Publication Details (including relevant citation information):
Yuki Takechi-Haraya, Kenzo Aki, Yumi Tohyama, Yuichi Harano, Toru Kawakami, Hiroyuki Saito, Emiko Okamura. Pharmaceuticals 2017, 10, 42; doi:10.3390/ph10020042
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 unsolved. 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 N terminus of R8, the non-endocytic membrane translocation of 19F-labeled R8 into a human myeloid leukemia cell line was observed at 4 °C with a time resolution of minute-order. 19F 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 to quantify 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.
Address (URL): http://www.mdpi.com/1424-8247/10/2/42/htm