Nakai Wataru, Yoshida Takeshi, Diez Diego, Miyatake Yuji, Nishibu Takahiro, Imawaka Naoko, Naruse Ken, Sadamura Yoshifusa, Hanayama Rikinari
Laboratory of Immune Network, WPI Immunology Frontier Research Center (IFReC), Osaka University, 3-1 Yamada-oka, Suita, Osaka 565-0871, Japan.
Department of Immunology, Kanazawa University Graduate School of Medical Sciences, 13-1 Takara, Kanazawa, Ishikawa 920-8640, Japan.
Sci Rep. 2016 Sep 23;6:33935. doi: 10.1038/srep33935.
Extracellular vesicles (EVs) such as exosomes and microvesicles serve as messengers of intercellular network, allowing exchange of cellular components between cells. EVs carry lipids, proteins, and RNAs derived from their producing cells, and have potential as biomarkers specific to cell types and even cellular states. However, conventional methods (such as ultracentrifugation or polymeric precipitation) for isolating EVs have disadvantages regarding purity and feasibility. Here, we have developed a novel method for EV purification by using Tim4 protein, which specifically binds the phosphatidylserine displayed on the surface of EVs. Because the binding is Ca-dependent, intact EVs can be easily released from Tim4 by adding Ca chelators. Tim4 purification, which we have applied to cell conditioned media and biofluids, is capable of yielding EVs of a higher purity than those obtained using conventional methods. The lower contamination found in Tim4-purified EV preparations allows more EV-specific proteins to be detected by mass spectrometry, enabling better characterization and quantification of different EV populations' proteomes. Tim4 protein can also be used as a powerful tool for quantification of EVs in both ELISA and flow cytometry formats. Thus, the affinity of Tim4 for EVs will find abundant applications in EV studies.
细胞外囊泡（EVs），如外泌体和微囊泡，充当细胞间网络的信使，使细胞之间能够交换细胞成分。EVs携带源自其产生细胞的脂质、蛋白质和RNA，具有作为特定于细胞类型甚至细胞状态的生物标志物的潜力。然而，用于分离EVs的传统方法（如超速离心或聚合物沉淀）在纯度和可行性方面存在缺点。在这里，我们开发了一种使用Tim4蛋白的新型EVs纯化方法，Tim4蛋白能特异性结合EVs表面展示的磷脂酰丝氨酸。由于这种结合依赖于钙离子，通过添加钙离子螯合剂，完整的EVs可以很容易地从Tim4上释放出来。我们将Tim4纯化方法应用于细胞条件培养基和生物流体，能够产生比使用传统方法获得的纯度更高的EVs。在Tim4纯化的EV制剂中发现的较低污染使得通过质谱法能够检测到更多EV特异性蛋白质，从而能够更好地表征和定量不同EV群体的蛋白质组。Tim4蛋白还可以用作ELISA和流式细胞术形式中EVs定量的强大工具。因此，Tim4对EVs的亲和力将在EV研究中得到广泛应用。
