Development of endogenous protein probes for characterizing surface proteins and cellular interactors of extracellular vesicles

Extracellular vesicles (EV) surface proteins have important extracellular functions and determine cellular tropism; however, characterizing the EV surfaceome remains challenging with available methods. EV-mediated intercellular communication takes place primarily through interactions at the recipient cell membrane, underscoring the importance of methodological advances to map this interplay. Here, we leverage the proximity labeling enzyme APEX2 (Apurinic/Apyrimidinic Endodeoxyribonuclease 2) for high-fidelity analysis of the EV surfaceome and cellular tropism. Surface display of APEX2 on EVs is achieved through its genetic fusion with EV-sorting domains, such as CD63 and TSPAN2. Upon adding the substrates biotin-phenol and hydrogen peroxide, vesicle surface APEX2 enables biotinylation of EV integral and corona proteins as well as target cells in vitro. Further data mining of the EV surfaceome reveals potential scaffolds for the bioengineering of EVs. Altogether, we introduce a robust tool for EV surfaceome and target cell mapping and uncover novel EV-sorting domains for bioengineering.

细胞外囊泡（Extracellular vesicles, EV）表面蛋白具有重要的细胞外生物学功能，并可决定细胞嗜性；然而，现有方法仍难以实现EV表面组的精准表征。EV介导的细胞间通讯主要通过与受体细胞膜的相互作用完成，这凸显了开发可解析该相互作用的方法学的重要性。本研究利用邻近标记酶APEX2（Apurinic/Apyrimidinic Endodeoxyribonuclease 2），对EV表面组与细胞嗜性开展高保真分析。通过将APEX2与EV分选结构域（如CD63、TSPAN2）进行基因融合，可实现APEX2在EV表面的展示。在添加底物生物素酚（biotin-phenol）与过氧化氢后，囊泡表面的APEX2可在体外对EV整合蛋白、蛋白冠以及靶细胞进行生物素标记。进一步对EV表面组进行数据挖掘，可获得用于EV生物工程的潜在支架。综上，本研究开发了一种可用于EV表面组与靶细胞定位的可靠工具，并发现了可用于生物工程的新型EV分选结构域。