title: Interaction of amphiphilic α-helical cell-penetrating peptides with heparan sulfate.
abstract: Cell-penetrating peptides (CPPs) are able to be taken up by cells and can deliver macromolecular cargos. However, the mechanism of this internalization is not yet fully understood. Recent theories suggest that the binding of cationic CPPs to negatively charged extracellular glycosaminoglycans, such as heparan sulfate (HS), is a possible mechanism of cellular uptake (CU). Our group has screened the CU activities of 54 systematically designed amphiphilic α-helical peptides in HeLa cells. Notably, a mutation in even a single residue significantly alters the CU ability of a peptide. To determine the structure-CU activity relationship of CPPs, four peptides, which contain a difference in one or two amino acids (i.e., Arg/Glu and Ala/Phe), were chosen from our CPP library to examine their interactions with HS. Fluorescence spectroscopy, isothermal titration calorimetry (ITC) and dynamic light scattering analysis indicated that the HS-binding affinities and HS-clustering abilities of the four CPPs correlated well with their CU activities in HeLa and A549 cells. The heat capacities of the CPPs, determined using ITC and binding free energy decomposition analyses in molecular dynamics simulations, revealed that electrostatic interactions were more dominant in the HS-binding processes of Arg-containing peptides in comparison to Glu-containing peptides, whereas hydrophobic contributions were the primary mode of interaction of Phe-containing peptides in comparison to Ala-containing peptides. Furthermore, it was implied that hydrophobic interactions may be more favourable than electrostatic interactions during the CU process.
authors.affiliation: Department of Bioengineering, Graduate School of Bioscience and Biotechnology, Tokyo Institute of Technology, Nagatsuta-cho 4259 B-40, Midori-ku, Yokohama 226-8501, Japan. hmihara@bio.titech.ac.jp.
authors.forename: Ji
authors.initials: J
authors.lastname: Yang
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Tsutsumi
authors.forename: Tadaomi
authors.initials: T
authors.lastname: Furuta
authors.forename: Minoru
authors.initials: M
authors.lastname: Sakurai
authors.forename: Hisakazu
authors.initials: H
authors.lastname: Mihara
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D002151:Calorimetry; D057846:Cell-Penetrating Peptides / Q000737:chemistry* / Q000378:metabolism*; D002942:Circular Dichroism; D006367:HeLa Cells; D006497:Heparan Sulfate / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D016018:Least-Squares Analysis; D008027:Light; D056004:Molecular Dynamics Simulation; D008969:Molecular Sequence Data; D017711:Nonlinear Dynamics; D017433:Protein Structure, Secondary; D012542:Scattering, Radiation; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013501:Surface-Active Agents / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D000596:Amino Acids; D057846:Cell-Penetrating Peptides; D006497:Heparan Sulfate; D013501:Surface-Active Agents