Investigation of Intercellular Trafficking of Global Glycosylphosphatidylinositol‐Anchored Proteins Using Cell Metabolic Engineering and Transwell Coculture

Glycosylphosphatidylinositols (GPIs) are essential for GPI-anchored proteins (GPI-APs), as the loose association of GPIs with the cell membrane is thought to facilitate GPI-AP shedding and intercellular trafficking, which dictates cell communication. To explore this property, a novel method was developed based on metabolic engineering of GPI-APs in live cells and coculture of labelled donor cells along with recipient cells in a Transwell system for straight observation of intercellular GPI-AP transfer. Studies using this method have provided direct proof of intercellular GPI-AP trafficking and its dependence on cell types. Notably, there is a preference for GPI-AP transfer from cancer to normal cells. Lipid raft-mediated endocytosis and macropinocytosis play a critical role in GPI-AP trafficking and incorporation, with extracellular vesicles (EVs) involved as GPI-AP transporters. It is further revealed that depleting cholesterol in the donor cell can enhance GPI-AP shedding and trafficking. The method also allowed for in-depth studies to gain insights into the regulatory mechanisms of GPI-AP trafficking, such as its association with phospholipase A2 (PLA2), EV biogenesis, and membrane-adjacent Ras and Rho signaling pathways. The results are important not only for understanding GPI biology but also for its biomedical applications, for example, GPI-based cancer therapy.