细粒棘球蚴原头节分泌的细胞外囊泡中人源蛋白质组学分析

doi:10.12140/j.issn.1000-7423.2020.06.004

中国寄生虫学与寄生虫病杂志 ›› 2020, Vol. 38 ›› Issue (6): 695-701.doi: 10.12140/j.issn.1000-7423.2020.06.004

细粒棘球蚴原头节分泌的细胞外囊泡中人源蛋白质组学分析

史春丽¹(), 杨慧²^,³, 潘雯⁴, 张馨⁴, 朱晓庭⁴, 赵嘉庆¹^,²^,³^,^*()

1 宁夏医科大学基础医学院,银川 750004
2 宁夏医科大学医学科学技术研究中心银川 750004
3 宁夏回族自治区医学科学研究所,银川 750004
4 宁夏医科大学临床医学院,银川 750004

收稿日期:2020-08-07 出版日期:2020-12-30 发布日期:2021-01-12
通讯作者: 赵嘉庆
作者简介:史春丽（1995-）女,硕士研究生,从事寄生虫感染与免疫机制研究。E-mail: forever_lily@126.com
基金资助:
宁夏回族自治区重点研发计划项目(2020BEG03014);宁夏自然科学基金(2019AAC03266);宁夏自然科学基金(2020AAC03426)

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

SHI Chun-li¹(), YANG Hui²^,³, PAN Wen⁴, ZHANG Xin⁴, ZHU Xiao-ting⁴, ZHAO Jia-qing¹^,²^,³^,^*()

1 Basic Medical College of Ningxia Medical University, Yinchuan 750004, China
2 Science and Technology Center of Ningxia Medical University, Yinchuan 750004, China
3 Institute of Medical Sciences of Ningxia Hui Autonomous Region, Yinchuan 750004, China
4 Clinical Medical College of Ningxia Medical University, Yinchuan 750004, China

Received:2020-08-07 Online:2020-12-30 Published:2021-01-12
Contact: ZHAO Jia-qing
Supported by:
Ningxia Hui Autonomous Region Key Research Program(2020BEG03014);Ningxia Natural Science Found Project(2019AAC03266);Ningxia Natural Science Found Project(2020AAC03426)

摘要/Abstract

摘要：

目的蛋白质组学分析鉴定来源于细粒棘球蚴原头节分泌的细胞外囊泡(EVs)中人源蛋白质的组分。方法收集细粒棘球蚴病患者肝脏中的完整包囊,无菌条件下分离原头节,用含30%去外泌体的胎牛血清完全培养基体外培养原头节,每3天换1次培养基并收集培养上清,用EVs分离试剂盒分离培养上清中的EVs。透射电镜观察EVs的形态;纳米粒子跟踪分析EVs的粒径;采用液相色谱-质谱法对EVs进行蛋白质组学测序,二级质谱数据使用Maxquant (v1.5.2.8)进行蛋白检索与注释。将人源蛋白通过在线工具David进行组织、疾病及通路的富集比对与注释。结果体外培养原头节分泌的EVs为杯状双层膜性囊泡,直径小于200 nm。蛋白质组学测序共测出20 317条肽段,鉴定获得1 461个蛋白,其中328个为人源蛋白。原头节分泌的EVs中高度富集的外泌体标志蛋白有细丝蛋白、14-3-3蛋白、热休克蛋白90、热休克蛋白70和丙酮酸激酶。EVs的人源蛋白中,组织富集程度居前3位的为血浆、Cajal-Retzius细胞、肝脏。疾病富集中涉及感染、免疫和癌症等相关疾病。Bio Carta通路富集结果显示,富集程度居前3位的分别为替代补体通路、凝集素补体通路和补体通路;KEGG通路富集程度居前3位的为黏着斑黏附信号通路、细胞外基质受体相互作用通路和蛋白酶体相互作用通路。结论体外培养原头节分泌的EVs中的人源蛋白与人体血浆和肝脏等组织相关,提示原头节通过分泌EVs与人体进行物质交换。

关键词: 细粒棘球蚴, 原头节, 细胞外囊泡, 免疫逃逸, 蛋白质组学

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

中图分类号:

R383.33

史春丽, 杨慧, 潘雯, 张馨, 朱晓庭, 赵嘉庆. 细粒棘球蚴原头节分泌的细胞外囊泡中人源蛋白质组学分析[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(6): 695-701.

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.

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参考文献 20

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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

细粒棘球蚴原头节分泌的细胞外囊泡中人源蛋白质组学分析

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

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