A novel amphipathic cell-penetrating peptide based on the N-terminal glycosaminoglycan binding region of human apolipoprotein E

https://doi.org/10.1016/j.bbamem.2018.12.010Get rights and content
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Highlights

  • An amphipathic cell-penetrating peptide, A2-17, is newly designed.

  • A2-17 binds to both heparin and lipid membranes with high affinity.

  • A2-17 forms amphipathic α-helix upon binding to heparin and lipid membranes.

  • A2-17 efficiently penetrates cell membranes even at low peptide concentrations.

Abstract

In the direct cell membrane penetration, arginine-rich cell-penetrating peptides are thought to penetrate into cells across the hydrophobic lipid membranes. To investigate the effect of the amphipathic property of arginine-rich peptide on the cell-penetrating ability, we designed a novel amphipathic cell-penetrating peptide, A2-17, and its derivative, A2-17KR, in which all lysine residues are substituted with arginine residues, based on the glycosaminoglycan binding region in the N-terminal α-helix bundle of human apolipoprotein E. Isothermal titration calorimetry showed that A2-17 variants have a strong ability to bind to heparin with high affinity. Circular dichroism and tryptophan fluorescence measurements demonstrated that A2-17 variants bind to lipid vesicles with a structural change from random coil to amphipathic α-helix, being inserted into the hydrophobic membrane interiors. Flow cytometric analysis and confocal laser scanning microscopy demonstrated the great cell penetration efficiency of A2-17 variants into CHO-K1 cells when incubated at low peptide concentrations (2 μM or less), suggesting that the increased amphipathicity with α-helix formation enhances the cell membrane penetration ability of arginine-rich peptides. Interestingly, A2-17KR exhibited lower efficiency of cell membrane penetration compared to A2-17 despite of their similar binding affinity to lipid membranes. Since high peptide concentrations (typically >10 μM) are usually prerequisite for efficient cell penetration of arginine-rich peptides, A2-17 is a unique amphipathic cell-penetrating peptide that exhibits an efficient cell penetration ability even at low peptide concentrations.

Abbreviations

CD
circular dichroism
CHO
Chinese hamster ovary
CLSM
confocal laser scanning microscopy
EPC
egg phosphatidylcholine
EPG
egg phosphatidylglycerol
FAM
5(6)-carboxyfluorescein
FBS
fetal bovine serum
GAG
glycosaminoglycan
GUV
giant unilamellar vesicle
ITC
isothermal titration calorimetry
PBS
phosphate-buffered saline
SUV
small unilamellar vesicle

Keywords

Arginine-rich peptide
Amphipathicity
Glycosaminoglycan
Lipid membrane
Cell membrane penetration

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Both authors contributed equally to this work.