research groups and pharmaceutical industry to overcome this limitation. Recombinant expression in microorganisms is often the first choice, since bacteria and yeast systems combine high level of recombinant protein expression, fast cell growth and multiplication and simple media requirement. Herein, we present a comprehensive revision, which describes the scorpion venom components that were produced in their recombinant forms using microbial systems. In addition, we highlight the pros and cons of performing the heterologous expression of these compounds, regarding the particularities of each microorganism and how these processes can affect the application of these venom components. The most used microbial system in the heterologous expression of scorpion venom components is Escherichia coli (85%), and among all the recombinant venom components produced, 69% were neurotoxins. This review may light up future researchers in the choice of the best expression system to produce scorpion venom components of interest.

Proteopeptidomic, Functional and Immunoreactivity Characterization of Bothrops moojeni Snake Venom: Influence of Snake Gender on Venom Composition.

Venom composition varies across snakes from all taxonomic levels and is influenced by the snakes’ age, habitat, diet, and sexual dimorphism. The present study reports the first in-depth investigation of venom composition in male and female Bothrops moojeni (B. moojeni) snakes (BmooM and BmooF, respectively) through three proteomics approaches associated with functional, cytotoxic, and immunoreactivity characterization. Compared with BmooM venom, BmooF venom exhibited weaker hyaluronidase, metalloproteinase, and phospholipase activity; stronger recognition by anti-bothropic serum; 1.4-fold stronger cytotoxicity; and greater number of peptides. The increased L-amino acid oxidase expression probably accounted for the stronger immunoreactivity and cytotoxicity of BmooF venom. BmooF and BmooM venom shared only 19% peptides. Some venom components were gender-specific, such as phospholipases B, phospholipase inhibitor, and hyaluronidases in BmooM, and cysteine-rich secretory proteins in BmooF. In conclusion, we describe herein the first proteomics study of B. moojeni snake venom and an in-depth characterization of gender-specific differences in venom composition. Altogether, our findings not only stress the importance of considering the snake’s gender during antivenom production, but also help to identify new potential drugs and biotechnological tools.