In this study, five arginine-rich peptides YGR6, YGR6E6, YG(RE)6, YGR6G6 and YG(RG)6 were engineered for investigating the roles of positive charges and sequence of cationic amino acid residues in their membrane transduction activity. The results demonstrated that the positive charges are crucial factors to achieve high level of cellular uptake. YGR6E6, YG(RE) 6, YGR6G6 and YG(RG)6 showed less cellular uptake compared with YGR6. Especially, in the neutralized form, the surface-binding and internalization of YGR6E6 and YG(RE)6 dramatically decreased. However, YGR6G 6 and YG(RG)6 still showed comparable uptake profile in CHO cells as compared to YGR6. A comparison between YGR6E 6 and YG(RE)6 or YGR6G6 and YG(RG) 6 indicated that the clustering of positive charges was important for initiating the membrane transduction activity of arginine-containing peptides. Cellular uptake assay was also conducted to investigate the ratio of endocytosis versus membrane transduction. Results from this study indicated that all these peptides were mainly internalized by membrane transduction rather than endocytosis.
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