Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells
Lipidomic and proteomic analysis of exosomes from mouse cortical collecting duct cells
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DOI：
10.1096/fj.201700417r
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发表时间：
2017-12-01
期刊：
影响因子：
4.8
通讯作者：
Alli, Abdel A.
中科院分区：
文献类型：
--
作者：
Dang, Viet D.;Jella, Kishore Kumar;Alli, Abdel A.
外泌体是内体衍生的纳米囊泡，其参与细胞通讯和信号传导。外泌体由上皮细胞产生，并且在包括血液和尿液的生物流体中发现。外泌体内的包装材料包括蛋白质和脂质，但外泌体亚型内的分子比较在很大程度上是未知的。本研究的目的是研究来源于极化的小鼠皮质集合管主细胞的顶端质膜和基底侧质膜的外泌体之间的差异。纳米颗粒跟踪分析表明，顶侧和基底侧外泌体的大小和浓度在3种不同温度（23、37和42摄氏度）下保持相对稳定。液相色谱-串联质谱分析揭示了来自相同细胞的两种外泌体内包装的蛋白质之间的显著差异。在顶端质膜来源的外来体样品中鉴定了在质膜的内小叶处表达的几种蛋白质，包括α-辅肌动蛋白-1、膜突蛋白、14-3-3蛋白ζ/δ、膜联蛋白A1/A3/A4/A5/A6、网格蛋白重链1、甘油醛-3-磷酸脱氢酶、α-烯醇酶、细丝蛋白-A和热休克蛋白90，但在来自小鼠皮质集合管细胞的基底外侧质膜外泌体中没有。除了蛋白质水平的差异外，基于质谱的鸟枪脂质组学分析显示，两种类型的外泌体的脂质类别和脂肪酸组成存在显着差异。我们发现，与基底外侧质膜外泌体相比，在顶端质膜中的其他磷脂中，鞘磷脂水平较高，心磷脂水平较低。本文提出的外泌体亚型的分子分析将有助于我们理解外泌体生物发生，并且结果可能对生物标志物发现具有潜在意义。
Exosomes are endosome-derived nanovesicles that are involved in cellular communication and signaling. Exosomes are produced by epithelial cells and are found in biologic fluids including blood and urine. The packaged material within exosomes includes proteins and lipids, but the molecular comparison within exosome subtypes is largely unknown. The purpose of this study was to investigate differences between exosomes derived from the apical plasma membrane and basolateral plasma membrane of polarized murine cortical collecting duct principal cells. Nanoparticle tracking analysis showed that the size and concentration of apical and basolateral exosomes remained relatively stable across 3 different temperatures (23, 37, and 42 degrees C). Liquid chromatography-tandem mass spectrometry analysis revealed marked differences between the proteins packaged within the two types of exosomes from the same cells. Several proteins expressed at the inner leaflet of the plasma membrane, including alpha-actinin-1, moesin, 14-3-3 protein zeta/delta, annexin A1/A3/A4/A5/A6, clathrin heavy chain 1, glyceraldehyde-3-phosphate dehydrogenase, a-enolase, filamin-A, and heat shock protein 90, were identified in samples of apical plasma membrane-derived exosomes, but not in basolateral plasma membrane exosomes from mouse cortical collecting duct cells. In addition to differences at the protein level, mass spectrometry-based shotgun lipidomics analysis showed significant differences in the lipid classes and fatty acid composition of the two types of exosomes. We found higher levels of sphingomyelin and lower levels of cardiolipin, among other phospholipids in the apical plasma membrane compared to the basolateral plasma membrane exosomes. The molecular analyses of exosome subtypes presented herein will contribute to our understanding of exosome biogenesis, and the results may have potential implications for biomarker discovery.