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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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