The lipid ‐binding D4 domain of perfringolysin O facilitates the active loading of exogenous cargo into extracellular vesicles

This study investigates the role of domain 4 (D4), a cholesterol-binding domain as a viable candidate for extracellular vesicle (EV) protein loading and cargo delivery. D4 recognizes cholesterol in the EV membrane and recruits proteins of interest into EVs. Our experiments using split-nano luciferase technology enhanced by fusogenic vesicular stomatitis virus glycoprotein also demonstrate the capability of D4-engineered EVs for intracellular protein delivery. Whereas extracellular vesicles (EVs) have been engineered for cargo loading, innovative strategies for it can still be developed. Here, we describe domain 4 (D4), a cholesterol-binding domain derived from perfringolysin O, as a viable candidate for EV cargo loading. D4 and its mutants localized to the plasma membrane and the membranes of different vesicular structures in the cytoplasm, and facilitate the transport of proteins of interest (POIs) into EVs. D4-EVs were internalized by recipient cells analogous to EVs engineered with CD9. Intracellular cargo discharge from D4-EVs was successfully detected with the assistance of vesicular stomatitis virus glycoprotein. This study presents a novel strategy for recruiting POIs into EVsvia a lipid-binding domain that ensures content release in recipient cells.
Source: FEBS Letters - Category: Biochemistry Authors: Tags: Research Letter Source Type: research