Related Experiment Video
Updated: Jun 21, 2025
Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers
Published on: September 19, 2022
Cell-penetrating activity of a short-chain ε-poly-l-α-lysine
Kohei Kaneda1, Yamato Takeuchi1, Kazuya Yamanaka2
1Graduate School of Bioscience and Biotechnology, Fukui Prefectural University, Eiheiji-cho, Fukui 910-1195, Japan.
Abstract:
Bacteria produce polycationic homopoly(amino acid)s, which are characterized by isopeptide backbones. We previously demonstrated that two representative bacterial polycationic isopeptides, ε-poly-l-α-lysine consisting of 25-35 l-α-lysine residues (ε-PαL25-35) and ε-poly-l-β-lysine consisting of l-β-lysine residues (ε-PβL4-13), were internalized into mammalian cells by both energy-independent direct penetration and energy-dependent endocytosis/macropinocytosis, and then diffused throughout the cytosol. In this study, we investigated the cell-penetrating activity of an ε-PαL short-chain derivative consisting of 5-14 l-α-lysine residues (ε-PαL5-14) to gain insight into the relationship between the isopeptide-chain length and the manner of cellular internalization. We prepared a conjugate of ε-PαL5-14 and a fluorescent dye (FAM) by click chemistry, and incubated the resulting polymer, ε-PαL5-14-FAM, with HeLa cells. Unlike ε-PαL25-35-FAM, ε-PαL5-14-FAM was internalized into cells only by energy-dependent endocytosis/macropinocytosis. Furthermore, a high concentration (>50 μM) was required for the internalization events. ε-PαL5-14 has a chain length almost equal to that of the membrane permeable ε-PβL4-13, which can enter cells at low concentrations. Considering that the basicity of the β-amino group is higher than that of α-amino acid at physiological pH, ε-PβL is expected to have a greater cell-penetrating capacity than ε-PαL, provided their isopeptide-chain lengths are similar, suggesting that a more extended chain derivative of ε-PβL would be more advantageous for cellular internalization of cargo proteins than ε-PαL25-35.
Insights
Short-chain poly(l-α-lysine) (ε-PαL₅-₁₄) enters mammalian cells via endocytosis, unlike longer chains. This bacterial peptide requires high concentrations for cellular uptake, highlighting chain length
Area of Science:
- Biochemistry
- Cell Biology
- Polymer Science
Background:
- Bacteria synthesize polycationic homopoly(amino acid)s with isopeptide backbones.
- Previous studies showed ε-poly-l-α-lysine (ε-PαL₂₅-₃₅) and ε-poly-l-β-lysine (ε-PβL₄-₁₃) enter mammalian cells via direct penetration and endocytosis.
- Understanding the relationship between isopeptide chain length and cellular internalization mechanisms is crucial.
Purpose of the Study:
- To investigate the cell-penetrating activity of a short-chain ε-poly-l-α-lysine derivative (ε-PαL₅-₁₄).
- To elucidate the impact of isopeptide chain length on cellular internalization pathways.
- To compare the uptake mechanisms of ε-PαL₅-₁₄ with longer chain variants and ε-PβL.
Main Methods:
- Synthesis of a fluorescently labeled ε-PαL₅-₁₄ conjugate (ε-PαL₅-₁₄-FAM) using click chemistry.
- Incubation of ε-PαL₅-₁₄-FAM with HeLa cells to observe cellular internalization.
- Analysis of uptake mechanisms, distinguishing between energy-dependent and independent pathways.
Main Results:
- Unlike longer ε-PαL₂₅-₃₅, ε-PαL₅-₁₄-FAM was internalized solely through energy-dependent endocytosis/macropinocytosis.
- Significant concentrations (>50 μM) of ε-PαL₅-₁₄ were required for cellular entry.
- ε-PβL, despite similar chain length to ε-PαL₅-₁₄, exhibits greater cell penetration capacity due to higher β-amino group basicity.
Conclusions:
- Short-chain ε-PαL derivatives exhibit distinct, energy-dependent cellular internalization mechanisms compared to longer chains.
- Chain length and amino acid type (α- vs. β-lysine) significantly influence the cell-penetrating efficiency of bacterial polycationic isopeptides.
- ε-PβL derivatives may offer advantages over ε-PαL for cargo protein delivery due to superior cellular uptake.
More Related Videos
07:33Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
Published on: December 19, 2020
12:02An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity
Published on: November 2, 2016
Related Concept Videos
01:25Selectins
01:31Lysosomes