title: Single chain variable fragment against nicastrin inhibits the gamma-secretase activity.
abstract: Gamma-secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid beta precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the gamma-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the gamma-secretase activity. Biochemical analyses revealed that the scFv disrupted the proper folding and the appropriate glycosyl maturation of the endogenous NCT, which are required for the stability of the gamma-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the gamma-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the gamma-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins.
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Ikuo
authors.initials: I
authors.lastname: Hayashi
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Sho
authors.initials: S
authors.lastname: Takatori
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Yasuomi
authors.initials: Y
authors.lastname: Urano
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan; Perseus Proteomics, Inc., 4-7-6 Komaba, Meguro-ku, Tokyo 153-0041, Japan.
authors.forename: Hiroko
authors.initials: H
authors.lastname: Iwanari
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Noriko
authors.initials: N
authors.lastname: Isoo
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Satoko
authors.initials: S
authors.lastname: Osawa
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Maiko A
authors.initials: MA
authors.lastname: Fukuda
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Tatsuhiko
authors.initials: T
authors.lastname: Kodama
authors.affiliation: Laboratory for Systems Biology and Medicine, Research Center for Advanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan.
authors.forename: Takao
authors.initials: T
authors.lastname: Hamakubo
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Tong
authors.initials: T
authors.lastname: Li
authors.affiliation: Department of Pathology, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: Philip C
authors.initials: PC
authors.lastname: Wong
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Electronic address: taisuke@mol.f.u-tokyo.ac.jp.
authors.forename: Taisuke
authors.initials: T
authors.lastname: Tomita
authors.affiliation: Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan; Department of Neuropathology, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Iwatsubo
mesh_terms: D000595:Amino Acid Sequence; D053829:Amyloid Precursor Protein Secretases / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000276:immunology*; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000378:metabolism; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000378:metabolism*; D007212:Indolizines / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000737:chemistry* / Q000235:genetics / Q000276:immunology*; D008969:Molecular Sequence Data; D011485:Protein Binding; D011487:Protein Conformation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D054836:Indolizidines
chemical_list: D053829:Amyloid Precursor Protein Secretases; D000906:Antibodies; D004791:Enzyme Inhibitors; D007135:Immunoglobulin Variable Region; D007212:Indolizines; D008562:Membrane Glycoproteins; C413604:nicastrin protein; C037806:castanospermine; D054836:Indolizidines
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