Abstract
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 19F-labeled R8 (19F-R8) into a human myeloid leukemia cell line was observed at 4 °C with a time resolution in the order of minutes. 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 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.
Highlights
Drug delivery using cell-penetrating peptides (CPPs) is one of the most powerful strategies to resolve the poor cell membrane permeability of new bioactive molecules such as oligonucleotides, plasmids, peptides and proteins for therapeutic pharmaceuticals [1]
The direct membrane translocation is an alternative to endocytosis in order to avoid the lysosomal degradation, how cationic CPPs traverse hydrophobic cell membranes is still controversial [10]
CPPs bind to and translocate into GAG-deficient cells and enzymatically GAG-removed cells [12,13,18,19], we have recently reported that the efficiency of the direct membrane translocation of arginine-rich CPPs is correlated with the favorable enthalpy of binding to heparin, of which the binding could be derived from formation of multidentate hydrogen bonding of the arginine residue with sulfate group of heparin [20]
Summary
Drug delivery using cell-penetrating peptides (CPPs) is one of the most powerful strategies to resolve the poor cell membrane permeability of new bioactive molecules such as oligonucleotides, plasmids, peptides and proteins for therapeutic pharmaceuticals [1]. The second is that the 19F NMR signal moves to a high magnetic field where 19F-R8 interacts with cell membrane, in contrast to the electrostatic 19F-R8 binding to GAG. The amount of 19F-R8 bound to GAG, at first, increased but Phdaermcraeceaustiecdalst2o01l7e,s1s0,t4h2an 5 μM within a short period of 4–6 min This quick decrease is thought otof 1b8e due to the transfer of 19F-R8 to the membrane from the cell surface. The value is consistent with the result of the real-time in-cell NMR measurement showing about 65 μM (81%) of 19F-R8 is remaining in a free state after the equilibrium is attained (Figure 6). Undesirable loss of peptide may be induced by the extensive solubilization and centrifugation for cell fractionation, almost no appearance of the membrane Pfrharcmtiaocenutiicsalsp2r0o1b7a, 1b0l,y42due to the signal broadening, as seen in spectra of 19F-R8 bound to GA10Gof o18r EPC/EPG LUV (Figure 3a,b). The present study shows a potential for elucidating direct membrane translocation mechanism of CPPs with minimal perturbation
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