The Mediterranean scorpion Mesobuthus gibbosus (Scorpiones, Buthidae): transcriptome analysis and organization of the genome encoding chlorotoxin-like peptides
© Diego-García et al.; licensee BioMed Central Ltd. 2014
Received: 7 November 2013
Accepted: 9 April 2014
Published: 21 April 2014
Transcrof toxin genes of scorpion species have been published. Up to this moment, no information on the gene characterization of M. gibbosus is available.
This study provides the first insight into gene expression in venom glands from M. gibbosus scorpion. A cDNA library was generated from the venom glands and subsequently analyzed (301 clones). Sequences from 177 high-quality ESTs were grouped as 48 Mgib sequences, of those 48 sequences, 40 (29 “singletons” and 11 “contigs”) correspond with one or more ESTs. We identified putative precursor sequences and were grouped them in different categories (39 unique transcripts, one with alternative reading frames), resulting in the identification of 12 new toxin-like and 5 antimicrobial precursors (transcripts). The analysis of the gene families revealed several new components categorized among various toxin families with effect on ion channels. Sequence analysis of a new KTx precursor provides evidence to validate a new KTx subfamily (α-KTx 27.x). A second part of this work involves the genomic organization of three Meg-chlorotoxin-like genes (ClTxs). Genomic DNA sequence reveals close similarities (presence of one same-phase intron) with the sole genomic organization of chlorotoxins ever reported (from M. martensii).
Transcriptome analysis is a powerful strategy that provides complete information of the gene expression and molecular diversity of the venom glands (telson). In this work, we generated the first catalogue of the gene expression and genomic organization of toxins from M. gibbosus. Our result represents a relevant contribution to the knowledge of toxin transcripts and complementary information related with other cell function proteins and venom peptide transcripts. The genomic organization of the chlorotoxin genes may help to understand the diversity of this gene family.
KeywordsScorpion transcriptome Chlorotoxin Genomic organization Mesobuthus gibbosus Venom glands Scorpion toxin
The evolutionary history of the scorpions begun around 425–450 million years ago, in the middle Silurian  and these animals are therefore often considered “living fossils”. Scorpions are morphologically conservative organisms  and approximately 1500 species are recognized and classified in different families [1, 3]. The family Buthidae is geographically distributed worldwide and is the largest of the scorpion families, comprising 81 genera and 570 species . Moreover, from a clinical perspective, Buthidae is the most important scorpion family . Several members of this family are toxic to mammals and can be dangerous to humans . Stings by scorpion species dangerous to humans can induce different levels of toxicity and sometimes have lethal consequences. Scorpion venom consists of a mixture of biologically active compounds: (poly-) peptide toxins that specifically target ion channels (Na+, Cl−, K+ and Ca2+) and other cellular receptors . In terms of venom, scorpion biodiversity is reflected in more than 134,000 – 1050,000 distinct natural ligands. This value considers the number of described species and the data of the different venom analyses yielding the characterization of approx. 100–700 different venom components (e.g. Buthidae family: 383–632 peptides in some species of Tityus and Leiurus genera ; 87–144 venom components in species of the genus Tityus; Scorpionidae family: Pandinus cavimanus 393 venom components ; Urudacus yaschenkoi 274 unique molecular masses ). Advanced methods of venom fractionation, chromatography, mass spectrometry and peptide sequencing allow the characterization of the components in scorpion venom. However, the identification of a large number of animal toxins is often also based on information obtained via transcriptome analyses. Expressed sequence tags (ESTs) from venom glands provide complementary information and often reveal not yet described components related to the biological activity of the venoms. Until now (November, 2013), 10171 scorpion nucleotide sequences were described (EST and nucleotide sequences from the databases) and only 2569 were identified as scorpion toxin or toxin-like (UniprotKB). As yet, we have discovered less than 1% of all venom components, despite the strong efforts made in this vast field to get knowledge about its considerable diversity.
Mesobuthus gibbosus (Brullé, 1832) is one of the most important health-threatening scorpions in Turkey. This species is considered an old species living in the Mediterranean shore of the Aegean region, including Anatolia, Greece and Aegean islands . Information related to the toxin and venom compounds from M. gibbosus is restricted to one report , which describes the mRNA precursors and peptides of three alpha-potassium channel toxins (α-KTxs) . No data has been reported regarding the toxin genes or genomic organization in this species.
In the present work, we described 1) the first catalogue of gene expression by transcriptome analysis of venom gland (telson) and 2) the genomic organization of the chlorotoxin genes. In order to generate the transcriptome data a cDNA library from M. gibbosus scorpion was constructed. The non-amplified cDNA library was randomly screened and the positive colonies carrying a DNA insert corresponding to ≥500 bp of the putative toxin transcripts were subsequently DNA sequenced and analyzed by bioinformatics tools. Our results reveal information of genes related to some cellular processes (e.g. NADH dehydrogenase, cytochrome, ribosomal protein, ribonuclease) and genes involved in venom gland functions (e.g. toxins, antimicrobial peptides, phospholipases and other putative venom peptides). We performed a comparative sequence analysis of the obtained toxin-like transcripts and the related toxin families. Three chlorotoxin-like genes from M. gibbosus (MegClTxs) were detected and the genomic organization of MegClTxs genes allowed us to describe a new group of the chlorotoxin family. Comparative sequence analysis with the genome of M. martensii and MegClTxs genes provide evidence of two ClTxs groups.
Results and discussion
Analysis of cDNA sequences and identification of new genes
Annotations list of the precursor sequences deduced from the cDNA
Potassium channel toxin alpha-KTx 10.1 [Centruroides noxius], 34%. 6 Cys
Depressant insect toxin BmK ITa1 [Mesobuthus martensii], 80%. 8 Cys
Potassium channel blocker alpha-KTx 26.1 [Mesobuthus martensii], 70%. 6 Cys
Calcium channel toxin BmCa1, 58%. 6 Cys
Sodium channel toxin-4 [Mesobuthus eupeus], 85%. Partial gene, ≥8 Cys
putative potassium channel toxin Tx771 [Buthus occitanus israelis], 57%. 8 Cys
Potassium channel toxin BmTXK-beta-2 [Mesobuthus martensii], 99%. 6 Cys
Potassium channel toxin alpha-KTx 14.2 [Mesobuthus martensii], 91%. 6 Cys
Depressant insect toxin BmK ITa1 [Mesobuthus martensii], 80%. 8 Cys
venom chloride channel toxin-1 [Mesobuthus eupeus], 83%. 8 Cys
putative potassium channel toxin Tx771 [Buthus occitanus israelis], 54%. Partial gene ≥8 Cys
Sodium toxin peptide BmKTb' [Mesobuthus martensii], 44%. 9 Cys
Antimicrobial and Cytolytic
antimicrobial peptide marcin-18 [Mesobuthus martensii], 81%.
defensin [Medicago truncatula], 31%. 8 Cys
Non-disulfide-bridged peptide 6.2 [Mesobuthus martensii], 93%.
Bradykinin-potentiating peptide NDBP6 [Lychas mucronatus], 85%.
Non-disulfide-bridged peptide 6.2 [Mesobuthus martensii], 94%.
Other venom components
venom protein Txlp2 [Hottentotta judaicus], 79%.
venom peptide [Hottentotta judaicus], 28%.
phospholipase A2D precursor [Tribolium castaneum], 49%. Partial gene.
ribonuclease R [Coxiella burnetii RSA 331], 33%. Partial gene
zinc finger matrin-type protein 2-like [Oryzias latipes] Actinopterygii, 69%.
NADH dehydrogenase subunit 3 [Mesobuthus gibbosus], 90%.
Monogalactosyldiacylglycerol synthase, partial [Megasphaera sp. NM10], 43%
cytochrome b [Mesobuthus gibbosus], partial, 95%
Adhesive plaque matrix protein, partial [Bos grunniens mutus], 20%
putative 40S ribosomal protein S25 [Dolomedes mizhoanus], 87%
Blo t profilin allergen [Latrodectus hesperus], 84%
transposase of Tn10 [Shigella flexneri 2b], 100%
Unknown (Hypothetical proteins)
hypothetical protein 11, partial [Urodacus yaschenkoi], 92%.
hypothetical secreted protein [Hottentotta judaicus], 45%.
hypothetical secreted protein [Hottentotta judaicus], 77%.
hypothetical protein [Plasmodium berghei strain ANKA], 46%.
conserved hypothetical protein [Ixodes scapularis], 42%.
hypothetical protein [Capitella teleta] Polychaeta, 25% .
hypothetical protein [Pandinus cavimanus], 27%.
hypothetical protein [Vibrio splendidus]
hypothetical protein 11, partial [Urodacus yaschenkoi], 92%.
Normally, a single-pass read of Mgib cDNA sequences includes the complete coding sequence (CDS) that corresponds with the sequence of amino acids in a peptide or protein. Mgib ESTs contain single-pass reads of the cDNA (transcript) sequence, encoding a complete precursor sequence which includes a signal peptide, mature sequence and depending of the transcript, an additional pro-peptide region. CAP3 may yield conflicting bases in the sequence generated for the contig. In order to confirm the precursors deduced from our Mgib singleton and contigs sequences and to be deposited in the GenBank database, we performed additional DNA sequencing of all obtained plasmids. Confirmed sequences, which were constructed by alignment of the group of one or more DNA sequences, was called “singleton” (named Mgib sequence) and “contig” or clusters (also named MgibClusters or MgibC) to follow the sequence analysis previously described in the transcript categories. The obtained nucleotide sequences were deposited in the GenBank database [accession numbers KF770797-KF770827, KF743063]. The annotation was based on the best match in the consulted databases (Table 1). However, some of the Mgib plasmids could not provide additional DNA sequence of high quality to complete the information of the corresponding single-pass read of cDNA sequence (see sequence Mgib EST in Table 1). These sequences were deposited in a division of the GenBank Database to the Expressed Sequence Tags (dbEST).
Scorpion venoms contain several structurally distinct families of peptidyl modulators of ion channels . In accordance with the ion channel specificity, these peptides can be divided into four categories: 1) peptides of 60–70 amino acids linked by 4 disulfide bridges that modulate sodium channel activity; 2) short and long peptides of 30–76 residues with 3 or 4 disulfide bridges that block potassium channels; 3) short-chain peptides of 34–39 amino acids with four disulfide bridges and putative venom chloride channel toxin that blocks small-conductance chloride channels (ClTx) and 4) short peptides with 3 disulfide bridges that modulate ryanodine receptors (ion channels that are responsible for the release of calcium). The transcriptome analysis of M. gibbosus reveals a total of 12 new toxin transcripts included in the four categories of peptidyl modulators. We identified six transcripts that encode new members of the scorpion toxins specific to potassium channels belonging to the α-KTx and β-KTx families. The α-KTxs transcripts encode new toxin-like sequences of different subfamilies (by similarity with α-KTx3.x, α-KTx14.x and α-KTx26.x). Mgib24 corresponds to a new β-KTx transcript. Four different sequences encoding sodium channel toxins (NaTxs) were identified and its sequence analysis showed a match with α and β-NaTx classes. In addition, we identified a putative calcium channel toxin (Mgib3) similar to BmCa1 toxin (58% identity, E-values 7e-15) and a putative chloride channel toxin or chlorotoxin-like transcript (Mgib88). Our results indicate that the transcripts bear a relation to toxins from diverse scorpion genera targeting different ion channels.
Potassium channel toxins
Scorpion toxins specific to potassium channels have been classified into families as alpha, beta, gamma (α- β- γ-KTx)  and kappa (κ-KTx) on the basis of the alignment of cysteines and conserved residues . The α-KTx family is considered as the largest potassium channel toxin family . Until now, the α-family included short-chain toxins (23–42 residues) with a total of around 150 different peptides, comprising 27 subfamilies and new peptides and precursors being continuously described (http://www.uniprot.org/docs/scorpktx). The β-KTx family, also known as long-chain potassium channel toxins (47–76 residues), has been organized into 3 groups , later denominated class I, II and III . Peptide scorpion toxins that block the voltage-gated Shakers (Kv1.x) channels typically consist of 30–40 residues and have a molecular weight of about 4 kDa . However, β-KTxs have shown effects on some Kv1.x channels and some members show a relation to scorpion defensins with antimicrobial activity.
New subfamily α-KTx 27.x
Mgib23 encodes a precursor of a toxin-like peptide similar to the putative potassium channel toxin Tx771 from Buthus occitanus Israelis (precursor sequence shows an identity of 57%, E-value 1e-14), to the putative neurotoxin B and C precursors from Lychas mucronatus (identity of 46% and 45%, E-value 2e-07) and lower identity with members of α-KTx12.x and α-KTx3.x families (Figure 2). Meg113 is a partial gene that probably corresponds to the same complete amino acid sequence from Mgib23. However, the differences in the nucleotide sequence can be taken as evidence to consider it a different transcript. The presence of 8 cysteines in the predicted mature sequence from Mgib23 and Mgib113, does not show a close relationship with subfamily α-KTx6.x members, that also possess 8 cysteines (see α-KTx6.1 sequence in the top of Figure 2). Members of the α-KTx12.x subfamily possess 8 cysteines and differ from the α-KTx6.x in the cysteines organization. Precursors of Mgib23, Tx771 and the putative neurotoxins B and C can be considered as members of the same KTx group (Additional file 2: Figure S1). Despite the lack of information related to the biological activity of members of this group, we believe that this group can be considered as a new α-KTx subfamily. According to the nomenclature for short-chain peptides, the percent of identity between α-KTxs subfamilies and database information (http://www.uniprot.org/docs/scorpktx) they correspond to α-KTx27.x . The geographic distribution of scorpions is traditionally organized into two groups, namely the Old and the New World scorpions. α-KTx12.5, α-KTx12.6 and α-KTx12.7 precursors from the “Old World” (only precursors from the genus Lychas are described) show also differences between the “New World” α-KTx12.x members (only precursors from the genus Tityus are described) (Additional file 2: Figure S1). All “New World” α-KTx12.x members show a consensus sequence: WC2STC4XC10XC16XC20XC31XC36XC38YT (8 cysteines) while “Old World” members show a predicted mature sequence: QKXC8XC14XC18XC29XC34TC36YY. Perhaps, some of the “Old World” α-KTx12.x members can be reclassified since the cystine arrangement is different in the first two members of this subfamily (only 6 cysteines in the predicted mature sequence of α-KTx12.5 and α-KTx12.7). α-KTx12.6 precursor shows similar cystine arrangement to the new α-KTx27.x family members (Additional file 2: Figure S1). Mgib23 (α-KTx27.4) displays a match with α-KTx3.10 and α-KTx3.6 toxins (Figure 2 and Additional file 2: Figure S1). All described α-KTx3.x toxins correspond only to toxins belonging to scorpion species of the Buthidae family and show an effect on potassium current and specific channels . However, the low identity of the Mgib23 and α-KTx3.x toxins precursors (around 30%, E-value 5e-05) and the discrepancy of the number of cysteines (6 cysteines in α-KTx3.x toxins) support the idea of a new KTx subfamily.
Sodium channel toxins
Antimicrobial precursor and other venom components
Transcripts related to cellular functions and unknown genes
We will only mention the number of transcripts obtained in the “CellPro” and “Unknown” transcript categories. Nine transcripts encoding common cellular proteins match proteins involved in diverse cell functions such as ribonuclease, NADH dehydrogenase or cytochrome b. Additional file 1: Table S1 shows the deduced amino acid sequence of CellPro and unknown transcripts. In addition, Clusters MgibC3, MgibC4 and clones Mgib1, Mgib45, Mgib72, Mgib95, Mgib99, Mgib222 and Mgib267 are similar to other scorpion hypothetical proteins with a function that remains unknown. Lastly, two “No match” ORFs were found (Additional file 1: Table S1).
The profile of gene expression in the venom glands from Buthidae family
In recent times, the genome of M. martensii revealed 32,016 protein-coding genes . The authors described a total of 116 neurotoxin genes located in this genome (of which 45 were unknown), consisting of 61 NaTxs, 46 KTxs, 5 ClTxs and 4 CaTx or toxins for ryanodine receptors. In addition, Cao and colleagues  confirmed 109 expressed neurotoxin genes in the transcriptome analysis by next generation sequencing (NGS). The advantage and the limitations of the sequencing technology depends on factors such as the sample (venom glands) amount, focus of the study or the cost. But, all sequencing techniques allow to explore different transcriptomes from venomous species. High throughput sequencing or next generation sequencing platforms offer the possibility of generating thousands of sequences that contribute to the study of different conditions and provide a “complete” catalogue of the gene expression (e.g. the 72 toxin-like isogroups from C. noxius represent only 0.4% of the total number of assembled transcripts). In this sense, our low-throughput sequencing is far from a complete catalogue of the gene expression. However, Sanger sequencing in transcriptome is the approach often used for the screening of the cDNA libraries in the follow conditions: i) limiting sample amount (e.g. one or two specimens) ii) transcripts sequencing for a future characterization (e.g. cDNA into the vector to future recombinant protein expression) and iii) general catalogues with focus in toxin or venom component transcripts (e.g. selection of the estimated toxin genes by length of the PCR fragments). Our results by Sanger sequencing provided a total of 12 “toxin transcripts” (from 301 clones in the cDNA library) corresponding to 10% of the neurotoxin genes located in the M. martensii genome (or 11% of the expressed neurotoxins by Illumina). Rendon-Anaya et al., identified 72 different toxin-like isogroups from C. noxius analysis by 454 sequencing (e.g. toxins, proteases, antimicrobial peptides) but only 48 toxin-like isogroups correspond to ion channel specific toxins. Our results of toxin–like transcripts to specific ion channel correspond to 25% of toxin transcripts obtained by 454 sequencing platform. The number of transcripts and information provided by the transcriptomes by Sanger sequencing is still important for the contribution to the scorpion transcripts.
Chlorotoxin-like genes and the first scorpion genome genomic organization
This report revels part of the diversity of genes expressed in the venom glands from M. gibbosus. We identified several transcripts of toxic relevance as evidenced by orthologous genes. Furthermore, ribosomal and housekeeping transcripts were obtained. The transcriptome analysis revealed new putative peptides and may help to identify putative post-translational modifications in the deduced amino acid precursor sequences of the transcripts. In addition, for the chlorotoxin family genes, we described the genomic organization of three new genes and confirmed the corresponding expressed sequences for two of them. This information may contribute to the classification of chlorotoxin genes into two groups for the genus Mesobuthus. This transcriptome contribution can be useful for further studies and to help discovery new gene families, toxins and other venom components.
Biological materials and cDNA library construction
A cDNA library of M. gibbosus venom glands was generated using the conditions previously described . A random sequencing strategy was used to screen the cDNA library. In order to select the positive colonies, random screening using blue/white colony selection (by non-functional β–galactosidase activity, consequence of the LacZ gene disruption by Mgib sequences or transcripts) and colony polymerase chain reaction (colony PCR) was performed. The PCR fragments selected correspond to the expected length of toxin and venom components transcripts (around 500–1000 bp). The selection of positive clones by colony PCR was done using forward and reverse primer screening (sites flanking pSMART21F inserts). The plasmid DNA of selected colonies was obtained by mini-prep kit preparations (Roche) and sequenced by Sanger method from both ends by GATC Biotech sequencing service (Germany).
Genomic DNA was obtained from the legs and tail of two specimens of M. gibbosus following the protocol described by Rodriguez de la Vega and colleagues . To obtain ClTxs genes from genomic DNA, we designed specific primers based in the information obtained by conserved signal peptide sequences (Figure 7) and the DNA sequence of Mgib88, the putative chlorotoxin from M. gibbosus (GeneBank: KF770800): 5’- ATG AAG TTC CTC TAT GGA ATC GTT TTC −3’ and 5’- TCA GTC ATA GCC ACA CAG ACA TTG TGG −3’. PCR products were amplified using the conditions described Diego-Garcia et al.. High Fidelity Taq polymerase (Roche) was used in the PCR reactions. PCR products were cloned in a pGEM vector (Promega) and sequenced by the Sanger method.
Bioinformatic analysis of DNA sequences
DNA sequences were analyzed by electropherogram quality analysis via the PHRED web service  and assembled in clusters using the CAP3 program . Sequences were processed as follows: unique sequences are considered singletons or singlets. An assembly of contiguous sequences is considered a contig. Additionally, all the plasmids included in the singletons and contigs were reverse strand sequenced to confirm the final deposited sequence in the GenBank database. Confirmed sequences were called “singleton” (named Mgib sequence) and “contig” or cluster (also named MgibCluster or MgibC). Each sequence was searched against the GenBank database with algorithms BLASTX and Protein BLAST to identify homologous sequences for comparison . All DNA sequences were manually inspected with DNA Strider 1.4f6 to identify open reading frames (ORF), 4Peaks 1.7.2 tools to confirm the nucleotide sequence and the multiple sequence alignment program and Clustalx 1.83.1 or ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2). SignalP 4.0  and ProP 1.0  servers were used for the prediction of the presence and location of signal peptide and pro-peptide cleavage sites respectively. All Mgib sequences that encode for toxin-like, antimicrobial and venom compounds were fully sequenced by reverse and forward DNA strand to be submitted to GenBank [KF770797-KF770827, KF743063]. Singletons were submitted to EST database from GenBank. Splice sites predictions to identify the exon-intron regions were obtained by using NetGene2 v2.4 .
The authors and co-authors of this paper have acted ethically in conducting the described research, having careful analysis of the data to avoid errors. Authors declare that the described work has not been published previously. All authors approve this manuscript.
Nucleotide or amino acid sequence deduced from cDNA
Toxin-like or precursor deduced of the cDNA sequence from Mesobuthus gibbosus
5’, Untranslational region
3’, Untranslational region
Expressed sequence tag
- MALDI-TOF MS:
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry
Sodium channel toxin
Voltage-gated sodium channels
Potassium channel toxin
Voltage-gated potassium channels.
The authors would like to thank Bea Garcia Mille for the technical assistance in the preparation of bacterial growth medium and some molecular biology material used in part of this work. The authors are indebted to Dr. Hakan Caliskan from the Biology Department of Eskisehir Osmangazi University (ESOGU) for the capturing and collect of scorpions used in this work. The authors would like to thank Edith Coronado for the English writing comments and the Reviewers of the manuscript for their suggestions and comments. This work was supported by the following grants: G.0433.12, G.A071.10 N and G.0257.08 (F.W.O. Vlaanderen), IUAP 7/10 (Inter-University Attraction Poles Program, Belgian State, Belgian Science Policy) and OT/12/081 (KU Leuven). FC was partially supported by a grant from the Scientific Research Projects Commission of ESOGU (number 19020).
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