Journal
PHARMACEUTICALS
Volume 10, Issue 2, Pages -Publisher
MDPI
DOI: 10.3390/ph10020042
Keywords
glycosaminoglycan; heparin; cell penetrating peptide; octaarginine; non-endocytic membrane translocation; in-cell nuclear magnetic resonance spectroscopy
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Funding
- Japan Society for the Promotion of Science [24550035, 15K05401]
- Hyogo Science and Technology Association [25073]
- Grants-in-Aid for Scientific Research [24550035, 15K05401, 17H03979, 15K05254] Funding Source: KAKEN
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Glycosaminoglycans (GAGs), which are covalently-linked membrane proteins at the cell surface have recently been suggested to involve in not only endocytic cellular uptake but also non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides (CPPs). However, in-situ comprehensive observation and the quantitative analysis of the direct membrane translocation processes are challenging, and the mechanism therefore remains still unresolved. In this work, real-time in-cell NMR spectroscopy was applied to investigate the direct membrane translocation of octaarginine (R8) into living cells. By introducing 4-trifluoromethyl-L-phenylalanine to the N terminus of R8, the non-endocytic membrane translocation of F-19-labeled R8 (F-19-R8) into a human myeloid leukemia cell line was observed at 4 degrees C with a time resolution in the order of minutes. F-19 NMR successfully detected real-time R8 translocation: the binding to anionic GAGs at the cell surface, followed by the penetration into the cell membrane, and the entry into cytosol across the membrane. The NMR concentration analysis enabled quantification of how much of R8 was staying in the respective translocation processes with time in situ. Taken together, our in-cell NMR results provide the physicochemical rationale for spontaneous penetration of CPPs in cell membranes.
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