title: A novel cell-penetrating peptide derived from human eosinophil cationic protein.
abstract: Cell-penetrating peptides (CPPs) are short peptides which can carry various types of molecules into cells; however, although most CPPs rapidly penetrate cells in vitro, their in vivo tissue-targeting specificities are low. Herein, we describe cell-binding, internalization, and targeting characteristics of a newly identified 10-residue CPP, denoted ECP(32-41), derived from the core heparin-binding motif of human eosinophil cationic protein (ECP). Besides traditional emphasis on positively charged residues, the presence of cysteine and tryptophan residues was demonstrated to be essential for internalization. ECP(32-41) entered Beas-2B and wild-type CHO-K1 cells, but not CHO cells lacking of cell-surface glycosaminoglycans (GAGs), indicating that binding of ECP(32-41) to cell-surface GAGs was required for internalization. When cells were cultured with GAGs or pre-treated with GAG-digesting enzymes, significant decreases in ECP(32-41) internalization were observed, suggesting that cell-surface GAGs, especially heparan sulfate proteoglycans were necessary for ECP(32-41) attachment and penetration. Furthermore, treatment with pharmacological agents identified two forms of energy-dependent endocytosis, lipid-raft endocytosis and macropinocytosis, as the major ECP(32-41) internalization routes. ECP(32-41) was demonstrated to transport various cargoes including fluorescent chemical, fluorescent protein, and peptidomimetic drug into cultured Beas-2B cells in vitro, and targeted broncho-epithelial and intestinal villi tissues in vivo. Hence this CPP has the potential to serve as a novel vehicle for intracellular delivery of biomolecules or medicines, especially for the treatment of pulmonary or gastrointestinal diseases.
authors.affiliation: Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan, Republic of China.
authors.forename: Shun-lung
authors.initials: SL
authors.lastname: Fang
authors.forename: Tan-chi
authors.initials: TC
authors.lastname: Fan
authors.forename: Hua-Wen
authors.initials: HW
authors.lastname: Fu
authors.forename: Chien-Jung
authors.initials: CJ
authors.lastname: Chen
authors.forename: Chi-Shin
authors.initials: CS
authors.lastname: Hwang
authors.forename: Ta-Jen
authors.initials: TJ
authors.lastname: Hung
authors.forename: Lih-Yuan
authors.initials: LY
authors.lastname: Lin
authors.forename: Margaret Dah-Tsyr
authors.initials: MD
authors.lastname: Chang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D001693:Biological Transport, Active; D016466:CHO Cells; D002460:Cell Line; D057846:Cell-Penetrating Peptides / Q000138:chemical synthesis / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D047150:Eosinophil Cationic Protein / Q000737:chemistry*; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism*; D019812:Heparan Sulfate Proteoglycans / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D021962:Membrane Microdomains / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D010873:Pinocytosis; D011485:Protein Binding; D014364:Tryptophan / Q000737:chemistry / Q000378:metabolism
chemical_list: D057846:Cell-Penetrating Peptides; D019812:Heparan Sulfate Proteoglycans; D014364:Tryptophan; D047150:Eosinophil Cationic Protein; D003545:Cysteine