DataSheet1_Distinguishing two distinct types of salivary extracellular vesicles: a potential tool for understanding their pathophysiological roles.docx

Extracellular vesicles (EVs), which are found in almost all cells and human body fluids, are currently being studied as a source of pathophysiological information. Previously, we demonstrated that at least two types of EVs can be isolated from human whole saliva (WS) using enzymatic activity of dipeptidyl peptidase IV (DPP IV) as a marker for differentiating the EV subsets. In the present study, EV fractions, termed EV-I 20 k-ppt and EV-II 100 k-ppt, were prepared by a combination of size-exclusion chromatography of improved condition and sequential centrifugation. The EV-I 20 k-ppt fraction contained medium/large EVs with a diameter of 100–1,000 nm, including aminopeptidase N (APN), mucin 1, ezrin, and Annexin A1. EV-II 100 k-ppt contained small EVs with a diameter of 20–70 nm, with DPP IV and CD9, programmed cell death 6-interacting protein, and tumor susceptibility gene 101 as characteristic proteins. Proteomic analyses also revealed distinctive repertoires of constituent proteins. Immunoprecipitation of several membrane proteins of the EVs with respective antibodies suggested their differential local membrane environment between the two types of salivary vesicles. Thus, we identified two distinctive types of EVs, one is APN/MUC1- rich EVs (EV-I, large/medium EVs) and the other is DPP IV/CD9-rich EVs (EV-II, small EVs). Furthermore, analysis of the binding of the EVs to coronavirus spike proteins showed that EV-II 100 k-ppt, but not EV-I 20 k-ppt, significantly bound to the spike protein of Middle East respiratory syndrome coronavirus (MERS-CoV). Finally, we developed a simple method to prepare two distinctive EVs from only 1 mL of human WS using sequential immunoprecipitation. Elucidating the features and functions of these two types of salivary EVs may help us understand their pathophysiological roles in the oral cavity and gastrointestinal tract.

几乎所有细胞及人体体液中均存在细胞外囊泡（Extracellular Vesicles，EVs），目前其作为病理生理信息来源已成为研究热点。既往本团队已证实，可借助二肽基肽酶IV（Dipeptidyl Peptidase IV，DPP IV）的酶活性作为亚型区分标志物，从人类全唾液（Whole Saliva，WS）中分离出至少两种EV亚型。本研究中，我们采用优化条件下的尺寸排阻色谱联合连续超速离心法，制备了两种EV组分，分别命名为EV-I 20 k-ppt与EV-II 100 k-ppt。EV-I 20 k-ppt组分包含直径100~1000 nm的中/大型EV，其标志性蛋白包括氨肽酶N（Aminopeptidase N，APN）、黏蛋白1、埃兹蛋白及膜联蛋白A1。EV-II 100 k-ppt组分则包含直径20~70 nm的小型EV，其特征蛋白为DPP IV、CD9、程序性细胞死亡6互作蛋白及肿瘤易感基因101。蛋白质组学分析亦显示两种组分的蛋白组成谱存在显著差异。针对EV数种膜蛋白的免疫沉淀实验结果提示，这两种唾液EV的局部膜微环境存在差异。综上，本研究鉴定出两种特征迥异的EV：一类为富含APN/MUC1的EV（即EV-I，中/大型EV），另一类为富含DPP IV/CD9的EV（即EV-II，小型EV）。此外，针对EV与冠状病毒刺突蛋白结合能力的分析显示，EV-II 100 k-ppt可显著结合中东呼吸综合征冠状病毒（Middle East Respiratory Syndrome Coronavirus，MERS-CoV）的刺突蛋白，而EV-I 20 k-ppt无此结合活性。最后，本团队开发了一种简便方法，仅需1 mL人类全唾液即可通过连续免疫沉淀法制备这两种特征迥异的EV。阐明这两种唾液EV的特征与功能，将有助于解析其在口腔及胃肠道中的病理生理作用。