Comparative venom gland transcriptome analysis of the scorpion Lychas mucronatus reveals intraspecific toxic gene diversity and new venomous components
- Zhao Ruiming†1,
- Ma Yibao†1,
- He Yawen1,
- Di Zhiyong1,
- Wu Yingliang1,
- Cao Zhijian1Email author and
- Li Wenxin1Email author
© Ruiming et al; licensee BioMed Central Ltd. 2010
Received: 6 May 2010
Accepted: 28 July 2010
Published: 28 July 2010
Lychas mucronatus is one scorpion species widely distributed in Southeast Asia and southern China. Anything is hardly known about its venom components, despite the fact that it can often cause human accidents. In this work, we performed a venomous gland transcriptome analysis by constructing and screening the venom gland cDNA library of the scorpion Lychas mucronatus from Yunnan province and compared it with the previous results of Hainan-sourced Lychas mucronatus.
A total of sixteen known types of venom peptides and proteins are obtained from the venom gland cDNA library of Yunnan-sourced Lychas mucronatus, which greatly increase the number of currently reported scorpion venom peptides. Interestingly, we also identified nineteen atypical types of venom molecules seldom reported in scorpion species. Surprisingly, the comparative transcriptome analysis of Yunnan-sourced Lychas mucronatus and Hainan-sourced Lychas mucronatus indicated that enormous diversity and vastly abundant difference could be found in venom peptides and proteins between populations of the scorpion Lychas mucronatus from different geographical regions.
This work characterizes a large number of venom molecules never identified in scorpion species. This result provides a comparative analysis of venom transcriptomes of the scorpion Lychas mucronatus from different geographical regions, which thoroughly reveals the fact that the venom peptides and proteins of the same scorpion species from different geographical regions are highly diversified and scorpion evolves to adapt a new environment by altering the primary structure and abundance of venom peptides and proteins.
More than 400 million years of evolution does not make the scorpions alter their morphology. However, although this one of the oldest arachnid is highly conserved in shape all along, they occupy vast territory of the world from Africa to Asia, Australia and America. Such powerful adaptability mostly owes to their highly specialized venom apparatus which consists of the vesicle holding a pair of venom glands connected to the stinger used to inject the venom. The extant scorpions can be phylogenetically divided into 14 families based on a morphological cladistic analysis, among which the Buthidae is considered to be the largest and the most medically important family. As a result, this enormous family has attracted the biggest scientific interest and been extensively studied. Till now, approximate 800 scorpion species have been classified into the Buthidae family. Previous study confirmed that each one of the species may typically contain more than one hundred different peptides in venom ranging in mass from 1,000-9,000 Da[3–5]. Among the venom peptides in this range, two classes are most regarded. One of them is small neurotoxic proteins that recognize ion channels and receptors in membranes of excitable cells, and thus toxic to different organisms[6, 7]. The other is antimicrobial peptides which are important defensive molecules of the ancient innate immunity.
It is acknowledged that the Buthidae family possesses a very different venom arsenal comparing to other non-Buthidae families. Moreover, even within the Buthidae family, vast abundance difference can be observed in venom compositions between genus to genus, different species within a genus and individuals within a species[10–12]. As a result, previous study estimated that approximately 150,000 distinct polypeptides presented in about 1500 known scorpion species in the world. Numerous peptides in scorpion venoms are a big treasury waiting for exploitation. Proteomic and transcriptomic approaches have already helped us to draw a rough picture of the molecular diversity of the scorpion venom components. To the best of our knowledge, the transcriptomic approach is more effective in getting an overview of scorpion venom. Not only because it can reflect the biological processes inside the venom gland cells, but also provides clues to the research of evolutionary path leading to scorpion toxin diversification directly.
Morphological comparison of two Lychas mucronatus populations
Lychas mucronatus is one scorpion species widely distributed in Southeast Asia. In China, they chiefly exist in Yunnan, Guangxi and Hainan provinces. Our group gets two populations of the scorpion Lychas mucronatus successively from Hainan and Yunnan provinces with distinct ecotopes (Figure 1). Based on Kovařík and Zhu's description[21, 22], the adults of Lychas mucronatus can be easily recognized based on coloration and morphological characteristics as following: (1) Generally, Lychas mucronatus is 40-65 mm long, carapace, mesosomal tergites and legs are yellow and blotched, metasoma segments are yellow to yellowish-brown from base to terminal, pedipalp, patella is predominantly dark, pedipalps manus is bright yellow with sparse, minute black spots and fingers are dark yellow brown; (2) Pedipalp fingers of males are curved, whereas those of female are straight; (3) Second segment of metasoma has 10 keels, and the third metasomal segment has 8 keels; (4) Sixth cutting edge on movable fingers of pedipalps has 3 granules; (5) Pectinal teeth number is 16-26 (frequently 19-23); (6) Metasoma of males has the same length as that of females. According to microscopic observation, both scorpion populations we collected possess the same characters mentioned above. So we convinced that two scorpion populations obtained from two provinces actually belong to the same species Lychas mucronatus.
EST sequencing and clustering
Distribution of 380 clusters assembled from the scorpion Lychas mucronatus collected from Yunnan province
Similar to venom peptide transcripts
Not similar to venom peptide transcripts
Known toxin types
Comparison of abundance of different toxin types between two populations of Lychas mucronatus
Atypical possible toxin types
Total Toxin-like peptides
NaTx (toxins specific for sodium channels)
Till now, NaTx were only found in the venom of the family Buthidae except one peptide phaiodotoxin from the family Iuridae. They are 6500-8500 Da (58-76 residues) polypeptides showing the conserved structure core constrained by three disulfide bridges: two disulfides link the α-helix to the anti-parallel β-sheet and the third disulfide links the β-sheet to an extended segment preceding the helix. But the fourth disulfide bridge, also called wrapper disulfide bridge, varies in position, and sometimes is even lost. These structure-conserved 'long-chain' peptides are considered to be the primary causes of the neurotoxic symptoms induced by scorpion envenomation.
We obtained 43 NaTxs transcripts from the venom gland cDNA library of Yunnan-sourced Lychas mucronatus, accounting for nearly 11% of whole toxin-like peptide sequences. These sequences were grouped in 12 clusters. According to sequence similarity and targeting receptor site, NaTxs are assorted into two patterns: α-NaTxs and β-NaTxs. 6 clusters putative α-NaTxs encoded by 11 ESTs were found in Yunnan-sourced population, whereas putative β-NaTxs possess of an obviously higher abundance which have 32 ESTs encoding 6 clusters.
One cluster of putative β-NaTxs (GT028621 and GT028626) encoded by 16 transcripts is the most abundant NaTx cluster in Yunnan-sourced population library. This cluster contains two almost identical sequences differing by only two amino acids. The transcript GT028621 encodes a mature peptide of 63 amino acid residues with 8 cysteines which would form a disulfide bridges pattern of "C-C-CX3C-C-CXC-C" (C, cysteine; X, amino acids of various types). This peptide shows homology to the beta-toxin Toxin CsEv1 identified from Centruroides sculpturatus. This homology may indicate that the most abundant 'long-chain' neurotoxin in the venom of Yunnan-sourced Lychas mucronatus can affect sodium channel activation by binding voltage-independently at site-4 of sodium channels. Although the most abundant NaTx transcripts (EU159276 and EU159292) from Hainan-sourced library have the same cysteine pattern as GT028621, obvious diversity can be observed in primary structures. Specially, GT028621 don't contain a basic amino acid residue at C-terminal region, which shows that C-terminal modification would not exist at the post-translational level.
α-KTx (α subfamily of toxins specific for potassium channels)
α-KTx is widely spread in all scorpion species ever studied. This main toxin type possesses from 25 to 45 amino acid residues well packed with three or four disulfide bridges. Their conserved three-dimensional structures are always constituted by a α-helix connected to a two or three-stranded β-sheet.
GT028876 represents a 3 ESTs cluster encoding a putative α-KTx. It has a mature peptide of 42 amino acid residues. Because of its unique primary structure, no homologies were shown after BLAST search. However, two clusters represented by EU163859 and EU163857 from Hainan-sourced population show some homology to GT028876. In respect that they have the same cysteine pattern as those function identified potassium channel toxins. GT028876 may be a new group of short chain K+ channel blockers.
GT028663 and GT028654 belong to the most abundant α-KTx cluster which is encoded by 70 ESTs. Both transcripts were supposed to encode a mature peptide with 49 amino acid residues. The mature peptides are constrained by 4 disulfide bridges, which is distinct to most other putative α-KTx toxins obtained from both scorpion populations. Blast search presented homologous Tx771 putative potassium channel toxin from Buthus occitanus Israelis and male-specific potassium channel inhibitor IsTX from Opisthacanthus madagascariensis which was proved to be a blocker to voltage-gated potassium channels Kv1.1 and Kv1.3. Surprisingly, the highest abundant toxin type from Yunnan-sourced Lychas mucronatus has no homologous transcript in Hainan-sourced population. Although there is still no clue on exact function of this α-KTx cluster, it is maybe very important for the survival of Yunnan-sourced Lychas mucronatus. Besides, another 2 ESTS cluster represented by GT028917 also possesses of the same cysteine pattern as above-mentioned sequences. These 72 ESTs form another new group of short chain K+ channel blockers together. EU163848 represents the most abundant α-KTx cluster in Hainan-sourced population. It possesses of a 32 amino acid mature peptide with a pI of 4.82. This 31 ESTs encoded acidic α-KTx cluster has homologous cluster represented by GT028769 in Yunnan-sourced population. But the abundance of these acidic putative K+ channel toxins is quite distinct between two populations and their functions are also to be identified.
Potassium scorpion toxins are usually classified into 4 subfamilies: α, β, γ and κ KTxs. Each subfamily consists of several groups. Based on similar analysis as described before, GT028811 and GT029234 belong to α-KTx12 group, GT028874 belongs to α-KTx17 group, and the other short chain KTxs constitute at least 4 new groups. α-KTx is the most abundant toxin type in Yunnan-sourced population. It takes parts of nearly 23% of total toxin-like peptides, which is nearly 2-fold higher than 13% in Hainan-sourced population. In a word, not only the primary structures, but also the abundance of α-KTxs between two scorpion populations displayed high diversity.
AMPs (antimicrobial peptides)
AMPs are a wide class of venom peptides with antimicrobial functions. It is an important defensive weapon of innate immunity for scorpion. AMPs take on great diversity in primary amino acid sequences. Most of them are short cationic peptides, and are structurally divided into three groups, namely linear peptides with an amphipathic α-helix, cysteine-rich peptides with one or several disulfide bridges, and peptides with a predominance of specific amino acids such as glycine, proline and histidine. In a word, AMPs form the first line of host defense against pathogenic infections.
In Yunnan-sourced Lychas mucronatus, 5 type peptides are presumed to belong to AMPs: Short cationic antimicrobial peptides (Scamp), Ponericin-like antimicrobial peptides (Plamp), bradykinin-potentiating peptide like (bpp like), Glycine-rich peptides and Anionic peptides.
Although there was still no consensus about the role of AMPs in scorpion venom, they were postulated to have the functions of protecting the scorpion from bacterial infection, depolarizing neural cells inducing immobilization of prey and potentiating the action of other neurotoxins within the venom. 8 clusters (86 ESTs) encode for AMPs were obtained from Yunnan-sourced Lychas mucronatus, which accounted for 21% of all toxin-like peptides. However, the abundance of AMPs reached remarkable 40% in Hainan-sourced Lychas mucronatus. The primary structure of AMPs from Hainan-sourced Lychas mucronatus showed more diversity. This comparison suggested that Hainan-sourced Lychas mucronatus definitely can protect itself more effectively from microorganisms.
Other venom components
Scorpion venom is a complex mixture of biologically active peptides with diverse physiological effects. Most of the peptides ever identified are disulfide-rich neurotoxins that specifically modulate various ion channels permeability of excitable and non-excitable cells. However, more and more other venom components have been characterized recently, including antimicrobial peptides without disulfide bridges and other functional molecules[32, 53]. In order to gain further insight into scorpion venom compositions, it is not enough to employ routine studies by protein chemistry always aiming at the isolation of specific active components. The introduction of powerful chromatographic techniques, followed by primary structure determination using automatic Edman degradation, makes it possible to produce an overview of scorpion venom compositions. Mass spectrometry was firstly used to get the mass fingerprinting of toxic fractions of Tityus serrulatus venom. Since then, overall venom compositions of ten scorpion species have been comprehensively investigated at the proteome level, including eight species from the family Buthidae. The powerful technique help to confirm the hypothesis that scorpion venom is a complex mixture of various distinct proteins with vastly abundant difference between families to families, genus to genus and different species within a genus. We chose transcriptomic approach to get an overview of the Yunnan-sourced Lychas mucronatus venom because it's not affected by the extraction of venom and the dynamic expression of the gland or peptide maturation like proteomic approach. The transcriptomic analysis is more likely to give a comprehensive comparison of the venom compositions from two Lychas mucronatus populations.
Some evidences already displayed the phenomenon of intraspecific diversity of scorpion venom peptides[12, 26]. According to SDS-PAGE and random amplified polymorphic DNA (RAPD) techniques, the venom of Scorpio maurus palmatus from four geographically isolated localities in Egypt was investigated. Both obvious morphological differences and protein molecular weights diversity were observed. But the particular distinctions such as primary structure and exact abundance of venom toxins among different populations of the same scorpion species are not clearly clarified. As two populations of the scorpion Lychas mucronatus successively obtained from Hainan and Yunnan provinces were convinced to be the same species according to morphological analysis, our study conduct on the venom components of Lychas mucronatus by a transcriptome approach may fitly figure out the question.
The experimental methods for constructing the venom gland cDNA library of Yunnan-sourced Lychas mucronatus was the same as Hainan-sourced Lychas mucronatus. 60 adult specimens from both scorpion populations were selected when venom gland cDNA library was constructed. The gender difference of scorpions is basically half to half. We cut off scorpion venom glands 2 days after extraction of their venom by electrical stimulation. Most temporary influences such as age, seasonal, sex, feeding behavior and time for RNA transcription can be excluded. Nevertheless, the experimental results confirmed our hypothesis about the diversity exist in nearly every toxin type (Table 2).
Almost all high-abundant toxin types were obtained from two scorpion populations. Great diversity was observed in these highly expressed venom compositions, especially neurotoxins and AMPs. Among neurotoxins, short chain α-KTx was highly expressed in Yunnan-sourced population, whereas long chain NaTx and β-KTx was highly expressed in Hainan-sourced population. Particularly, α-KTx in the venom of Yunnan-sourced population is not only 2-fold higher abundant than in Hainan-sourced population, but also more diversified in primary structures. GT028663 represents the most abundant α-KTx cluster in Yunnan-sourced population, but we can not find its homologous transcripts in the venom gland cDNA library of Hainan-sourced population. EU163848 represents the most abundant α-KTx cluster from Hainan-sourced population. Although EU163848 shows homology to GT028601 from Yunnan-sourced population, EU163848 obviously has a higher expression level. Taken together, Yunnan-sourced Lychas mucronatus possesses a higher abundant and more diversified α-KTxs in venom and the homologous transcripts of the most abundant α-KTx cluster in the venom of Hainan-sourced Lychas mucronatus have a relatively low expressed level in the venom of Yunnan-sourced Lychas mucronatus. All these obvious diversity should be intricately related to scorpions' adaption in different environment which primarily concern the scorpions' interaction with their prey and predators. On the other hand, Hainan-sourced Lychas mucronatus possesses a higher abundance of NaTxs in venom. Because the NaTxs can cause severe neurotoxic symptoms after scorpion envenomation, Hainan-sourced Lychas mucronatus could be a more dangerous killer, which is more efficient for not only preying but also defensing itself from other predators.
According to the transcriptome analysis, we can conclude that both scorpion populations possess relatively high abundant AMPs. But it's unexpected that the abundance of AMPs in the venom of Hainan-sourced population reached remarkable 40% of all toxin-like peptides. Till now, more and more AMPs have been isolated and characterized[56, 57]. AMPs can destroy bacteria, fungi, parasites, and even some viruses. They are important defensive weapons of scorpion innate immunity. So we can assume that the high expression levels of AMPs could protect Hainan-sourced Lychas mucronatus away from microbial infection, and it may also implicate other unascertained benefits for the survival of the population.
Hainan island locates in Southern China and is separated from mainland by Qiongzhou strait. Because surrounded by South China Sea, the climate of Hainan island is hot and humid all the year round. Yunnan locates in southwest China and is a mountainous region, whose rainfall is relatively less than Hainan province. Hainan island is an excellent living environment for Lychas mucronatus. In this biotope, Hainan-sourced Lychas mucronatus has enough prey. But this hot and humid environment may also bring powerful nature enemy and more pathogenicbacteria. In a word, the distinctive living environments probably relate to the adaptive evolution of two Lychas mucronatus populations.
Our work unravels a large number of venom molecules never identified in scorpion species. The result provides a comprehensive comparative analysis of venom transcriptomes of the same scorpion species from different geographical regions, which thoroughly reveals the fact that peptides and proteins of the same scorpion species from different geographical regions are highly diversified and the scorpion venom arsenal is a constantly evolving system to adapt the different biotopes. We can make a conclusion that there are far more peptides in one scorpion species venom than previous expectation considering of the geographical isolation.
cDNA library construction
The Yunnan-sourced Lychas mucronatus were collected from Shidian county of Yunnan province in September 2008. Venom glands of 60 wild specimens were cut off 2 days after extraction of their venom by electrical stimulation, and ground into fine powder in liquid nitrogen. Total RNA was isolated with TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA), and then mRNA was prepared with PolyATtract® mRNA Isolation Systems (Promega, Madison, WI, USA). SuperScript™ Plasmid System (Invitrogen) was used to construct a directional cDNA library from 6 μg mRNA. cDNA inserts were directionally cloned into the plasmids pSPORT 1 according to the supplier's instructions. The recombinant plasmids were transformed into electrocompenent cells.
After growing the clones overnight in appropriate Luria Broth culture medium containing 100 μg/ml of ampicillin, random colonies were selected in order to obtain an unbiased overview of the venom gland transcriptome. After overnight culture, plasmid DNA was isolated using alkaline lysis method. Purified plasmids were single-pass sequenced on ABI 3730 automated sequencers (Applied Biosystems, Foster City, CA, USA).
Phred program were used to examine the trace files of sequenced clones, the cutoff Phred score was set to 40 as before[30, 61]. Vector and adaptor sequences were removed using the program Cross Match. After removing the PolyA tail, we discarded those sequences shorter than 100 nt. High-quality sequences were processed on the website EGassembler http://egassembler.hgc.jp/ with the default parameter. The resulted sequences were deposited into the dbEST. Each cluster was annotated by being searched against SWISS-PROT http://www.expasy.org/tools/blast/ and GenBank NCBI database http://www.ncbi.nlm.nih.gov/blast using BLAST algorithms with an e-value cut-off set to <10-4. After BLAST search, the unmatched clusters were further identified for open reading frames using the ORFfinder http://www.ncbi.nlm.nih.gov/projects/gorf/. Considering the extreme diversity of scorpion toxins, those clusters putative to encode venom peptides was re-examined manually to pick out individual different isoforms. All clusters were checked for the existence of signal peptides using the SignalP 3.0 program http://www.cbs.dtu.dk/services/SignalP/. All the sequence alignment was performed by Clustal_X 1.83 software followed by manual adjustment, and viewed by the software Jalview.
This work was supported by grants from the Basic Project of Ministry of Science and Technology of China (No. 2007FY210800) and the Major State Basic Research Development Program of China (Nos. 2005CB522903, 2010CB529800, 2010CB530100), the China Specific Project for Developing New Drugs (Nos. 2009ZX09103-612 and 2008ZX10001-015-9), and the Program for Changjiang Scholars and Innovative Research Team in University (No. IRT0745).
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