Cell Surface Labeling by Engineered Extracellular Vesicles
Cell Surface Labeling by Engineered Extracellular Vesicles
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DOI：
10.1002/adbi.202000007
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发表时间：
2020-05-10
影响因子：
4.1
通讯作者：
Pucci, Ferdinando
中科院分区：
文献类型：
--
作者：
Hamilton, Nicklas;Claudio, Natalie M.;Pucci, Ferdinando
细胞外囊泡（EVs）可以通过细胞表面信号传导介导局部和远距离的细胞间通讯。为了对未经处理的内源性释放的ev进行体内研究，需要能够检测ev -细胞表面相互作用的敏感但严格的方法。然而，电动汽车的分离和回输可能会引入偏差。在体内研究ev的一种严格方法是通过基因工程将膜结合报告细胞植入亲代细胞。然而，电动汽车可以携带的报告分子数量相对较少，因此，该方法的灵敏度不是最佳的。这项工作解决了这个问题，通过工程ev来显示一种膜结合形式的排序酶a (SrtA)，一种细菌转肽酶，可以催化报告分子在ev结合细胞的更大表面上的转移。优化和验证了SrtA设计和反应要求。即使在细胞表面蛋白上仅存在一个n端甘氨酸，也可以实现对ev结合细胞的有效体外标记。与EV结合细胞的间接标记（例如，使用CD63-GFP融合）相比，基于srta的方法显示灵敏度提高了1-2倍，这取决于EV来源。这种新方法将有助于在体内鉴定和研究与天然ev相互作用的全套宿主细胞。
Extracellular vesicles (EVs) can mediate local and long-range intercellular communication via cell surface signaling. In order to perform in vivo studies of unmanipulated, endogenously released EVs, sensitive but stringent approaches able to detect EV-cell surface interactions are needed. However, isolation and reinfusion of EVs can introduce biases. A rigorous way to study EVs in vivo is by genetically engineering membrane-bound reporters into parental cells. Still, the amount of reporter molecules that EVs can carry is relatively small, and thus, the sensitivity of the approach is suboptimal. This work addresses this issue by engineering EVs to display a membrane-bound form of Sortase A (SrtA), a bacterial transpeptidase that can catalyze the transfer of reporter molecules on the much bigger surface of EV-binding cells. SrtA design and reaction requirements are optimized and validated. Efficient in vitro labeling of EV-binding cells is achieved, even in the presence of only one N-terminal glycine on cell surface proteins. As compared to indirect labeling of EV-binding cells (e.g., using CD63-GFP fusion), the SrtA-based approach shows 1-2 log increase in sensitivity, depending on the EV source. This novel approach will be useful to identify and study the full set of host cells interacting with native EVs in vivo.