墨江蜈蚣（<i>Scolopendra mojiangica</i>）毒素多样性的比较分析研究

Zi-Chao Liu; Jin-Yang Liang; Xin-Qiang Lan; Tao Li; Jia-Rui Zhang; Fang Zhao; Geng Li; Pei-Yi Chen; Yun Zhang; Wen-Hui Lee; Feng Zhao

doi:10.24272/j.issn.2095-8137.2020.019

墨江蜈蚣（Scolopendra mojiangica）毒素多样性的比较分析研究

doi: 10.24272/j.issn.2095-8137.2020.019

刘子超^1,3,,
梁津旸^2,5,,
兰新强^1,2,5,
李涛^1,4,
张家瑞^2,7,
赵芳^1,4,6,
李庚^1,4,
陈佩仪^1,4,
赵云^2, ,,
李文辉^2, ,,
赵锋^1,4,6, ,

1.
普洱学院生物与化学学院云南省高校民族医药资源研究与东南亚国际合作重点实验室，云南普洱 665000，中国
2.
中国科学院昆明动物研究所中国科学院/云南省动物模型与人类疾病机理重点实验室，云南昆明 650223，中国
3.
昆明学院农学与生命科学学院云南省高校蛭类资源开发利用工程研究中心, 云南昆明 650214, 中国
4.
普洱学院生物活性分子与药物研发重点实验室，云南普洱 665000，中国
5.
中国科学院大学昆明生命科学学院，云南昆明 650204，中国
6.
复旦大学中西医结合研究院传统药物比较研究所，上海 200032，中国
7.
广州医科大学南山学院，广东广州 511436，中国

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出版历程
- 收稿日期: 2019-10-15
- 刊出日期: 2020-03-01

Comparative analysis of diverse toxins from a new pharmaceutical centipede, Scolopendra mojiangica

Zi-Chao Liu^1,3,,
Jin-Yang Liang^2,5,,
Xin-Qiang Lan^1,2,5,
Tao Li^1,4,
Jia-Rui Zhang^2,7,
Fang Zhao^1,4,6,
Geng Li^1,4,
Pei-Yi Chen^1,4,
Yun Zhang^{2
, ,},
Wen-Hui Lee^{2
, ,},
Feng Zhao^{1,4,6
, ,}

1.
Key Laboratory of Ethnic Medical Resources Research and Southeast Asian International Cooperation of Yunnan Universities, Department of Biology and Chemistry, Puer University, Puer, Yunnan 665000, China
2.
Key Laboratory of Animal Models and Human Disease Mechanisms of the Chinese Academy of Sciences & Yunnan Province, Kunming Institute of Zoology, Chinese Academy of Sciences, Kunming, Yunnan 650223, China
3.
Engineering Research Center for Exploitation and Utilization of Leech Resources in Universities of Yunnan Province, School of Agronomy and Life Sciences, Kunming University, Kunming, Yunnan 650214, China
4.
Key Laboratory of Active Molecules and Drug Development, Puer University, Puer, Yunnan 665000, China
5.
Kunming College of Life Science, University of Chinese Academy of Sciences, Kunming, Yunnan 650204, China
6.
Institute of Comparative Study of Traditional Materia Medica, Institute of Integrative Medicine of Fudan University, Shanghai 200032, China
7.
Nanshan College, Guangzhou Medical University, Guangzhou, Guangdong 511436, China

Funds: This work was supported by grants from the Chinese National Natural Science Foundation (81860696, 31560596, 81373945, and 31360516), Yunnan Applied Basic Research Projects (2016FD076), Key Research Program of the Chinese Academy of Sciences (KJZD-EW-L03), "Yunling Scholar" Program, Yunnan Provincial Training Programs of Youth Leader in Academic and Technical Reserve Talent (2019HB058), and Puer University (2017XJKT12 & CXTD011)

More Information

Corresponding author: E-mail: zhangy@mail.kiz.ac.cn; leewh@mail.kiz.ac.cn; zhaofeng@mail.kiz.ac.cn

#Authors contributed equally to this work

摘要

摘要:
在我国传统中医药记载中，蜈蚣的药用历史有上千年。蜈蚣是一类最古老的有毒动物，其分泌的毒液是重要的捕食、防御的武器，含有大量生物及药理活性分子，极具药用价值。墨江蜈蚣（Scolopendra mojiangica）是一种较为常用的备选药材。本研究通过比较分析，揭示了墨江蜈蚣与药典收录药材—少棘蜈蚣（S. subspinipes mutilans）之间的毒素分子多样性及差异。通过ESI-MS/MS鉴定，并结合毒腺转录组数据分析，在墨江蜈蚣蛋白质组中鉴定了六千多种肽、246种蛋白质分子，其中毒素样蛋白、功能未知的毒素占五分之一。虽然少棘蜈蚣中，毒素蛋白占总蛋白的比例更高，但是通过转录组学比较分析发现，两种蜈蚣毒素转录本的组成非常相似。同时，墨江蜈蚣转录组中鉴定的毒素样蛋白数量是毒液蛋白质组中数量的10倍，由此我们可以推导出更多毒素样多肽的前体结构。墨江蜈蚣毒液的毒性实验发现，其粗毒同样具有很强的溶血活性及杀昆虫活性。我们的研究表明墨江蜈蚣的毒素样蛋白具有丰富的多样性，将为蜈蚣毒素样蛋白的药物开发和利用提供了新的基础。
- 蜈蚣 /
- 毒素 /
- 药用价值 /
- 蛋白组-转录组关联分析
Abstract:
As the oldest venomous animals, centipedes use their venom as a weapon to attack prey and for protection. Centipede venom, which contains many bioactive and pharmacologically active compounds, has been used for centuries in Chinese medicine, as shown by ancient records. Based on comparative analysis, we revealed the diversity of and differences in centipede toxin-like molecules between Scolopendra mojiangica, a substitute pharmaceutical material used in China, and S. subspinipes mutilans. More than 6 000 peptides isolated from the venom were identified by electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and inferred from the transcriptome. As a result, in the proteome of S. mojiangica, 246 unique proteins were identified: one in five were toxin-like proteins or putative toxins with unknown function, accounting for a lower percentage of total proteins than that in S. mutilans. Transcriptome mining identified approximately 10 times more toxin-like proteins, which can characterize the precursor structures of mature toxin-like peptides. However, the constitution and quantity of the toxin transcripts in these two centipedes were similar. In toxicity assays, the crude venom showed strong insecticidal and hemolytic activity. These findings highlight the extensive diversity of toxin-like proteins in S. mojiangica and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins.
- Centipede /
- Toxins /
- Pharmaceutical use /
- Proteotranscriptomic analysis
#Authors contributed equally to this work
注释:

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Figure 1. Proteomic and transcriptomic analyses of new pharmaceutical centipede

A: Molecular phylogenetic analysis of centipede, S. mojiangica, by maximum likelihood based on COI genes. Red labels correspond to two centipedes in our study, and posterior probabilities are assigned to nodes. B: Workflow for proteomic and transcriptomic analyses of centipede, S. mojiangica. Venom was processed and subjected to SDS-PAGE followed by in-gel digestion. Samples were then analysed in a separate ESI-MS/MS assay. For transcriptomic analysis, venom glands (not venom) were used for high-throughput sequencing. Functional analysis was combined with proteomic and transcriptomic data.

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Figure 2. Profiles of S. mojiangica proteome

A: Cumulative distribution of protein peptide coverage. Horizontal axis shows protein peptide coverage, and vertical axis shows protein ratio. B: Pie chart of identified proteins from our S. mojiangica proteome. C: Comparison of toxin-like proteins determined by proteomic analysis among three centipedes: i.e., S. mojiangica, S. viridis (Gonzalez-Morales et al., 2014), and S. mutilans (Zhao et al., 2018a). D: Distribution of molecular weights of proteome proteins.

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Figure 3. Identification of toxins from transcriptome of venom gland in centipedes

A: Comparison of transcripts identified in venom glands from two centipedes, S. mojiangica and S. mutilans, with transcriptomic analysis. B: Expression of all transcripts in venom glands of S. mojiangica and S. mutilans. Read counts reflect quantification accuracy of differential expression by mapping reads to transcripts and read counting. C: Pie chart of venom toxin-like proteins/peptides identified in transcriptomes of S. mojiangica and S. mutilans. In total, 410 and 342 venom toxin-like proteins/peptides were identified from S. mojiangica and S. mutilans, respectively, using transcriptomic analysis.

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Figure 4. Comparison of toxin-like molecules distributed in centipedes S. mojiangica and S. mutilans

A: Distribution of identified toxin-like molecules in S. mojiangica and S. mutilans. Toxin-like transcripts (n=410) in S. mojiangica were divided into 34 categories. Blue dots represent transcripts of S. mutilans and red dots represent transcripts of S. mojiangica. B: Main components of toxin-like molecules expressed in S. mojiangica and S. mutilans. Transcriptomic analysis showed only four types of toxin-like molecules with differential gene expression between S. mojiangica and S. mutilans.

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Figure 5. Insecticidal activity of crude centipede venom

A: Insecticidal activity of crude centipede venom. B: Hemolytic activity of elution of crude centipede venom. Peaks 1, 3, 5, and 6 at concentrations of 1 mg/mL were incubated with human red blood cells for 30 min at 37 °C, and absorbance of supernatant was measured at 540 nm.

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Table 1. Toxin-like proteins/peptides identified from venom proteome of S. mojiangica centipede

Sequence ID	GenBank accession No.	Sequence description	Category	Peptides	E-Value	MW (kD)	Calc. pI	FPKM
ScoMo_singlet48841	AT0003236	Blarina toxin precursor (EC 3.4.21.-)	Blarina toxin	9	1.00E-37	21.61	4.15	92.54
ScoMo_singlet50899	AT0003766	Mucrofibrase-5 precursor (EC 3.4.21.-)	Mucrofibrase-5	11	4.00E-16	14.40	9.93	3 454.74
ScoMo_singlet71394	AT0002263	Pseudechetoxin-like protein precursor	Pseudechetoxin	276	9.00E-42	28.74	9.86	7 195.57
ScoMo_contig2076	gi\|429840589	K+ channel inhibitor	Channel inhibitor	617	4.00E-164	62.76	9.15	1.37
ScoMo_singlet78309	AT0000117	Latisemin precursor	Latisemin	412	2.00E-22	20.89	7.96	0.00
ScoMo_contig4762	AT0003236	Blarina toxin precursor (EC 3.4.21.-)	Blarina toxin	108	1.00E-44	28.58	6.5	15 173.32
ScoMo_singlet45908	AT0003741	Thrombin-like enzyme contortrixobin (EC 3.4.21.-)	Serine proteinase	109	1.00E-41	44.94	5.08	1 685.57
ScoMo_singlet67462	AT0000120	Pseudecin precursor	Pseudechetoxin	66	5.00E-32	23.71	8.91	14 111.58
ScoMo_singlet72573	AT0000552	Hopsarin-D (EC 3.4.21.6)	Hopsarin-D	93	1.00E-121	85.15	6.53	132.70
ScoMo_singlet76606	AT0000554	Trocarin precursor (EC 3.4.21.6)	Trocarin	38	3.00E-138	84.92	6.17	60.34
ScoMo_singlet25641	AT0000552	Hopsarin-D (EC 3.4.21.6)	Hopsarin-D	46	5.00E-20	27.21	4.6	184.53
ScoMo_singlet69905	AT0000554	Trocarin precursor (EC 3.4.21.6)	Trocarin	14	4.00E-107	40.69	5.28	1 245.366
ScoMo_singlet57737	AT0003404	Zinc metalloproteinase fibrolase (EC 3.4.24.72)	Metalloproteinase	20	4.00E-16	35.21	8.13	48.71
ScoMo_singlet8256	AT0000762	Alpha-latrocrustotoxin	Alpha-latrocrustotoxin	10	0	50.48	6.79	136.27
ScoMo_singlet68890	AT0000552	Hopsarin-D (EC 3.4.21.6)	Hopsarin-D	13	5.00E-75	42.03	7.88	161.84
ScoMo_singlet7846	gi\|392295725	Omega-slptx-ssm2a neurotoxin precursor	Neurotoxin	11	8.00E-36	8.56	4.93	16 647.01
ScoMo_singlet55496	gi\|501293796	Cathepsin L	Cathepsin L	180	1.00E-155	37.30	6.35	2.83
ScoMo_singlet39956	AT0000554	Trocarin precursor (EC 3.4.21.6)	Trocarin	12	4E-09	4.64	3.79	5.55
MW: Molecular Weight; Calc. pI: The calculated isoelectric point (pI); FPKM: Fragments Per Kilobase of exon model per Million mapped fragments.

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参考文献(36)

[1]	Chen K, Yu B. 1999. Certain progress of clinical research on Chinese integrative medicine. Chinese Medical Journal (Engl), 112(10): 934−937.
[2]	Chen M, Li J, Zhang F, Liu Z. 2014. Isolation and characterization of SsmTx-I, a Specific Kv2.1 blocker from the venom of the centipede Scolopendra Subspinipes Mutilans L. Koch. Journal of Peptide Science, 20(3): 159−164. doi: 10.1002/psc.2588
[3]	Conesa A, Gotz S, Garcia-Gomez JM, Terol J, Talon M, Robles M. 2005. Blast2GO: a universal tool for annotation, visualization and analysis in functional genomics research. Bioinformatics, 21(18): 3674−3676. doi: 10.1093/bioinformatics/bti610
[4]	Edgar RC. 2010. Quality measures for protein alignment benchmarks. Nucleic Acids Research, 38(7): 2145−2153. doi: 10.1093/nar/gkp1196
[5]	Edgecombe GD, Giribet G. 2007. Evolutionary biology of centipedes (Myriapoda: Chilopoda). Annual Review of Entomology, 52: 151−170. doi: 10.1146/annurev.ento.52.110405.091326
[6]	Gonzalez-Morales L, Pedraza-Escalona M, Diego-Garcia E, Restano-Cassulini R, Batista CV, Gutierrez Mdel C, Possani LD. 2014. Proteomic characterization of the venom and transcriptomic analysis of the venomous gland from the Mexican centipede Scolopendra viridis. Journal of Proteomics, 111: 224−237. doi: 10.1016/j.jprot.2014.04.033
[7]	Hakim MA, Yang S, Lai R. 2015. Centipede venoms and their components: resources for potential therapeutic applications. Toxins (Basel), 7(11): 4832−4851. doi: 10.3390/toxins7114832
[8]	Harvey AL. 2014. Toxins and drug discovery. Toxicon, 92: 193−200. doi: 10.1016/j.toxicon.2014.10.020
[9]	He QY, He QZ, Deng XC, Yao L, Meng E, Liu ZH, Liang SP. 2008. ATDB: a uni-database platform for animal toxins. Nucleic Acids Research, 36.
[10]	Hou H, Yan W, Du K, Ye Y, Cao Q, Ren W. 2013. Construction and expression of an antimicrobial peptide scolopin 1 from the centipede venoms of Scolopendra subspinipes mutilans in Escherichia coli using SUMO fusion partner. Protein Expression and Purification, 92(2): 230−234. doi: 10.1016/j.pep.2013.10.004
[11]	Jiang H, Wong WH. 2009. Statistical inferences for isoform expression in RNA-Seq. Bioinformatics, 25(8): 1026−1032. doi: 10.1093/bioinformatics/btp113
[12]	Kalia J, Milescu M, Salvatierra J, Wagner J, Klint JK, King GF, Olivera BM, Bosmans F. 2015. From foe to friend: using animal toxins to investigate ion channel function. Journal of Molecular Biology, 427(1): 158−175. doi: 10.1016/j.jmb.2014.07.027
[13]	King G. 2013. Venoms to drugs: translating venom peptides into therapeutics. Australian Biochemist, 44(3): 13−16.
[14]	King GF. 2011. Venoms as a platform for human drugs: translating toxins into therapeutics. Expert Opinion on Biological Therapy, 11(11): 1469−1484. doi: 10.1517/14712598.2011.621940
[15]	Kumar S, Stecher G, Tamura K. 2016. MEGA7: molecular evolutionary genetics analysis version 7.0 for bigger datasets. Molecular Biology and Evolution, 33(7): 1870−1874. doi: 10.1093/molbev/msw054
[16]	Langmead B, Trapnell C, Pop M, Salzberg SL. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10(3): R25. doi: 10.1186/gb-2009-10-3-r25
[17]	Liu ZC, Zhang R, Zhao F, Chen ZM, Liu HW, Wang YJ, Jiang P, Zhang Y, Wu Y, Ding JP, Lee WH, Zhang Y. 2012. Venomic and transcriptomic analysis of centipede Scolopendra subspinipes dehaani. Journal of Proteome Research, 11(12): 6197−6212. doi: 10.1021/pr300881d
[18]	Peng K, Kong Y, Zhai L, Wu X, Jia P, Liu J, Yu H. 2010. Two novel antimicrobial peptides from centipede venoms. Toxicon, 55(2-3): 274−279. doi: 10.1016/j.toxicon.2009.07.040
[19]	Pertea G, Huang X, Liang F, Antonescu V, Sultana R, Karamycheva S, Lee Y, White J, Cheung F, Parvizi B, Tsai J, Quackenbush J. 2003. TIGR Gene Indices clustering tools (TGICL): a software system for fast clustering of large EST datasets. Bioinformatics, 19(5): 651−652. doi: 10.1093/bioinformatics/btg034
[20]	Rong M, Yang S, Wen B, Mo G, Kang D, Liu J, Lin Z, Jiang W, Li B, Du C, Yang S, Jiang H, Feng Q, Xu X, Wang J, Lai R. 2015. Peptidomics combined with cDNA library unravel the diversity of centipede venom. Journal of Proteomics, 114: 28−37. doi: 10.1016/j.jprot.2014.10.014
[21]	Savitski MM, Nielsen ML, Kjeldsen F, Zubarev RA. 2005. Proteomics-grade de novo sequencing approach. Journal of Proteome Research, 4(6): 2348−2354. doi: 10.1021/pr050288x
[22]	Smith JJ, Herzig V, King GF, Alewood PF. 2013. The insecticidal potential of venom peptides. Cellular and Molecular Life Sciences: CMLS, 70(19): 3665−3693. doi: 10.1007/s00018-013-1315-3
[23]	Trapnell C, Pachter L, Salzberg SL. 2009. TopHat: discovering splice junctions with RNA-Seq. Bioinformatics, 25(9): 1105−1111. doi: 10.1093/bioinformatics/btp120
[24]	Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, Van Baren MJ, Salzberg SL, Wold BJ, Pachter L. 2010. Transcript assembly and quantification by RNA-Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nature Biotechnol, 28(5): 511−515. doi: 10.1038/nbt.1621
[25]	Undheim EA, Fry BG, King GF. 2015. Centipede venom: recent discoveries and current state of knowledge. Toxins (Basel), 7(3): 679−704. doi: 10.3390/toxins7030679
[26]	Undheim EA, Jenner RA, King GF. 2016. Centipede venoms as a source of drug leads. Expert Opinion on Drug Discovery, 11(12): 1139−1149. doi: 10.1080/17460441.2016.1235155
[27]	Undheim EA, King GF. 2011. On the venom system of centipedes (Chilopoda), a neglected group of venomous animals. Toxicon, 57(4): 512−524. doi: 10.1016/j.toxicon.2011.01.004
[28]	Wang K, Fang H, Ye M, Chen H, Zhu Y, Fang H. 1997. Investigation on the resources of medicinal centipedes and identification on their commodities. Journal of Chinese Medicinal Materials, 20(9): 450−452.
[29]	Yang S, Liu Z, Xiao Y, Li Y, Rong M, Liang S, Zhang Z, Yu H, King GF, Lai R. 2012. Chemical punch packed in venoms makes centipedes excellent predators. Molecular and Cellular Proteomics, 11(9): 640−650. doi: 10.1074/mcp.M112.018853
[30]	Yang S, Xiao Y, Kang D, Liu J, Li Y, Undheim EA, Klint JK, Rong M, Lai R, King GF. 2013. Discovery of a selective NaV1.7 inhibitor from centipede venom with analgesic efficacy exceeding morphine in rodent pain models. Proceedings of the Natlional Academy of Sciences of the Untied States of America, 110(43): 17534−17539. doi: 10.1073/pnas.1306285110
[31]	Yang S, Yang F, Wei N, Hong J, Li B, Luo L, Rong M, Yarov-Yarovoy V, Zheng J, Wang K, Lai R. 2015. A pain-inducing centipede toxin targets the heat activation machinery of nociceptor TRPV1. Nature Communications, 6: 8297. doi: 10.1038/ncomms9297
[32]	Zhang Y. 2015. Why do we study animal toxins?. Zoological Research, 36(4): 183−222.
[33]	Zhao F, Guo X, Wang Y, Liu J, Lee WH, Zhang Y. 2014a. Drug target mining and analysis of the Chinese tree shrew for pharmacological testing. PLoS One, 9(8): e104191. doi: 10.1371/journal.pone.0104191
[34]	Zhao F, Lan X, Li T, Xiang Y, Zhao F, Zhang Y, Lee WH. 2018a. Proteotranscriptomic analysis and discovery of the profile and diversity of toxin-like proteins in centipede. Molecular and Cellular Proteomics, 17(4): 709−720. doi: 10.1074/mcp.RA117.000431
[35]	Zhao F, Lan XQ, Du Y, Chen PY, Zhao J, Zhao F, Lee WH, Zhang Y. 2018b. King cobra peptide OH-CATH30 as a potential candidate drug through clinic drug-resistant isolates. Zoological Research, 39(2): 87−96. doi: 10.24272/j.issn.2095-8137.2018.025
[36]	Zhao F, Yan C, Wang X, Yang Y, Wang G, Lee W, Xiang Y, Zhang Y. 2014b. Comprehensive transcriptome profiling and functional analysis of the frog (Bombina maxima) immune system. DNA Research, 21(1): 1−13. doi: 10.1093/dnares/dst035