Glycosaminoglycan Binding and Non-Endocytic Membrane Translocation of Cell-Permeable Octaarginine Monitored by Real-Time In-Cell NMR Spectroscopy

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.

Understanding Cell Penetration of Cyclic Peptides

“…195 Takechi-Haraya et al employed real-time in-cell NMR spectroscopy and R 8 N-terminally modified with 19 F-4trifluoromethyl-L-phenylalanine ( 19 F-R 8 ) as a probe to monitor direct translocation into HL60 myeloid leukemia cells at 4 °C. 196 Rapid nonendocytic internalization was observed at 80−100 μM concentrations, which proceeded through initial association with cell-surface GAGs, followed by permeation through the membrane into the cytosol. Time-dependent measurements indicated that extracellular R 8 rapidly reached an equilibrium concentration in ∼5 min, with a transient increase in GAG-bound peptide that then rapidly diminished and gradual increases in membrane and cytosolic peptide until an equilibrium was reach in ∼10 min.…”

Cell Membrane Penetration without Pore Formation: Chameleonic Properties of Dendrimers in Response to Hydrophobic and Hydrophilic Environments

“…The energetic cost to transfer positively charged Arg from water to the interior of a membrane suggests that this process should be highly unfavourable, in striking contrast with the experimental evidence that CPPs cross the membrane quite easily on a timescale of minutes 68 . Our model reconciles a non-disruptive penetration mechanism with the apparently modest translocation energy barrier inferred from experiment, suggesting that once the peptide is deprotonated the hydrophobic barrier is much lower than previously thought.…”

Glycosaminoglycans and Proteoglycans

“…GAGs interact with proteins in many biological systems, and as a consequence they have numerous biological and therapeutic functions [ 15 , 16 , 17 , 18 , 19 , 20 , 21 ]. In fact, GAGs can be considered the most exploited carbohydrates in the pharmaceutical market [ 14 ].…”

“…GAGs are also involved in the cell uptake of potentially useful drug-delivery systems. Takechi-Haraya and associates have in this issue described the contribution of GAGs in the non-endocytic direct cell membrane translocation of arginine-rich cell-penetrating peptides by studying the cell-permeation of octaarginine monitored through real-time in-cell 19 F NMR spectroscopy [ 21 ].…”

“…This special issue on “ Glycosaminoglycans and Proteoglycans ” contains a diverse collection of articles, illustrating the range of biological systems in which GAGs and PGs operate and can be considered in the design of pharmaceutical interventions. They range from the analysis of GAG pharmaceutical products [ 32 ] and Hp neutralization [ 26 ] through Hp mimetics [ 27 ], GAG-mediated uptake of cell penetrating peptides [ 21 ], to the development of PG mimics for endothelial repair [ 20 ]. Review articles cast a new light on regenerative medicine [ 18 ], chemokine-GAG interactions [ 16 ], the DS-PG related human genetic disorders, and Flavivirus interactions with host cells [ 17 ].…”