Different isolation approaches lead to diverse glycosylated extracellular vesicle populations
Different isolation approaches lead to diverse glycosylated extracellular vesicle populations
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DOI：
10.1080/20013078.2019.1621131
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发表时间：
2019-12-01
影响因子：
16
通讯作者：
Reis, Celso A.
中科院分区：
文献类型：
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作者：
Freitas, Daniela;Balmana, Meritxell;Reis, Celso A.
细胞外囊泡（EV）是一组参与细胞间通讯并介导广泛生物学功能的小分泌颗粒。EV货物由大量分子组成，包括糖缀合物。在此，我们报告了关于分离策略对EV群体糖基化谱的影响的第一项研究。使用不同的现有技术方案，即差异超离心（UC）、总外泌体分离（TEI）、OptiPrep密度梯度（ODG）和尺寸排阻色谱（SEC），导致EV群体显示出不同的糖缀合物组。通过UC、ODG和SEC方法获得的EV群体显示出相似的蛋白质和聚糖谱，而TEI方法分离出最不同的EV群体。此外，ODG和SEC分离方案提供了增强的EV糖蛋白检测。值得注意的是，展示肿瘤相关聚糖唾液酸-Tn（STn）的蛋白质被鉴定为包装到EV中的货物，与分离方法无关。通过UC、ODG和SEC分离的携带STn的EV样品呈现了在通过TEI分离的EV中未检测到的相当多的癌症相关蛋白。我们的工作证明了在获得的EV群体中使用不同分离方法的影响，以及分离群体的糖基化谱的结果。此外，我们的研究结果强调了选择适当的EV分离方案和细胞培养条件以确定EV糖缀合物的结构和功能复杂性的重要性。
Extracellular vesicles (EVs) are a heterogeneous group of small secreted particles involved in intercellular communication and mediating a broad spectrum of biological functions. EVs cargo is composed of a large repertoire of molecules, including glycoconjugates. Herein, we report the first study on the impact of the isolation strategy on the EV populations' glycosylation profile. The use of different state-of-the-art protocols, namely differential ultracentrifugation (UC), total exosome isolation (TEI), OptiPrepdensity gradient (ODG) and size exclusion chromatography (SEC) resulted in EV populations displaying different sets of glycoconjugates. The EV populations obtained by UC, ODG and SEC methods displayed similar protein and glycan profiles, whereas TEI methodology isolated the most distinct EV population. In addition, ODG and SEC isolation protocols provided an enhanced EV glycoproteins detection. Remarkably, proteins displaying the tumour-associated glycan sialyl-Tn (STn) were identified as packaged cargo into EVs independently of the isolation methodology. STn carrying EV samples isolated by UC, ODG and SEC presented a considerable set of cancer-related proteins that were not detected in EVs isolated by TEI. Our work demonstrates the impact of using different isolation methodologies in the populations of EVs that are obtained, with consequences in the glycosylation profile of the isolated population. Furthermore, our results highlight the importance of selecting adequate EV isolation protocols and cell culture conditions to determine the structural and functional complexity of the EV glycoconjugates.