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

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

Cloning and heterologous expression of hyaluronidase and/or novel toxins obtained from the transcriptome of Tityus serrulatus\' venom gland

As hialuronidases de peçonhas animais são capazes de clivar o hialuronan presente na matriz extracelular, facilitando a difusão das toxinas no tecido da vítima. Essas enzimas têm sido negligenciadas, devido à instabilidade enzimática e à baixa concentração na peçonha. Assim, a expressão heteróloga das hialuronidases permite a sua obtenção em quantidades que viabilizam seu estudo estrutural e funcional. Associado a isso, o transcriptoma propicia a identificação de novas toxinas e componentes de baixa proporção na peçonha. Portanto, o presente trabalho realizou o transcriptoma da glândula de peçonha do escorpião Tityus serrulatus e a clonagem e expressão heteróloga da hialuronidase. No transcriptoma foram obtidos 558 ESTs, dos quais 61,8% correspondem às toxinas, dentre elas neurotoxinas com ação em canais iônicos, metaloproteinas, hipotensinas, peptídeos antimicrobianos, dentre outros. Foram também identificadas novas toxinas como o neuropeptídeo e Ts16.1, descritos pela primeira vez para o gênero Tityus. Dentre os transcritos obtidos, foi identificado apenas um clone correspondente ao C-terminal incompleto de uma hialuronidase de T. serrulatus. Consequentemente, foi produzido o gene sintético contendo a sequência da TsHyal-1, obtida em bancos de dados, no vetor de expressão pPICZ?A para expressão heteróloga em P. pastoris. A rTsHyal-1 foi expressa em escala laboratorial em meio não suplementado (BMM) em pH 7,0 durante 96 h da indução com alimentação diária de metanol a 0,75%. A rTsHyal-1 foi produzida na sua forma solúvel e ativa (838,31 UTR/mg) e resultou em um rendimento proteico de 250 mg/L de material expresso. O secretoma do meio de expressão mostrou que além da rTsHyal-1, a P. pastoris também secreta proteínas nativas associadas à ligação do ATP, metabolismo de carboidrato e resposta ao estresse oxidativo. A rTsHyal-1 foi parcialmente purificada em troca catiônica fraca e apresentou atividade específica de 1.097,45 UTR/mg. A rTsHyal-1 apresenta massa molecular de 49,5 kDa e o tratamento com PNGase F seguido da análise por espectrometria de massas (MALDI-TOF) indicou que uma possível N-glicosilação de 4,5 kDa. Adicionalmente, o sequenciamento dos digestos trípticos da enzima realizado no MALDI-TOF e pelo Q-Exactive resultaram em 46,8% de cobertura da sequencia da proteína. A rTsHyal-1 apresenta especificidade pelo substrato hialuronan, seguido da condroitina C, A e B e apresentou atividade ótima em pH 6,0 e a 40°C. Adicionalmente, o ensaio do MTT indicou que a enzima recombinante não apresenta citotoxicidade in vitro. Os resultados obtidos determinaram as melhores condições para a expressão heteróloga da rTsHyal-1, que corresponde à primeira hialuronidase recombinante de escorpião expressa em P. pastoris com atividade enzimática preservadaThe venom hyaluronidases of animals are able to cleave hyaluronan present in the extracellular matrix, acting as a spreading factor for the toxins into the tissues of the victim. These enzymes have been neglected due to their instability and low concentration in the venom. Thus, heterologous expression of hyaluronidases permits the obtainment of sufficient amount for structural and functional studies. Moreover, the transcriptome allows the identification of new toxins or components with low proportion in the venom. In this way, this study aimed the construction of the transcriptome of Tityus serrulatus venom gland and cloning/heterologous expression of hyaluronidase. In the transcriptome, 558 ESTs were identified and 61.8% corresponded to toxins, such as neurotoxins with action on ion channels, metalloproteins, hypotensins, antimicrobial peptides, among others. In addition, new toxins were identified, comprising one neuropeptide and Ts16.1, described for the first time to the genus Tityus. Among the obtained transcripts, one identified clone corresponded to an incomplete C-terminal of a hyaluronidase. Consequently, a synthetic gene was synthesized containing the sequence of TsHyal-1 (obtained from databases) in the pPICZ?A vector for heterologous expression in P. pastoris. The rTsHyal-1 was expressed at laboratorial scale in unsupplemented medium (BMM) at pH 7.0 for 96 h, after induction time, with daily feeding of 0.75% methanol. The rTsHyal-1 was produced in soluble and active form (838.31 UTR/mg) and resulted in a protein yield of 0,266 mg/mL in final expressed material. Besides, the secretome of the medium showed that P. pastoris also secretes native proteins bound with ATP, proteins related to carbohydrate metabolism and oxidative stress response. The rTsHyal-1 was partially purified in a weak cation exchange and presented specific activity of 1097.45 UTR/mg. The rTsHyal-1 has molecular mass of 49.5 kDa and the treatment with PNGase F and analysis by mass spectrometry (MALDI-TOF) indicated a potential N-glycosylation of 4.5 kDa. Additionally, the sequencing of tryptic digests performed in the MALDI-TOF and Q-Exactive resulted in 46.8% of protein sequence coverage. The rTsHyal-1 presents substrate specificity for hyaluronan followed by chondroitin C, A and B and showed an optimum activity at pH 6.0 and 40°C. Furthermore, the MTT assay indicated that the recombinant enzyme does not display in vitro cytotoxicity. These results validate the biotechnological process of the heterologous expression of rTsHyal-1. This is the first recombinant hyaluronidase from scorpions expressed in P. pastoris system with enzymatic activity preserved

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

Antivenomics by mass spectrometry : use of magnetic beads.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenom, which is antitoxin antibodies (Igs) collected from immunized animals. However, these treatments may induce immunological reactions including severe adverse reaction to the patient. Moreover, venom compositions strongly differ from species, gender and habitat, explaining why providing broadly effective antivenoms is a real challenge. In this context, quantitatively evaluating the toxin-binding capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called ¿antivenomics¿ methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant, but their preparation and their lifetime represent constraints to the throughput of antivenom evaluation. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper, Echis ocellatus

Steatoda nobilis : A comparative study including full body MALDI Imaging and application to Venomics field.

The Noble false widow spider Steatoda nobilis is a member of the Therididae family, akin the ¿true¿ Black widows of the genus Latrodectus. Sn is rapidly expanding its geographic range throughout Europe and parts of the Americas, particularly in and around human dwellings. Sn has also been shown to be of medical significance in the UK and in Ireland, where a growing number of severe cases of envenomation has occurred over the past five years [Dunbar, 2021]. To illustrate the comparison of both male and female Sn profiles, we additionally present the first whole-body imaging of a spider using MALDI mass spectrometry. This proof-of concept allows to compare the anatomy of females and males based on molecular markers (specific m/z distribution on spider slices)

Development of an innovative methodology of antivenomics approach, combining the use of magnetic beads and mass spectrometry.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenom, which is antitoxin antibodies (Igs) collected from immunized animals. However, these treatments may induce immunological reactions including severe adverse reaction to the patient. Moreover, venom compositions strongly differ from species, gender and habitat, explaining why providing broadly effective antivenoms is a real challenge. In this context, quantitatively evaluating the toxin-binding capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called “antivenomics” methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant, but their preparation and their lifetime represent constraints to the throughput of antivenom evaluation. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper, Echis ocellatus

Antivenomics by mass spectrometry: use of magnetic beads.

Snakebite is classified as a Category A neglected tropical disease by the WHO, as it causes the death of about 150,000 people every year, and up to 4 times more people suffer long-term morbidity, mostly in rural and poor areas of the world. Snake envenoming is classically treated by injecting antivenoms containing antitoxin antibodies (Igs), produced by hyperimmunized animals. As antitoxin Igs represent only 10-15% of the total antivenom Ig content, such treatments have poor efficacy and the administration of animal Igs may induce immunological adverse reactions, possibly severe for the patient. Moreover, venom compositions strongly differ from species, gender, and habitat, explaining why providing broadly effective antivenoms is a real challenge. An effort has been made to reduce snakebite deaths and disability by 50% by 2030 [1]. In this context, quantitatively evaluating the toxin-binding (/neutralizing) capability of any antivenom is crucial to improve the production of effective snakebite therapeutics. In this study, we propose an alternative methodology for the so-called ‘antivenomics’ methodology. Indeed, affinity columns coupled to mass spectrometry have been demonstrated performant [2], but their preparation and their lifetime represent strong constraints to the increase of antivenom evaluation throughput. In this work, we exploit the potential of magnetic beads, LC-MS and shotgun proteomics mass spectrometry to speed up antivenom efficacy characterization. The antivenom Igs are bound to magnetic beads, before being incubated in the presence of various venoms. Comparative MS analysis of the toxins remaining in suspension (not recognized by Ig) and those remaining on the beads (recognized by Ig) allows the binding selectivity of the antivenom to be determined. The strategy is demonstrated here with venom from the medically important African viper Echis ocellatus