Heparin-Promoted Cellular Uptake of the Cell-Penetrating Glycosaminoglycan Binding Peptide, GBP_ECP, Depends on a Single Tryptophan

Abstract

A 10-residue, glycosaminoglycan-binding peptide, GBP, 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 GBP solution. 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. GBP bound 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. GBP has been proven to enter cells through macropinocytosis. The GBP treatment inhibited A549 lung cancer cell migration and invasion, implying that the cellular microenvironment would be modulated by GBP internalization. The intracellular penetration of GBP leading 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, GBP shows substantial potential as a novel delivery therapeutic through rapid and effective internalization and interference with cell mobility.

Cell Penetrating Peptide Derived from Human Eosinophil Cationic Protein Decreases Airway Allergic Inflammation

“…In our previous studies we have clearly demonstrated the HSPG binding and internalization of CPPecp in BEAS-2B cell 7 , 8 , 10 ; and the in vivo data showed venous injected CPPecp deposited in respiratory epithelia and intestinal villi 8 . It should be noted that our CPPecp possessing unique features in HS binding and membrane interaction among peptides of comparable sequences have been clearly demonstrated by nuclear magnetic resonance study 18 , together with in vitro and in vivo data in hand strongly supporting novel cytokine modulation activity of this multi-functional peptide.…”

“…Among various GAGs HS has been identified in all animal tissues. As a consequence of binding to a receptor, HS can recruit GAG-binding natural receptor ligands, for example, IL-4 13 , IL-5 14 , IL-6 15 , 16 and IL-13 17 to promote downstream bioactivities 18 .…”

Increasing C-Terminal Hydrophobicity Improves the Cell Permeability and Antiproliferative Activity of PACE4 Inhibitors against Prostate Cancer Cell Lines

“…Hydrophobic residues, when present in a CPP, increase the interactions with lipid bilayer and thus increasing translocation of peptides through membrane . Among hydrophobic residues, inclusion of Trp within basic peptides is a molecular determinant for enhancement of cell uptake efficiency. − For instance, more abundancy of Trp residues in amphiphilic helical CPPs increased their uptake in A549 cell lines . The exclusive role of tryptophan in cell penetration of polyarginine peptides is ascribed to the interaction with sulfated glycosaminoglycans possibly through hydrophobic and π–anion interactions in addition to its important hydrophobic interactions with lipid bilayer. , …”

Heparan sulfate targeting strategy for enhancing liposomal drug accumulation and facilitating deep distribution in tumors

“…Unlike other CPPs, GBP recognizes heparin by 2 charged residues, Arg 5 and Lys 7 , and inserts the membrane by an irreplaceable moiety of an aromatic residue, Trp 4 . Both of these residues coordinately interact with HS and the lipid membrane on the cell surface, contributing to the induction of epithelial cell uptake by macropinocytosis (Hung et al, 2017). In this study, the Cys 6 residue of GBP was utilized to covalently conjugate with lipids to insert into liposomes.…”

“…In this study, the Cys 6 residue of GBP was utilized to covalently conjugate with lipids to insert into liposomes. Cys 6 is not involved in HS and membrane binding properties, and replacement with serine resulted in over 70% cell-penetrating and cargo delivery activities in vitro (Fang et al, 2013;Hung et al, 2017). Consumption of the Cys 6 residue efficiently avoids GBP dimerization.…”