Transcriptomic And Proteomic Study Of Venom Peptides From The Scorpion Androctonus Bicolor, And Molecular Evolution Analysis Of The Scorpion K~+- Channel Toxin Genes

Androctonus bicolor, which belongs to genus Androctonus, is one of the most medically-significant scorpion species in North Africa. It was found that the venom from A, bicolor was highly toxic to mammals and dangerous to human. The LD50 value of the A. bicolor venom in mice was 1.21 mg/kg as tested using intravenous injection. However, little has been known about the venom components from A. bicolor so far. To better understand the diversity of the venom peptides from the scorpion, and lay foundation for their applications in both basic research and clinic therapeutics, we employed transcriptomic approaches combined with mass spectrometry to dissect the venom compositions of A. bicolor.We obtained 730 ESTs code for venom peptides (toxins) from the cDNA library of scorpion A. bicolor venom. Bioinformatics analysis indicated that we identified 106 different venom peptides from the scorpion, including 103 novel peptides and 3 peptides that are identical to the described toxins from other species of scorpions. On the basis of protein sequence similarity, these peptides could be classified into 23 distinct families, including Na+-channel toxin (NaTx)-like peptides (33.5% of the total venom peptide transcripts), short-and long-chain K+-channel toxin (ScKTx and LcKTx)-like peptides (32.56%), Ca2+-channel toxin (CaTx)-like peptides (0.32%), Kunitz-type toxins (4.80%), putative antimicrobial peptides (4.20%), Defensin-like peptides (3.50%), Bradykinin-potentiating peptide (BPP)-like peptides (3.20%), BmKa2-like peptides (2.60%), new-type NDBPs (2.88%), as well as many new-type DBPs (7.36%), including venom peptides cross-linked with one, two, three, five or six disulfide bridges, respectively. We identified 24 novel NaTx-like peptides from the scorpion. These peptides displayed 90%-66% sequence identities to the known scorpion toxins. The transcripts of AbNaTxs account for 33.5% of the venom peptide transcriptome. We identified a total of 15 new a-KTxs from the scorpion, and these peptides can be assigned into 6 subfamilies of KTxs:a-KTx3, α-KTx8, α-KTx14, α-KTx14, a-KTx16, a-KTx31 and a-KTx32. We identified 10 unique β-KTxs from the scorpion A. bicolor. The transcripts of these peptides account for 7.96% of the venom peptide transcriptome, and can be grouped into 3 subfamilies. We identified a novel transcript encoding the precursor of a putative calcium channel toxin (CaTx) that was referred to as AbCaTxl. We also identified two putative antimicrobial peptides (AMPs), which were referred to as Androcin18-1 and Androcin18-3, respectively. Defensins account for 3.5% of the total venom peptide transcripts. We found the transcripts of 4 unique novel defensins, which were referred to as AbDef-1, AbDef-3, AbDef-4 and AbDef-5, respectively. AbDef-3 differs from AbDef-4 in 8 amino acid residues, and displays 86% sequence identity to AbDef-4. It is interesting to see that AbDef-3 is cross-linked by three disulfide bridges, whereas AbDef-4 is stabilized by four disulfide bridges. BLAST searches against GenBank indicated that both AbDef-3 and AbDef-4 displayed no identities to any other known peptides; this strongly suggests that the two peptides represent two novel classes of defensins from arthropods. We identified two different Bpps from the scorpion, which were referred to as AbBpp-1 and AbBpp-2, respectively. Aba-1 and Aba-2 were two different acidic NDBPs, and the two peptides showed 69% sequence identity to each other. The D+E contents in Aba-1 and Aba-2 molecules are 36% and 37%, respectively. We identified five unique Kunitz-type toxins from A. bicolor, and they were classified into two groups:toxins with three disulfide bridges (AbKci-2, AbKci-3, AbKci-4 and AbKci-5), and those with four disulfide bridges (AbKci-1). AbNtl, AbNt2, AbNt3 and AbNt4 constitute a unique novel class of venom NDBPs. based on their characterization of a repetitive sequence of 11 residues (RFD (A) RAAE (D) AS (G) PG). They represent a novel class of scorpion venom peptides, and were thus named as NDBP8.1, NDBP8.2, NDBP8.3 and NDBP8.4, respectively. AbNt5, AbNt6 and AbNt7 are three novel-type NDBPs that have unique amino acid compositions. It is interesting to see that the amino acid compositions of the three peptides are so unique. AbNt5 is rich in the residues G and Y that account for 59.2% and 14.3% of the total residues, respectively. AbNt6 is rich in the residues G and A, which account for 24.6% and 29.8% of the total residues, respectively. In contrast, AbNt7 is rich in Y and G that account for 43.2% and 21.6% of the total residues, respectively. AbNt8, a highly hydrophilic peptide, is rich in charged residues. AbNt9 is a novel NDBP that has unique amino acid composition and sequence, and contains a repetitive sequence of 10 residues:YLPTLQKXNR (K) (X stands for any amino acid). We identified 7 novel venom peptides that stabilized with only a disulfide bridge. These peptides can be grouped into two classes. The first class is a-TxC2 including AbTxC2l, AbTxC22, AbTxC23, AbTxC24 and AbTxC25, and the other is P-TxC2 including AbTxC210 and AbTxC211. The five a-TxC2 peptides are rich in the basic amino acid residues Arg and Lys, which account for 39.6%-37.3% of total residues, respectively. It is noteworthy that the two P-TxC2 peptides are highly rich in amino acid residues G and Y, and share a conserved cysteine motif:CX4CX39. AbTxC41 and AbTxC42 were two novel peptides stabilized with two disulfide bridges. They are rich in the positively charged amino acid residues Arg or Lys, making the two molecules highly basic. We discovered four novel venom peptides that were cross-linked with three disulfide bridges. The four peptides can be classified into two different groups: AbTxC61 and AbTxC62 belong to a subfamily; AbTxC63 and AbTxC64 belong to another subfamily. It is interesting to see that the Astakine-like peptides are also present in the venom glands of A. bicolor. We identified three unique Astakine-like peptides from the scorpion, which were stabilized by five disulfide bridges, and referred to as AbAsl, AbAs2 and AbAs3, respectively. To the best of our knowledge, this is the first report of Astakine-like peptides presenting in the venom glands of scorpions. We discovered two novel venom peptides that were cross-linked with six disulfide bonds. They were referred to as AbTxC121 and AbTxC122, respectively. The two peptides possess different cysteine patterns. Taken together, these data highlight the unique diversity of the venom peptides from the scorpion A. bicolor.We used LC-MS/MS to analysis the venom composition of A. bicolor. The LC-MS/MS results of all fractions were used to search the four different protein/peptide database. We identified 16 venom peptides from the venom of A. bicolor, of which 15 matched with the peptides deduced from transcripts,1 matched with a known Na+-channel toxin, Ab8. The detected peptides include one CaTx (AbCaTx1), one α-KTxs (AbTx4 and AbTx10), two β-KTx (AbKTx-2 and AbKTx-6), four α-NaTxs (Ab8, AbNaTx2, AbNaTx3 and AbNaTx18), five β-NaTxs (AbNaTx6, AbNaTx7, AbNaTx12, AbNaTx15, and AbNaTx25), one novel-type toxin (AbTxC61) and one Kunitz-type toxin (AbKci5). A total of 10 NaTxs were detected with high sequence coverage, suggesting that NaTxs are the most abundant in the venom of the scorpion. It’s noteworthy that AbTxC61 accounts for 34.39% of the identified mass spectra, suggesting its important role in the resting gland. By comparing the number and annotations of matched mass spectra, we found that A. bicolor had a closer similarity relationship to the old world scorpion Mesobuthus martensii than the new world scorpion Centruroides exilicauda. Our results reveal that the venom peptides are highly diverse with various physiological factors. Cellular associated proteins are highly conserved across different species, while venom peptides show species specificity.K+-channel specific toxins from scorpions are powerful probes used in the structural and functional characterization of different subfamilies of K+-channels which are thought to be the most diverse ion channels. However, only a limited number of K+-channel toxins have been identified from scorpions so far; moreover, little is known about the mechanisms for the generation of a combinatorial peptide library in a venom gland of a scorpion. To identify new K+-channel toxins, we constructed a cDNA library from the venom glands of scorpion animals which were at the very early stage of secretory phase after extraction of their venom. Sequencing for 200 clones with inserts of 300-400 bp showed that three new K+-channel specific toxin-like peptides (referred to as BmKcugx, BmKcugl and BmKcug2 respectively) were obtained. BmKcugl and BmKcug2 are two new members of α-KTx1 subfamily, and have been classified as α-KTx1.14 and α-KTx1.15, respectively. BmKcugx represents a new subfamily of K+-channel specific toxins which was classified into α-KTx22. BmKcugx was thus classified as α-KTx22.1. It is striking to find that the genomic gene of BmKcugla (a close homologue of BmKcug1 obtained)/BmKcug2 has two introns, one inserted in 5’UTR of the gene, and the other one inserted in the nucleotide sequence coding for C-terminal region of the signal peptide; in contrast, BmKcugx gene possesses only one inton inserted in the nucleotide sequence encoding C-terminal region of the signal peptide. Transcriptomic analysis for the venom glands of M. martensii Karsch indicated that the abundances of the transcripts of BmKcug1a and BmKcug2 are much higher than that of BmKcugx. Moreover, we identified an alternative splicing event occurred at the intron 1 and exon 2 junction within the 5’UTR of BmKcug2 transcript. Our data provide new knowledge for better understanding of the mechanism by which the combinatorial peptide library is generated in the scorpion venom gland.It was shown that the majority of scorpion venom peptides have evolved from a common ancestor by gene duplication and subsequent sequence mutations. Therefore, the genes of scorpion toxins may provide an excellent model for the investigations of intron evolution in response to selective forces after gene duplications. However, such investigations are generally hampered by the limited availability for high-quality data of conserved genomic sequences, and thus yet little have been known about the dynamic evolution of the introns from scorpion toxin genes so far. To address this issue, we performed evolutionary analysis for the genes coding for the 13 different K+-channel specific toxins from the scorpion M. martensii Karsch and A. bicolor. Further analysis revealed that the genes of BmTxl, BmKcug2, BmTx4, BmTx3B, BmTx2, BmKcugla, BmP08, sKTx2, BmTxKS3 contain two conserved introns (referred to as intron-1 and intron-2) located in the 5’UTR and signal peptide encoding regions, respectively. However, the genes of BmK38, BmK39, BmKcugx and abTxl only have a conserved intron corresponding to intron-2 inserted in the signal peptide encoding region. These observations, combined with the rooted phylogenetic tree analysis supports a conclusion that the genes of BmTxl, BmKcug2, BmTx4, BmTx3B, BmTx2, BmKcugla, BmP08, sKTx2 and BmTxKS3 have gained an intron in their 5’UTR. It has been established that intron gain in 5’UTR markedly enhances gene expression level. Our findings suggest that selective force has driven 5’UTR intron gain for the genes of the K+-channel specific toxins, which should markedly increase the toxin dose that a scorpion ejects per sting, and thus enable the scorpions to better adapt the environmental pressures, such as the increase of scorpion venom-resistance of prey and predators, and significant reduction of the natural availability of prey of scorpions.In summary, this study combined transcriptomic and proteomic approaches to systematically analysis the venom components of scorpion A. bicolor. Our efforts led to a discovery of 103 novel putative venom peptides. These newly identified venom peptides could be used as potential lead for drug design and development. The presence of a total of 16 different venom peptides was further confirmed by LC-MS/MS analysis. These data highlight the unique diversity of the venom peptides from A. bicolor. To the best of our knowledge, A. bicolor is the first species from the genus Androctonus, of which transcriptomic has been characterized so far. The adaptation analysis of scorpion short linear cationic peptides provides new insights to design enhanced potency antimicrobial peptides. Molecular evolution analysis of the short chain potassium channel toxins reveals that the scorpion venom repertoire is a dynamic evolving system. Our results provide insights to study the adaptive evolution of scorpion venom peptide.