title: Syndecan-4 Is a Receptor for Clathrin-Mediated Endocytosis of Arginine-Rich Cell-Penetrating Peptides.
abstract: Arginine-rich cell-penetrating peptides (CPPs) such as Tat and oligoarginine peptides have been widely used as carriers for intracellular delivery of bioactive molecules. Despite accumulating evidence for involvement of endocytosis in the cellular uptake of arginine-rich CPPs, the primary cell-surface receptors for these peptide carriers that would initiate endocytic processes leading to intracellular delivery of bioactive cargoes have remained poorly understood. Our previous attempt to identify membrane receptors for octa-arginine (R8) peptide, one of the representative arginine-rich CPPs, using the photo-cross-linking probe bearing a photoreactive diazirine was not successful due to considerable amounts of cellular proteins nonspecifically bound to the affinity beads. To address this issue, here we developed a photo-cross-linking probe in which a cleavable linker of a diazobenzene moiety was employed to allow selective elution of cross-linked proteins by reducing agent-mediated cleavage. We demonstrated that introduction of the diazobenzene moiety into the photoaffinity probe enables efficient purification of cross-linked proteins with significant reduction of nonspecific binding proteins, leading to successful identification of 17 membrane-associated proteins that would interact with R8 peptide. RNAi-mediated knockdown experiments in combination with the pharmacological inhibitors revealed that, among the proteins identified, syndecan-4, one of the heparan sulfate proteoglycans, is an endogenous membrane-associated receptor for the cellular uptake of R8 peptide via clathrin-mediated endocytosis. This syndecan-4-dependent pathway was also involved in the intracellular delivery of bioactive proteins mediated by R8 peptide. These results reveal that syndecan-4 is a primary cell-surface target for R8 peptide that allows intracellular delivery of bioactive cargo molecules via clathrin-mediated endocytosis.
authors.affiliation: Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.
authors.forename: Yoshimasa
authors.initials: Y
authors.lastname: Kawaguchi
authors.affiliation: Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.
authors.forename: Toshihide
authors.initials: T
authors.lastname: Takeuchi
authors.affiliation: Institute of Transformative Bio-Molecules (WPI-ITbM), Nagoya University , Furo-cho, Chikusa-ku, Nagoya 464-8602, Japan.
authors.forename: Keiko
authors.initials: K
authors.lastname: Kuwata
authors.affiliation: Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama , Sugitani, Toyama 930-0194, Japan.
authors.forename: Junya
authors.initials: J
authors.lastname: Chiba
authors.affiliation: University Office, University of Toyama , Gofuku 3190, Toyama 930-8555, Japan.
authors.forename: Yasumaru
authors.initials: Y
authors.lastname: Hatanaka
authors.affiliation: Nanoscience and Nanotechnology Research Center, Research Organization for the 21st Century, Osaka Prefecture University , Naka-ku, Sakai, Osaka 599-8570, Japan.
authors.forename: Ikuhiko
authors.initials: I
authors.lastname: Nakase
authors.affiliation: Institute for Chemical Research, Kyoto University , Uji, Kyoto 611-0011, Japan.
authors.forename: Shiroh
authors.initials: S
authors.lastname: Futaki
mesh_terms: D001120:Arginine / Q000378:metabolism*; D004705:Endocytosis / Q000502:physiology*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism; D053671:Syndecan-4 / Q000378:metabolism* / Q000502:physiology
chemical_list: D008565:Membrane Proteins; D053671:Syndecan-4; D001120:Arginine