| Scorpion is one of the most dangerous venomous animals and is widespread all over the world.400 million years of evolution equipped scorpion with highly specialized venom gland and vastly abundant venom components. Most of these active venom components are proteins and peptides, which are very important for the survival of scorpion and also provide valuable candidate molecules for drug design and development. However, although scorpion has been used as drug for 2000 years in our country, our knowledge on venom toxins is still limited in a few scorpion species and their high abundance toxin types, enormous number of new toxins are urgently waiting for exploring. Besides, vast abundance difference between scorpion species has already been proved, but intraspecific diversity still need more comprehensive analysis. Our work unravels a large number of venom molecules never identified in scorpion species according to cDNA library constructing. The result provides a comprehensive comparative analysis of venom transcriptomes of the same scorpion species from different geographical regions. We also cloned and characterized the first Kunitz-type toxin gene SdPI from the venom gland cDNA library of the scorpion Lychas mucronatus.Lychas mucronatus is one scorpion species widely distributed in southern China. Anything is hardly known about its venom components, despite the fact that it can often cause human accidents. We obtained two populations of Lychas mucronatus successively from Hainan and Yunnan province, which subsequently identified to be the same species with Hainan-sourced population according to morphological identification. According to perform a comparative venomous gland transcriptome analysis by constructing and screening the venom gland cDNA library of the scorpion Lychas mucronatus from different geographical regions, a total of 158 novel toxins were characterized in this work. Based on sequence similarity, these toxins can be categorized into 20 different types, including NaTx (toxins specific for sodium channels), a-KTx (a subfamily of toxins specific for potassium channels),β-KTx(βsubfamily of toxins specific for potassium channels), LVP (lipolysis activating peptides), calcine (toxins specific for ryanodine receptors), scamp(small cationtic antimicrobial peptides), ponericin like peptides, BmKbpp like peptides, anionic peptides, GrTx (glycine-rich toxin), SPSV (serine protease from scorpion venom), PLA2,8C-toxin, salivary protein, La1 like peptides, Metalloprotease, Trypsin inhibitor like peptide, cytotoxic peptides and Atypical toxins. Among these toxin types, GrTx, salivary protein and 8C-toxin are novel toxin types seldom reported before. These results reveal extreme diversity of scorpion toxins.Secondly, 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.16 known toxin types existed in both scorpion populations venom were compared, including NaTx, a-KTx,β-KTx, LVP, calcine, scamp, ponericin like peptides, BmKbpp like peptides, anionic peptides, GrTx, SPSV, PLA2,8C-toxin, salivary protein, La1 like peptides, Metalloprotease and Trypsin inhibitor like peptide.Great diversity was observed in all highly expressed venom compositions, especially neurotoxins and AMPs. Among neurotoxins, short chainα-KTx was highly expressed in Yunnan-sourced population, whereas long chain NaTx andβ-KTx was highly expressed in Hainan-sourced population. These diversities may indicate that scorpion evolves to adapt a new environment by altering the primary structure and abundance of venom peptides and proteins.Besides, based on EST analysis and Blast, we also identified a novel Kunitz-type toxin gene SdPI from the venom gland cDNA library of Yunnan-sourced Lychas mucronatus. The full-length cDNA sequence of SdPI is 364 nt,243 nt open reading frame of SdPI encodes 80 amino acid residues, including signal peptide of 21 amino acids and mature peptide of 59 amino acids. The mature peptide of SdPI contains six cysteine residues and possesses a unique cysteine framework different from any other reported Kunitz-type toxins. The cDNA sequence encoding the mature peptide of SdPI was amplified by PCR. After digested by Nde I and Xho I,it was inserted into the pET28a expression vector. The recombination vector was transformed to E. coli Rosetta strain and induced with IPTG in LB culture. Because the expressed SdPI protein mainly existed in the form of inclusion body, the recombined SdPI protein inclusion body was treated with the method of denaturation and refolding and finally purified by RP-HPLC. We finally obtained chromatography pure SdPI peptide with 5.5 mg/L LB medium. The recombined SdPI peptide can inhibit the activity of trypsin with high potency and its Ki is 1.6×10-7 M. It also showed thermostability in test. Further structural modeling and mutation experiments confirmed that SdPI kept a similar serine protease inhibitory reactive site like other Kunitz-type toxins which used K14 as the PI position.In conclusion, 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. Besides, we also firstly characterized the function of Kunitz-type toxin derived from scorpion venoms which not only provides some clues on identified the unknown atypical types of venom molecules in scorpion venom, but also open a new avenue for developing drugs with scorpion venom derived protease inhibitor peptides. |
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