title: Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBPECP, Depends on a Single Tryptophan.
abstract: A 10-residue, glycosaminoglycan-binding peptide, GBPECP, derived from human eosinophil cationic protein has been recently designated as a potent cell-penetrating peptide. A model system containing peptide, glycan, and lipid was monitored by nuclear magnetic resonance (NMR) spectroscopy to determine the cell-penetrating mechanism. Heparin octasaccharide with dodecylphosphocholine (DPC) lipid micelle was titrated into the GBPECPsolution. Our data revealed substantial roles for the charged residues Arg5 and Lys7 in recognizing heparin, whereas Arg3 had less effect. The aromatic residue Trp4 acted as an irreplaceable moiety for membrane insertion, as the replacement of Trp4 with Arg4 abolished cell penetration, although it significantly improved the heparin-binding ability. GBPECPbound either heparin or lipid in the presence or absence of the other ligand indicating that the peptide has two alternative binding sites: Trp4 is responsible for lipid insertion, and Arg5 and Lys7 are for GAG binding. We developed a molecular model showing that the two effects synergistically promote the penetration. The loss of either effect would abolish the penetration. GBPECPhas been proven to enter cells through macropinocytosis. The GBPECPtreatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBPECPinternalization. The intracellular penetration of GBPECPleading to inhibition of epithelial cell migration and invasion depends on the presence of the tryptophan residue in its sequence compared with similar derivative peptides. Therefore, GBPECPshows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Li-Chun
authors.initials: LC
authors.lastname: Hung
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Ingjye
authors.initials: I
authors.lastname: Jiang
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Chien-Jung
authors.initials: CJ
authors.lastname: Chen
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Jia-Yin
authors.initials: JY
authors.lastname: Lu
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Yi-Fen
authors.initials: YF
authors.lastname: Hsieh
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Ping-Hsieh
authors.initials: PH
authors.lastname: Kuo
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.|Instrumentation Center, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Yi-Lin
authors.initials: YL
authors.lastname: Hung
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.|Department of Medical Science, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Lily Hui-Ching
authors.initials: LH
authors.lastname: Wang
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.|Department of Life Science, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
authors.affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University , Hsinchu 30013, Taiwan.|Department of Life Science, National Tsing Hua University , Hsinchu 30013, Taiwan.
authors.forename: Shih-Che
authors.identifier: 0000-0002-2169-0157
authors.initials: SC
authors.lastname: Sue
mesh_terms: D000818:Animals; D066241:Carbon-13 Magnetic Resonance Spectroscopy; D045744:Cell Line, Tumor; D057846:Cell-Penetrating Peptides / Q000378:metabolism*; D006025:Glycosaminoglycans / Q000378:metabolism*; D006493:Heparin / Q000378:metabolism*; D006801:Humans; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D066244:Proton Magnetic Resonance Spectroscopy; D014364:Tryptophan / Q000378:metabolism*
chemical_list: D057846:Cell-Penetrating Peptides; D006025:Glycosaminoglycans; D014364:Tryptophan; D006493:Heparin