Hamilton Nicklas, Claudio Natalie M, Armstrong Randall J, Pucci Ferdinando
Department of Otolaryngology-Head and Neck Surgery, Oregon Health and Science University, Portland, OR, USA.
Department of Cell, Developmental & Cancer Biology, Oregon Health and Science University, Portland, OR, USA.
Adv Biosyst. 2020 Dec;4(12):e2000007. doi: 10.1002/adbi.202000007. Epub 2020 May 10.
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.
细胞外囊泡（EVs）可通过细胞表面信号传导介导局部和远距离的细胞间通讯。为了对未受操纵的内源性释放的EVs进行体内研究，需要能够检测EV-细胞表面相互作用的灵敏且严格的方法。然而，EVs的分离和再输注可能会引入偏差。在体内研究EVs的一种严格方法是将膜结合报告基因导入亲代细胞。尽管如此，EVs能够携带的报告分子数量相对较少，因此该方法的灵敏度并不理想。这项工作通过对EVs进行工程改造，使其展示一种膜结合形式的分选酶A（SrtA）来解决这个问题，SrtA是一种细菌转肽酶，能够催化报告分子转移到EV结合细胞的更大表面上。对SrtA的设计和反应要求进行了优化和验证。即使在细胞表面蛋白仅存在一个N端甘氨酸的情况下，也能实现对EV结合细胞的高效体外标记。与EV结合细胞的间接标记（例如使用CD63-GFP融合蛋白）相比，基于SrtA的方法灵敏度提高了1至2个数量级，具体取决于EV的来源。这种新方法将有助于识别和研究体内与天然EVs相互作用的全套宿主细胞。
