Proteomic analysis of human proteins in extracellular vesicles secreted by protoscoleces of Echinococcus granulosus

doi:10.12140/j.issn.1000-7423.2020.06.004

Abstract

Abstract:

Objective To identify the protein of human source in extracellular vesicles (EVs) secreted by protoscoleces of Echinococcus granulosus by proteomic analysis. Methods Liver intact cysts were collected from cystic echinococcosis patients, and the protoscoleces were separated under aseptic conditions and then cultured in 30% exosome-free fetal bovine serum in vitro, with medium replacement every three days. The supernatant was collected, from which EVs were isolated by a simple and rapid extracellular vesicle separation kit. A transmission electron microscope was employed to observe and identify the morphology of EVs, and the concentration and size of EVs were evaluated by nanoparticle tracking analysis. The EVs underwent Liquid chromatography-mass spectrometry analysis for identification and characterization of their protein content. The secondary mass spectrometry data were used to retrieve and annotated with Maxquant (v1.5.2.8). The enrichment analysis of tissues, disease and pathway for the human source proteins in EVs were were carried out using online tool David. Results The EVs secreted by protoscoleces in vitro were cup-shaped bilayer membranous vesicles with a diameter less than 200 nm. The proteomic analysis found a total of 20 317 peptides, and identified 1 461 proteins, including 328 human source proteins. Exosome marker proteins, including filament protein, 14-3-3 protein, heat shock protein 90, heat shock protein 70 and pyruvate kinase, are highly enriched in EVs secreted by protoscolex. The top 3 tissues in enrichment extent were plasma, Cajal-Retzius cells and liver. The disease enrichment found involving infection, immunity and cancer. The Bio Carta pathways analysis indicated top 3 enriched pathways, which were the alternative complement pathway, lectin complement pathway and complement pathway; the top 3 of KEGG pathways enrichment were adhesion plaque, extracellular matrix receptor-related action, and proteasome. Conclusion The proteins of human source in EVs secreted by protoscoleces cultured in vitro are related to human plasma and liver, indicating that the protoscoleces exert substance exchange with human via seretion of EVs.

Key words: Echinococcus granulosus, Protosoleces, Extracellular vesicle, Immune escape, Proteomics

CLC Number:

R383.33

SHI Chun-li, YANG Hui, PAN Wen, ZHANG Xin, ZHU Xiao-ting, ZHAO Jia-qing. Proteomic analysis of human proteins in extracellular vesicles secreted by protoscoleces of Echinococcus granulosus[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2020, 38(6): 695-701.

Figures/Tables 5

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References 20

[1]	Wei YH, Hu Yuan, Cao JP. Advances in genetic polymorphism of Echinococcus granulosus[J]. Chin J Parasitol Parasit Dis, 2019,37(4):481-485. (in Chinese)
	( 魏玉环, 胡媛, 曹建平. 细粒棘球绦虫基因多态性的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2019,37(4):481-485.)
[2]	Heidari Z, Sharbatkhori M, Mobedi I, et al. Echinococcus multilocularis and Echinococcus granulosus in canines in North-Khorasan Province, Northeastern Iran, identified using morphology and genetic characterization of mitochondrial DNA[J]. Parasit Vectors, 2019,12(1):606. pmid: 31881913
[3]	Agudelo Higuita NI, Brunetti E, McCloskey C. Cystic echinococcosis[J]. J Clin Microbiol, 2016,54(3):518-523.
[4]	Jenkins DJ, Romig T, Thompson RCA. Emergence/re-emergence of Echinococcus spp.: a global update[J]. Int J Parasitol, 2005,35(11/12):1205-1219.
[5]	Hill AF. Extracellular vesicles and neurodegenerative diseases[J]. J Neurosci, 2019,39(47):9269-9273. pmid: 31748282
[6]	Shen H, Liu CY, Zhao YM. Research status and prospect of extracellular vesicles of parasites[J]. Chin J Parasitol Parasit Dis, 2018,36(4):413-417. (in Chinese)
	( 沈辉, 刘春英, 赵玉敏. 寄生虫细胞外囊泡的研究现状及展望[J]. 中国寄生虫学与寄生虫病杂志, 2018,36(4):413-417.)
[7]	Akbar N, Azzimato V, Choudhury RP, et al. Extracellular vesicles in metabolic disease[J]. Diabetologia, 2019,62(12):2179-2187. pmid: 31690986
[8]	Siles-Lucas M, Sánchez-Ovejero C, González-Sánchez M, et al. Isolation and characterization of exosomes derived from fertile sheep hydatid cysts[J]. Vet Parasitol, 2017,236:22-33.
[9]	Monteiro KM, de Carvalho MO, Zaha A, et al. Proteomic analysis of the Echinococcus granulosus metacestode during infection of its intermediate host[J]. Proteomics, 2010,10(10):1985-1999.
[10]	Marcilla A, Trelis M, Cortés A, et al. Extracellular vesicles from parasitic helminths contain specific excretory/secretory proteins and are internalized in intestinal host cells[J]. PLoS One, 2012,7(9):e45974.
[11]	Hidalgo C, García MP, Stoore C, et al. Proteomics analysis of Echinococcus granulosus protoscolex stage[J]. Vet Parasitol, 2016,218:43-45.
[12]	Aziz A, Zhang WB, Li J, et al. Proteomic characterisation of Echinococcus granulosus hydatid cyst fluid from sheep, cattle and humans[J]. J Proteom, 2011,74(9):1560-1572.
[13]	Deolindo P, Evans-Osses I, Ramirez MI. Microvesicles and exosomes as vehicles between protozoan and host cell communication[J]. Biochem Soc Trans, 2013,41(1):252-257.
[14]	Fan XB, He X, Pan WQ. Advances in the study of exocrine bodies from parasites and their functions[J]. Chin Trop Med, 2017,17(12):1267-1272. (in Chinese)
	( 范晓斌, 何兴, 潘卫庆. 寄生虫来源的外泌体及其功能研究进展[J]. 中国热带医学, 2017,17(12):1267-1272.)
[15]	Cheng WJ, Jiang H, Dong HF, et al. Progress of exocrine and other extracellular vesicles in the study of parasites and parasitic diseases[J]. Chin J Schisto Control, 2019,31(5):555-559. (in Chinese)
	( 程文君, 蒋洪, 董惠芬, 等. 外泌体及其他细胞外囊泡在寄生虫与寄生虫病研究中的进展[J]. 中国血吸虫病防治杂志, 2019,31(5):555-559.)
[16]	Frotscher M. Dual role of Cajal-Retzius cells and reelin in cortical development[J]. Cell Tissue Res, 1997,290(2):315-322.
[17]	Balaphas A, Meyer J, Sadoul K, et al. Platelets and platelet-derived extracellular vesicles in liver physiology and disease[J]. Hepatol Commun, 2019,3(7):855-866. doi: 10.1002/hep4.1358 pmid: 31304449
[18]	Conigliaro P, Triggianese P, Ballanti E, et al. Complement, infection, and autoimmunity[J]. Curr Opin Rheumatol, 2019,31(5):532-541. doi: 10.1097/BOR.0000000000000633 pmid: 31192812
[19]	Marshall S, Kelly PH, Singh BK, et al. Extracellular release of virulence factor major surface protease via exosomes in Leishmania infantum promastigotes[J]. Parasit Vectors, 2018,11(1):355. doi: 10.1186/s13071-018-2937-y
[20]	Zhou X, Wang W, Cui F, et al. Extracellular vesicles derived from Echinococcus granulosus hydatid cyst fluid from patients: isolation, characterization and evaluation of immunomodulatory functions on T cells[J]. Int J Parasitol, 2019,49(13/14):1029-1037.

编号 No.	登录号 Protein accession No.	蛋白名称 Protein name	基因名 Gene name	肽段覆盖率/% Coverage rate/%	相对分子质量 M_r	特异性肽段数 Unique pep count
1	P35443	凝血酶敏感蛋白-4 Thrombospondin-4	THBS4	16.6	105 870	9
2	P13591	神经细胞黏附分子-1 Neural cell adhesion molecule-1	NCAM1	19.0	94 573	14
3	P00742	凝血因子 X Coagulation factor X	F10	12.1	54 731	6
4	Q14515	半胱氨酸酸性蛋白-1 SPARC-like protein-1	SPARCL1	6.6	75 207	5
5	Q8NBJ4	高尔基膜蛋白-1 Golgi membrane protein-1	GOLM1	8.5	45 333	5
6	Q06828	纤维蛋白 Fibromodulin	FMOD	17.3	43 178	5
7	Q99983	骨调节蛋白 Osteomodulin	OMD	3.6	49 492	2
8	O60462	骨调节蛋白-2 Neuropilin-2	NRP2	25.9	104 830	19
10	Q7LGC8	碳水化合物磺基转移酶-3 Carbohydrate sulfotransferase 3	CHST3	15.7	54 705	7
11	P16070	CD44抗原 CD44 antigen	CD44	2.7	81 537	2
12	P23471	受体型酪氨酸蛋白磷酸酶 Receptor-type tyrosine-protein phosphatase zeta	PTPRZ1	3.9	254 580	8
13	P13639	伸长因子-2 Elongation factor-2	EEF2	10.7	95 337	8
14	Q6YHK3	CD109抗原 CD109 antigen	CD109	4.4	161 690	5
15	Q9UBP4	Dickkopf相关蛋白-3 Dickkopf-related protein-3	DKK3	18.9	38 390	4
16	Q92783	信号转导分子-1 Signal transducing adapter molecule-1	STAM	3.1	59 179	2
17	P16112	聚集素核心蛋白 Aggrecan core protein	ACAN	3.9	261 330	9
18	O14786	神经肽-1 Neuropilin-1	NRP1	18.4	103 130	12
19	P23470	受体型酪氨酸蛋白磷酸酶γ Receptor-type tyrosine-protein phosphatase gamma	PTPRG	6.2	162000	7
20	P20908	胶原α-1(V)链 Collagen alpha-1(V) chain	COL5A1	8.3	183 560	12