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The in-depth venome of the Brazilian rattlesnake Crotalus durissus terrificus: an integrative approach combining its venom gland transcriptome and venom proteome Gisele Adriano Wiezel, Priscila Yumi Tanaka Shibao, Camila Takeno Cologna, Romualdo Morandi Filho, Carlos Ueira-Vieira, Edwin De Pauw, Loïc Quinton, and Eliane C Arantes J. Proteome Res., Just Accepted Manuscript • DOI: 10.1021/acs.jproteome.8b00610 • Publication Date (Web): 01 Oct 2018 Downloaded from http://pubs.acs.org on October 3, 2018
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The in-depth venome of the Brazilian rattlesnake Crotalus durissus terrificus: an integrative approach combining its venom gland transcriptome and venom proteome
Gisele A Wiezela§, Priscila Y T Shibaoa§, Camila T Colognaa, Romualdo Morandi Filhob, Carlos Ueira-Vieirab, Edwin De Pauwc, Loïc Quintonc, Eliane C Arantesa*
a
Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeirão Preto,
University of São Paulo, Av. do Café, s/n, 14040-903, Ribeirão Preto, Brazil b
Laboratory of Genetics, Biotechnology Institute, Federal University of Uberlândia, Rua Acre, s/n,
38400-902, Uberlândia, Brazil c
Laboratory of Mass Spectrometry, MolSys Research Unit, Department of Chemistry, University of
Liège, Bat. B6c, 4000 Liège, Belgium
§ Both authors contributed equally to this work
*Corresponding author: Dr. Eliane Candiani Arantes School of Pharmaceutical Sciences of Ribeirão Preto - University of São Paulo Department of Physics and Chemistry Av. do Café, s/n, 14040-903, Ribeirão Preto-SP, Brazil. Tel: +55-16-3315-4275; FAX: +55-16-3315-4880 Email address:
[email protected] ACS Paragon Plus Environment
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ABSTRACT
Snake venoms are complex mixtures mainly composed of proteins and small peptides. Crotoxin is one of the most studied components from Crotalus venoms, but many other components are less known due to their low abundance. The venome of Crotalus durissus terrificus, the most lethal Brazilian snake, was investigated combining its venom gland transcriptome and proteome to create a holistic database of venom compounds unraveling novel toxins. We constructed a cDNA library from C. d. terrificus venom gland using the Illumina platform and investigated its venom proteome through high resolution LC-MS/MS. After integrating data from both datasets, more than 30 venom components classes were identified by the transcriptomic analysis and 15 of them were detected in the venom proteome. However, few of them (PLA2, SVMP, SVSP and VEGF) are relatively abundant. Furthermore, only 7 expressed transcripts contributed to ~82% and ~73% of the abundance in the transcriptome and proteome, respectively. Additionally, novel venom proteins are reported, and we highlight the importance of using different databases to perform the data integration and discuss the structure of the venom components-related transcripts identified. Concluding, this research paves the way to novel investigations and discovery of future pharmacological agents or targets in the antivenom therapy.
Keywords: snake venomics, venom proteome, venom gland transcriptome, RNA-seq, orbitrap, hyaluronidase, fibroblast growth factor, carboxypeptidase
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1. INTRODUCTION The rattlesnakes comprehend the genera Crotalus and Sistrurus (http://www.reptile-database.org/) but only Crotalus snakes are represented in Brazil. Furthermore, Crotalus genus exclusively includes the Crotalus durissus species, which is subdivided into 6 subspecies (http://www.reptile-database.org/). Their venoms are quite similar and have neurotoxic action, clotting activity on fibrinogen, myotoxic activity, activity of platelet aggregation and induction of edema1,2. C. d. terrificus and C. d. collilineatus are the prevalent subspecies in the Brazilian territory2. Crotoxin is the major toxin present in Crotalus venoms3, but other toxins were already described including, crotamine, L-amino acid oxidase (LAAO), hyaluronidase, lectin and serine and metalloproteases2, 4-7
. The difficulty on studying minor toxins is mainly due to the lessened yield of those components in the
venom. Thus the harsh traditional activity-guided methods of protein purification, in addition to the laborious work and time costing, lead to the characterization of the major venom components8. In this scenario, omics technologies are playing a key role by pointing the spotlight on toxins previously undetectable and shedding light on the venom composition and evolution9, 10. Venom gland transcriptome is a powerful tool for unraveling venom components. Up to now, there are more than 50 transcriptomes from different snake venom glands and venoms available10. This approach has already allowed the identification of novel components in the C. d. collilineatus venom gland, such as cardiotoxin, vespryn and phospholipase A2 inhibitor11. More recently, the Next Generation Sequencing (NGS) analysis, mainly RNA-seq, have been allowing the identification of poorly expressed RNA in a more sensitive, lower cost and faster way12, 13. Another approach to unravel the complex mixture secreted by venom gland is the proteome/peptidome, which was developed due to the advent of mass spectrometry (MS) technologies14, 15. Regarding snake venoms, the “bottom-up” strategy, in which protein/peptides are digested prior to the MS analysis, is used to create virtual libraries of natural compounds for biotechnological applications, improve antivenoms, foresee envenoming profile, and even analyze genetic divergence and geographical differentiation2, 7, 15, 16. Although some groups have already investigated the venom proteome of C. d. terrificus, no transcriptome analysis has been reported yet4-6. Currently, the state of the art of venom comprehension relies on “omics” technologies and their integration to obtain a holistic view of venoms. In this sense, the term ACS Paragon Plus Environment
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“venomics” was recently polished up and has been attributed to the use of these technologies to unveil the full composition of venoms16. Therefore, this research paper presents the analysis of RNA-seq transcriptome, the venom proteome and the integration of both libraries from one specimen of Crotalus durissus terrificus. This work paves the way to the discovery and study of protein that were never detected in C. d. terrificus venom before and are probably involved in the snake envenoming process.
2. MATERIAL AND METHODS
2.1. Crotalus durissus terrificus Crotalus durissus terrificus specimen (male) was captured in Ribeirão Preto region (21° 10′ 36″ S, 47° 49′ 15″ W) and the snake was kept in the central snake house (University of São Paulo, Ribeirão Preto, SP, Brazil) in accordance with the guidelines of the Brazilian Institute of Environment and Renewable Natural Resources (IBAMA). The venom and venom gland used in this study were obtained from this same specimen, which produces white venom. C. d. terrificus venom (CdtV) was milked 3 days prior the venom gland extraction and was desiccated and stored at -20 °C until used. For the venom gland extraction, the specimen was sacrificed following the ARRIVE guidelines. The experiments were reviewed and approved by the Animals Usage Ethics Committee from School of Pharmaceutical Sciences of Ribeirão Preto – University of São Paulo (process n. 15.5.256.60.2). For this purpose, a combined dose of the anesthetics ketamine (10 mg/kg) and pentobarbital (150 mg/kg) according to the snake’s body weight was applied through intravenous access to sacrifice the animal. The venom gland was immediately extracted.
2.2. C. d. terrificus venom gland transcriptome Total RNA was extracted from the venom gland using TRIzol® reagent (Life technologies, USA) according to the manufacturer’s protocol and its yield and integrity was accessed by 2100 Bioanalyzer (Agilent, USA). The cDNA library was prepared with the kit TruSeq Stranded mRNA (Illumina, USA). Reads were paired-end (2x100 bp) sequenced in duplicate by the Illumina 2500 HiSeq platform (Illumina) using the kits Truseq SBS v3-HS 200 cycles and Truseq PE cluster v3 cBot-HS (Illumina). Raw reads trimming was carried out in Galaxy17 – through the tool Trimmomatic18 – to remove adapters and discard ACS Paragon Plus Environment
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short (
