Open Journal of Genetics, 2012, 2, 210-220 OJGen http://dx.doi.org/10.4236/ojgen.2012.24027 Published Online December 2012 (http://www.SciRP.org/journal/ojgen/) Transcriptome analysis of the Tityus serrulatus scorpion venom gland Érika R. Alvarenga, Thaís M. Mendes, Bárbara F. Magalhães, Flávia F. Siqueira, Arthur E. Dantas, Tatiana M. Barroca, Carolina C. Horta, Evanguedes Kalapothakis Departamento de Biologia Geral, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil Email: [email protected] Received 20 October 2012; revised 21 November 2012; accepted 29 November 2012 ABSTRACT commonly as the Brazilian yellow scorpion) has been the subject of extensive research efforts to isolate and char- The Tityus serrulatus scorpion is considered the most acterize its toxins and other venom components for po- dangerous scorpion in Brazil and is responsible for tential clinical benefit [4-13]. several cases of human envenomation annually. In Analyses of T. serrulatus venom have revealed a com- this study, we performed transcriptome profiling of plex mixture of functionally diverse molecules, including the T. serrulatus venom gland. In addition to tran- neurotoxins, proteases, hyaluronidases, and hypotensins scripts with housekeeping functions, such as those [3-14]. Of these, the neurotoxins are believed to play a related to protein synthesis, energy supply and struc- key role in the pathogenesis of scorpionism through their tural processes, transcripts from thirty-five families interactions with sodium channels and potassium chan- of venom peptides or proteins were identified. These nels (as reviewed by Cologna et al. [3]). However, to transcripts included three new complete sequences of gain a more in-depth and comprehensive understanding toxins and more than a dozen putative venom gland of the molecular components of the T. serrulatus venom proteins/peptides. The venom gland transcriptome gland, the gene expression profiling is necessary. profile was verified by comparison with the previ- Transcriptomic analyses of the scorpion venom gland ously determined proteomic profile. In conclusion, have been performed recently for species belonging to this transcriptome data provides novel insights into various scorpion families, including Buthidae (T. stig- the putative mechanisms underlying the venomous murus and Centruroides noxius), Scorpiopidae (Scor- character of T. serrulatus. The collected data of scor- piops margerisonae and S. jendeki), Scorpionidae pion transcripts and proteins/peptides described (Pandinus cavimanus and Heterometrus petersii), Iuridae herein may be an important resource for identifying (Hadrurus gertschi), and Liochelidae (Opisthacanthus candidate targets of molecular therapies and preven- cayaporum) [15-26]. Transcriptomes have provided im- tative measures. portant insights into the biological processes that are taking place inside venom gland cells [15,18,19,26], as Keywords: Scorpions; Antimicrobial Peptides; well as identified new genes and expression patterns that Neutoxins; Venom Glands; Brazilian Yellow Scorpion form the basis of functional genomics studies [27,28]. Such data for the venom glands of different species re- 1. INTRODUCTION presents a resource of potential toxinological and clinical relevance [29,30]. In 2007, the World Health Organization (WHO) offi- In the current study, the molecular complexity of T. cially designated human envenomation by scorpion sting serrulatus venom was investigated by transcriptome pro- as a neglected public health issue and recommended ur- filing of the venom gland. This approach identified the gent international action with a focus on the tropical re- main cellular components and revealed new putative gions where these venomous animals are abundant. In venom constituents, some of which may be candidate tar- Brazil, scorpion stings have been recognized a serious gets of new therapeutic strategies to help promote the public health threat for many decades; yet, the annual health of sting victims. incidence of accidental stings remains high, with more than 50,000 cases reported in 2010 [1]. The most fre- 2. MATERIAL AND METHODS quent culprit among the cases of human envenomation 2.1. Library Construction by Brazilian scorpions is the Tityus serrulatus of the Buthidae family [2,3]. As such, this species (known A cDNA library was constructed from active venom OPEN ACCESS É. R. Alvarenga et al. / Open Journal of Genetics 2 (2012) 210-220 211 glands of 60 T. serrulatus scorpions that had been milked ment was performed by webPrank [42] and visualised by two days prior to the RNA extraction, as previously de- the Jalview program [43]. Peptide signal prediction was scribed [31]. carried out for putative venom components by using the SignalP 4.0 program [44]. 2.2. Expressed Sequence Tag (EST) Sequencing, Data Processing, and Bioinformatic Analysis 3. RESULTS For large-scale DNA sequencing (EST generation), ran- 3.1. EST Sequencing and Clustering dom clones were grown in antibiotic selective medium A total of 1629 high-quality ESTs were generated from for approximately 18 h. The plasmid DNA was then iso- the T. serrulatus venom glands. The average length of lated using the standard alkaline lysis method and se- these ESTs was 421 bp. TGICL-based clustering of the quenced on an ABI 3130 sequencer with reagents from 1629 ESTs yielded 185 contigs (= 1171 ESTs). The the BigDye Sequencing Kit (Applied Biosystems Inc., average length of these contigs was 625 pb. Four-hund- Foster City, CA, USA) and the standard M13 forward or red-and-fifty-eight ESTs showed no significant similarity reverse primers. to any other ESTs in the database and were identified as The resultant trace files of the sequenced clones were applied to the Phred program for base calling and quality singlets. scoring using a Phred score cut-off value of 20 [32]. The nucleotide sequences corresponding to vector, adaptors, 3.2. T. serrulatus Venom Gland Transcript and Escherichia coli DNA, and any short transcripts Profile (<100 pb) were removed by the SeqClean program [33] The BLASTx searches against the UniProt database (http://compbio.dfci.harvard.edu/tgi/software). The final indicated that among the 643 uniques (185 contigs and sequences were deposited in GenBank under accession 458 singlets), 54% (348 uniques representing 1259 ESTs) IDs JK731601-JK732954. The TGICL program [34] was encoded precursors of known proteins. The remaining 46% used to assemble high-quality ESTs into contigs (over- (295 uniques representing 370 ESTs) had e-values >1 × lapping ESTs that together represent a consensus se- 10–10 and were thus designated as having no match. quence). Any ESTs without significant similarity to any These no-match transcripts may represent new proteins other ESTs were classified as singlets. Considering the or peptides. diversity of scorpion toxins, those clusters putative to Cellular localisation and function analyses classified encode venom peptides were re-examined manually to the uniques- and ESTs-deduced products into 10 catego- pick out individual different isoforms. The electrophero- ries; the distributions of which are shown in Figures 1(a) gram of each putative isoform was visually inspected to and (b). The most frequently represented functional confirm the sequencing quality in the polymorphic re- categories among the cellular components in T. serrula- gion. tus venom gland were: Protein synthesis and processing, To annotate the contigs, the set of contigs plus singlets structural function, and energy supply. (collectively designated as “uniques”) were searched against the UniProt database [35] (March 2011) using the 3.3. Housekeeping Genes BLASTx [36] (e-values < 1 × 10–10) and nr database using BLASTx (e-values < 1 × 10–10) in the BLAST2GO When considering only the ESTs that presented simila- program [37]. Kyoto Encyclopedia of Genes and Ge- rity with sequences in the database, about 40% of ESTs nomes (KEGG) pathway analysis [38] was also perfor- were related to cellular components. KEGG analysis (cor- med. The toxin nomenclature was assigned according to roborated with manual annotation; Table 1) indicated the recommendations of King et al. [39] and Cologna et that the most frequently represented pathways were also al. [3]. involved in protein synthesis and processing (ribosome Amino acid sequence of the transcripts was deduced and protein processing in the endoplasmic reticulum), using the open reading frame (ORF) Finder program structural processes (cardiac muscle contraction—impor- (http://www.ncbi.nlm.nih.gov/projects/gorf/). Subsequen- tant to venom release; regulation of actin cytoskeleton), ty, the Seed Server (unpublished, Guedes et al.) was used and energy supply (oxidative phosphorylation). The to search for venom component orthologs. Briefly, the automated KEGG pathway analysis and manual annota- Seed Server methodology uses a protein of interest to tion method yielded similar but not identical results, like- group homologues by means of Seed Linkage software ly due to the different rules of each approach used to [40] and UniRef50 Enriched KEGG Orthology (UEKO) categorize the transcript products. clusters built with the procedure described by Fernandes By manual annotation, the most frequently represented et al. [41]. The sequences used for alignment analysis functional categories were protein synthesis and pro ces- were retrieved from the UniProt database and the align- sing (37 ESTs coding ribosomal proteins), eukaryotic Copyright © 2012 SciRes.