[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-140] Alkyl chain length-dependent membrane anchoring of artificial lipidated proteins controls extracellular vesicle uptake

○Nozomu Ogushi¹, Kazuki Uchida¹, Yoshirou Kawaguchi¹, Rie Wakabayashi¹, Masahiro Goto^1,2, Noriho Kamiya^1,2 (1. Department of Applied Chemistry, Graduate School of Engineering, Kyushu University (Japan), 2. Division of Biotechnology, Center for Future Chemistry, Kyushu University (Japan))

Keywords:

Extracellular vesicles,microbial transglutaminase,site-specific lipidation,lipid–protein conjugates,cellular uptake

[Purpose]
Extracellular vesicles (EVs) are promising drug delivery carriers; however, systematic and minimally invasive strategies for the display of functional proteins on EV membranes remain limited. In this study, we aimed to establish a new design principle for EV surface engineering by employing a site-specific artificial lipidation strategy based on microbial transglutaminase (MTG)-mediated formation of an ε-(γ-glutamyl)lysine isopeptide bond, enabling the preparation of artificial lipidated proteins with precisely controlled lipid chain lengths and the evaluation of their anchoring to native EV membranes.
[Method]
Artificial lipidated proteins, including muGFP-Cn and Z_HER2:342-muGFP-Cn (Z-muGFP-Cn), a human epidermal growth factor receptor 2 (HER2)-binding affibody genetically fused to muGFP, were prepared by microbial transglutaminase (MTG)-mediated site-specific conjugation of lipid peptides with different alkyl chain lengths (C14, C16, and C18) to LLQG-tagged proteins, yielding single-site mono-lipidated proteins. The anchoring efficiency of lipidated proteins to EV membranes was quantitatively evaluated by nano-flow cytometry (NanoFCM) after simple incubation of muGFP-Cn with EVs derived from Expi293 cells. Furthermore, cellular uptake of Z-muGFP-Cn-modified EVs was assessed by flow cytometry using HER2-positive SK-BR-3 cells and HER2-negative MDA-MB-231 cells to examine receptor-dependent internalization.
[Results]
Quantitative NanoFCM analysis revealed that anchoring of lipidated proteins to EV membranes strongly depended on lipid chain length, with longer alkyl chains resulting in higher fractions of protein-modified EVs. muGFP-C14 showed limited membrane association, whereas C16 and C18 variants exhibited efficient and stable EV anchoring. Furthermore, EVs modified with HER2-targeting Z-muGFP-Cn displayed enhanced cellular uptake in HER2-positive SK-BR-3 cells, while uptake was markedly suppressed in HER2-negative MDA-MB-231 cells. This receptor-dependent uptake behavior was most pronounced for EVs modified with longer-chain lipidated proteins.
[Consideration]
NanoFCM results indicate that alkyl chain length-dependent anchoring of muGFP-Cn is driven by enhanced hydrophobic interactions with the EV membrane, resulting in a higher fraction of muGFP-modified EVs. Consistent with this anchoring behavior, EV uptake was selectively enhanced for C16- and C18-lipidated variants in HER2-positive cells, reflecting improved ligand presentation and avidity-driven internalization. In contrast, in HER2-negative cells, EVs displaying lipidated proteins exhibited reduced uptake, suggesting masking of native nonspecific interaction sites in the absence of receptor-mediated binding.
[Conclusion]
In conclusion, we established a minimally invasive and tunable strategy for EV surface engineering based on artificial protein lipidation. Alkyl chain length was identified as a key design parameter governing EV membrane anchoring and cell-selective uptake behavior.