Single Chain Variable Fragment against Nicastrin Inhibits the γ-Secretase Activity
2009; Elsevier BV; Volume: 284; Issue: 41 Linguagem: Inglês
10.1074/jbc.m109.055061
ISSN1083-351X
AutoresIkuo Hayashi, Sho Takatori, Yasuomi Urano, Hiroko Iwanari, Noriko Isoo, Satoko Osawa, Maiko A. Fukuda, Tatsuhiko Kodama, Takao Hamakubo, Tong Li, Philip C. Wong, Taisuke Tomita, Takeshi Iwatsubo,
Tópico(s)Protein Structure and Dynamics
Resumoγ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-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 γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins. γ-Secretase is a membrane protein complex that catalyzes intramembrane proteolysis of a variety of substrates including the amyloid β precursor protein of Alzheimer disease. Nicastrin (NCT), a single-pass membrane glycoprotein that harbors a large extracellular domain, is an essential component of the γ-secretase complex. Here we report that overexpression of a single chain variable fragment (scFv) against NCT as an intrabody suppressed the γ-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 γ-secretase complex and the intrinsic proteolytic activity, respectively, implicating the dual role of NCT in the γ-secretase complex. Our results also highlight the importance of the calnexin cycle in the functional maturation of the γ-secretase complex. The engineered intrabodies may serve as rationally designed, molecular targeting tools for the discovery of novel actions of the membrane proteins. γ-Secretase catalyzes intramembrane proteolysis of a variety of substrates including amyloid β precursor protein (APP) 3The abbreviations used are: APPamyloid-β precursor proteinAβamyloid-β peptideCHAPSO3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonateCNXcalnexinCSTcastanospermineCTFC-terminal fragmentNCTnicastrinPSpresenilinEndo Hendoglycosidase HECDextracellular domainERendoplasmic reticulumscFvsingle chain variable fragment. to generate amyloid β peptide (Aβ), the latter being a major component of senile plaques in the brains of Alzheimer disease patients. Thus, agents that inhibit γ-secretase activity could serve as an effective therapeutics for Alzheimer disease, whereas the γ-secretase activity plays important roles in cell signaling pathways including Notch signaling (1Tomita T. Expert Rev. Neurother. 2009; 9: 661-679Crossref PubMed Scopus (120) Google Scholar, 2Wolfe M.S. Semin. Cell Dev. Biol. 2009; 20: 219-224Crossref PubMed Scopus (73) Google Scholar). γ-Secretase consists of at least four integral membrane proteins, i.e. presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, all of which are essential to the proteolytic activity (3Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 4Selkoe D.J. Wolfe M.S. Cell. 2007; 131: 215-221Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar, 5Spasic D. Annaert W. J. Cell Sci. 2008; 121: 413-420Crossref PubMed Scopus (75) Google Scholar). Molecular cellular and chemical biological analyses have revealed that PS forms a hydrophilic pore involving the transmembrane domain 6 and 7, where conserved catalytic aspartates reside to function as catalytic residues of γ-secretase complex (6Sato C. Morohashi Y. Tomita T. Iwatsubo T. J. Neurosci. 2006; 26: 12081-12088Crossref PubMed Scopus (134) Google Scholar, 7Tolia A. Chávez-Gutiérrez L. De Strooper B. J. Biol. Chem. 2006; 281: 27633-27642Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). APH-1 is a multipass membrane protein that plays a role in stabilization and trafficking of the γ-secretase complex (8Niimura M. Isoo N. Takasugi N. Tsuruoka M. Ui-Tei K. Saigo K. Morohashi Y. Tomita T. Iwatsubo T. J. Biol. Chem. 2005; 280: 12967-12975Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), and PEN-2 is a cofactor for the activation and the regulation of the γ-secretase activity (3Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 9Isoo N. Sato C. Miyashita H. Shinohara M. Takasugi N. Morohashi Y. Tsuji S. Tomita T. Iwatsubo T. J. Biol. Chem. 2007; 282: 12388-12396Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). amyloid-β precursor protein amyloid-β peptide 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate calnexin castanospermine C-terminal fragment nicastrin presenilin endoglycosidase H extracellular domain endoplasmic reticulum single chain variable fragment. NCT, which was identified as a PS-binding protein (10Yu G. Nishimura M. Arawaka S. Levitan D. Zhang L. Tandon A. Song Y.Q. Rogaeva E. Chen F. Kawarai T. Supala A. Levesque L. Yu H. Yang D.S. Holmes E. Milman P. Liang Y. Zhang D.M. Xu D.H. Sato C. Rogaev E. Smith M. Janus C. Zhang Y. Aebersold R. Farrer L.S. Sorbi S. Bruni A. Fraser P. St George-Hyslop P. Nature. 2000; 407: 48-54Crossref PubMed Scopus (827) Google Scholar), is a single-pass membrane protein that harbors an extracellular domain (ECD) with a number of N-glycosylation sites. In mammalian cells NCT undergoes Endo H-resistant complex glycosylation and acquires trypsin resistance during the assembly process of the γ-secretase complex (11Leem J.Y. Vijayan S. Han P. Cai D. Machura M. Lopes K.O. Veselits M.L. Xu H. Thinakaran G. J. Biol. Chem. 2002; 277: 19236-19240Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar, 12Kaether C. Lammich S. Edbauer D. Ertl M. Rietdorf J. Capell A. Steiner H. Haass C. J. Cell Biol. 2002; 158: 551-561Crossref PubMed Scopus (169) Google Scholar, 13Kimberly W.T. LaVoie M.J. Ostaszewski B.L. Ye W. Wolfe M.S. Selkoe D.J. J. Biol. Chem. 2002; 277: 35113-35117Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 14Tomita T. Katayama R. Takikawa R. Iwatsubo T. FEBS Lett. 2002; 520: 117-121Crossref PubMed Scopus (63) Google Scholar, 15Yang D.S. Tandon A. Chen F. Yu G. Yu H. Arawaka S. Hasegawa H. Duthie M. Schmidt S.D. Ramabhadran T.V. Nixon R.A. Mathews P.M. Gandy S.E. Mount H.T. St George-Hyslop P. Fraser P.E. J. Biol. Chem. 2002; 277: 28135-28142Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar, 16Herreman A. Van Gassen G. Bentahir M. Nyabi O. Craessaerts K. Mueller U. Annaert W. De Strooper B. J. Cell Sci. 2003; 116: 1127-1136Crossref PubMed Scopus (166) Google Scholar, 17Shirotani K. Edbauer D. Capell A. Schmitz J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: 16474-16477Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Molecular and cellular analyses revealed that the trypsin resistance, presumably indicating the proper structural folding of NCT, might be directly linked to the enzymatic activity, whereas the complex glycosylation is dispensable. Moreover, multiple sequence alignment analyses revealed that NCT ECD have a similarity to an aminopeptidase (18Fagan R. Swindells M. Overington J. Weir M. Trends Biochem. Sci. 2001; 26: 213-214Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), whereas certain catalytic residues are not conserved. Recently one study has suggested that NCT plays a critical role in substrate recognition (19Shah S. Lee S.F. Tabuchi K. Hao Y.H. Yu C. LaPlant Q. Ball H. Dann 3rd, C.E. Südhof T. Yu G. Cell. 2005; 122: 435-447Abstract Full Text Full Text PDF PubMed Scopus (388) Google Scholar). During the proteolytic process, NCT ECD captures the most N terminus of the substrate as a primary substrate receptor (i.e. exosite) for the γ-secretase via the aminopeptidase-like domain. However, this view has been recently challenged (20Chávez-Gutiérrez L. Tolia A. Maes E. Li T. Wong P.C. de Strooper B. J. Biol. Chem. 2008; 283: 20096-20105Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). Nevertheless, as structural information of NCT ECD is totally lacking, the functional role of the structural maturation of NCT in the formation and activity of the γ-secretase remains unclear. Molecular engineering of monoclonal antibodies opens a venue for the functional analyses of targeted molecule and the therapeutic intervention for several diseases (21Holliger P. Hudson P.J. Nat. Biotechnol. 2005; 23: 1126-1136Crossref PubMed Scopus (1514) Google Scholar). A single-chain antibody fragment (scFv) is comprised of heavy- and light-chain sequences of an antibody linked by a short linker and preserves binding abilities of its parental antibody. scFv can be expressed intracellularly as an intrabody (22Miller T.W. Messer A. Mol. Ther. 2005; 12: 394-401Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 23Stocks M. Curr. Opin. Chem. Biol. 2005; 9: 359-365Crossref PubMed Scopus (59) Google Scholar), which provides a powerful method for phenotypic knock-out of the genes. Intrabodies have been investigated as treatments for a variety of pathological conditions, including neurodegenerative diseases such as Parkinson disease and Huntington disease. Moreover, several recent publications have highlighted the therapeutic potential of intrabodies targeting intra- as well as extracellular epitopes (24Verbeke K. Gils A. Declerck P.J. J. Thromb. Haemost. 2004; 2: 298-305Crossref PubMed Scopus (11) Google Scholar, 25Arbel M. Yacoby I. Solomon B. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 7718-7723Crossref PubMed Scopus (89) Google Scholar, 26Gal-Tanamy M. Zemel R. Berdichevsky Y. Bachmatov L. Tur-Kaspa R. Benhar I. J. Mol. Biol. 2005; 347: 991-1003Crossref PubMed Scopus (18) Google Scholar, 27Paganetti P. Calanca V. Galli C. Stefani M. Molinari M. J. Cell Biol. 2005; 168: 863-868Crossref PubMed Scopus (98) Google Scholar, 28Farady C.J. Sun J. Darragh M.R. Miller S.M. Craik C.S. J. Mol. Biol. 2007; 369: 1041-1051Crossref PubMed Scopus (46) Google Scholar, 29Mikkelsen J.H. Gyrup C. Kristensen P. Overgaard M.T. Poulsen C.B. Laursen L.S. Oxvig C. J. Biol. Chem. 2008; 283: 16772-16780Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar). Here, we generated scFv against NCT from an anti-NCT monoclonal antibody. Unexpectedly, the overexpression of the anti-NCT scFv as an intrabody abolished the proteolytic activity by the destabilization of the γ-secretase complex and the inappropriate glycosylation of NCT. This is the first example showing that engineered antibody would be a useful tool for the direct modulation of the γ-secretase complex and its activity. C-terminal V5-His-tagged human NCT ECD inserted in pBlueBac4.5 (Invitrogen) was generated from NCT/V5-His in pBlueBac4.5 (30Hayashi I. Urano Y. Fukuda R. Isoo N. Kodama T. Hamakubo T. Tomita T. Iwatsubo T. J. Biol. Chem. 2004; 279: 38040-38046Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) by long PCR. Cytoplasmic RNA was prepared from 1 × 107 hybridoma cells by using Isogen reagent (Nippongene, Tokyo, Japan). The cells were lysed by mixing with Isogen and incubated at room temperature for 5 min. After centrifugation of the lysate, the RNA was precipitated and dissolved in distilled water. This RNA was used as a template for first-strand cDNA synthesis with 3′ primers specific for the mouse IgG genes (Novagen, Darmstadt, Germany). The cDNA fragments were then amplified by PCR with LA Taq (Takara, Shiga, Japan) using 3′ and 5′ primers from the mouse Ig primer set as per the manufacturer's instructions (Ig-Prime kit protocols; Novagen). The PCR products were subcloned into the pEF6/V5-His-TOPO vector (Invitrogen) by the TOPO cloning method. scFv cDNAs inserted into pSecTag2C (Invitrogen) were constructed as follows. The PCR-derived DNA fragments in pEF6/V5-His TOPO were subjected to splice overlapping extension PCR to connect heavy- and light-chain genes to give a single fusion protein gene. In the first round PCR, heavy- and light-chain genes were amplified by using the following primers: the variable region heavy-chain gene, 5′-ggggaattcGAAGTGAAGCTGGTGGAG-3′ (VHF#1) and 5′-caccacctccggaaccaccaccaccggaaccaccacctccGGCTGAGGAGACTGTGAGAGT-3′ (VHR#1); the variable region light-chain genes, 5′- ggtggttccggtggtggtggttccggaggtggtggttcaGACATTGTGCTGACACAGTCT-3′ (VLF#1) and 5′-cccgcggccgcTTTTATTTCCAGCTTGGT-3′ (VLR#1) or 5′-ggtggttccggtggtggtggttccggaggtggtggttcaGATATCCAGATGACACAGACT-3′ (VLF#2) and 5′- cccgcggccgcTTTGATTTCCAGCTTGGT-3′ (VLR#2). In the second round PCR, the amplified heavy- and light-chain fragments were linked by using VHF#1 and VLR#1 or #2. The amplified scFv cDNAs were digested with EcoRI and NotI to subclone into the EcoRI-NotI-digested pSecTag2C vector. Wild-type as well as mutant (i.e. Δ312, 648ATAA) human NCT inserted in the pEF6/V5-His-TOPO was generated as previously described (14Tomita T. Katayama R. Takikawa R. Iwatsubo T. FEBS Lett. 2002; 520: 117-121Crossref PubMed Scopus (63) Google Scholar). All cDNAs were sequenced by automated sequencer (LI-COR, Lincoln, NE). cDNAs encoding deletion mutants of human NCT fused with V5 tag were kindly gifted from Drs. Keiro Shirotani and Christian Haass (Ludwig-Maximilians-University, Munich, Germany) (17Shirotani K. Edbauer D. Capell A. Schmitz J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: 16474-16477Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Maintenance of Sf9 cells, transfection, and recombinant baculovirus preparation were done as previously described (30Hayashi I. Urano Y. Fukuda R. Isoo N. Kodama T. Hamakubo T. Tomita T. Iwatsubo T. J. Biol. Chem. 2004; 279: 38040-38046Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 31Ogura T. Mio K. Hayashi I. Miyashita H. Fukuda R. Kopan R. Kodama T. Hamakubo T. Iwatsubo T. Tomita T. Sato C. Biochem. Biophys. Res. Commun. 2006; 343: 525-534Crossref PubMed Scopus (85) Google Scholar). Hybridoma cells were maintained in RPMI 1640 medium supplemented with l-glutamine (Nikken Bio Medical Laboratory, Kyoto, Japan) containing 15% (v/v) fetal bovine serum, 100 international units/ml of penicillin, 100 μg/ml of streptomycin, and 1 mm sodium pyruvate (Sigma) at 37 °C in 5% CO2. All transfections were achieved by FuGENE 6 (Roche Applied Science) according to the manufacturer's instructions. HEK293 cell lines stably expressing scFv were selected by Zeocin (Invitrogen). Ncstn knock-out fibroblasts (NKO cells) (32Li T. Ma G. Cai H. Price D.L. Wong P.C. J. Neurosci. 2003; 23: 3272-3277Crossref PubMed Google Scholar) stably expressing wild-type and mutant NCT were selected by Blasticidin (Calbiochem). For NCT ECD production, Sf9 cells were infected with recombinant virus encoding NCT ECD at multiplicity of infection 2 and incubated for 72 h. For scFv, 5201F-expressing cells (clone 2) were incubated in regular media for 72 h. NCT ECD or scFv was recovered from the culture media by using a nickel-chelating column (GE Healthcare). Bound proteins were eluted by a stepwise gradient of imidazole (5–300 mm) in phosphate-buffered saline. Eluted fractions were analyzed by Coomassie or silver staining as well as immunoblotting. Purified NCT ECD or bovine serum albumin was coated on 96-well plates at the concentration of 2 μg/ml in a coating buffer (0.1 m sodium bicarbonate, pH 8.6), and the plates were incubated overnight at 4 °C. After the coating, the plates were blocked by a blocking buffer (phosphate-buffered saline containing 1× BlockAce (Dainippon Sumitomo Pharma, Osaka, Japan) and 0.02% (w/v) sodium azide) and stored at 4 °C until used. A5201A, anti-V5 antibody (Invitrogen), as a positive control or mouse IgG fraction (SIGMA) as a negative control was added at various concentrations to the wells, and the plates were incubated overnight at 4 °C. Binding of antibody was detected by anti-mouse IgG antibody conjugated with horseradish peroxidase (GE Healthcare) and tobacco mosaic virus substrate. A450 was measured and quantitated by SpectraMax M2 microplate reader (Molecular Devices, Sunnyvale, CA). Anti-G1Nr3, G1L3, and PNT3 polyclonal antibodies against glutathione S-transferase-fused human PS1 N terminus, cytoplasmic loop region, or synthetic peptide corresponding to the N-terminal 26 amino acids of human/mouse PEN-2, respectively, were previously described (30Hayashi I. Urano Y. Fukuda R. Isoo N. Kodama T. Hamakubo T. Tomita T. Iwatsubo T. J. Biol. Chem. 2004; 279: 38040-38046Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 33Watanabe N. Tomita T. Sato C. Kitamura T. Morohashi Y. Iwatsubo T. J. Biol. Chem. 2005; 280: 41967-41975Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 34Tomita T. Takikawa R. Koyama A. Morohashi Y. Takasugi N. Saido T.C. Maruyama K. Iwatsubo T. J. Neurosci. 1999; 19: 10627-10634Crossref PubMed Google Scholar, 35Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Anti-PS1NT polyclonal antibody was kindly gifted from Drs. Gopal Thinakaran and Sangram Sisodia (The University of Chicago, Chicago, IL). Other antibodies were purchased from Cell Signaling Technology (Danvers, MA) (anti- c-myc (9B11)), Covance (Princeton, NJ) (anti-APH-1aL (O2C2)), Santa Cruz Biotechnology (Santa Cruz, CA) (anti-NCT N terminus (N19)), Sigma (anti-NCT C terminus (N1660), anti-α-tubulin (DM1A)), or Stressgen (Ann Arbor, MI) (anti-Calnexin). Cells were solubilized with HEPES buffer (10 mm HEPES, pH 7.4, 150 mm NaCl) containing 1% (w/v) CHAPSO. Immunoprecipitation, immunoblot analysis, metabolic labeling, and enzymatic digestion experiments were previously described (14Tomita T. Katayama R. Takikawa R. Iwatsubo T. FEBS Lett. 2002; 520: 117-121Crossref PubMed Scopus (63) Google Scholar, 33Watanabe N. Tomita T. Sato C. Kitamura T. Morohashi Y. Iwatsubo T. J. Biol. Chem. 2005; 280: 41967-41975Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 34Tomita T. Takikawa R. Koyama A. Morohashi Y. Takasugi N. Saido T.C. Maruyama K. Iwatsubo T. J. Neurosci. 1999; 19: 10627-10634Crossref PubMed Google Scholar, 35Tomita T. Watabiki T. Takikawa R. Morohashi Y. Takasugi N. Kopan R. De Strooper B. Iwatsubo T. J. Biol. Chem. 2001; 276: 33273-33281Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 36Tomita T. Maruyama K. Saido T.C. Kume H. Shinozaki K. Tokuhiro S. Capell A. Walter J. Grünberg J. Haass C. Iwatsubo T. Obata K. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 2025-2030Crossref PubMed Scopus (355) Google Scholar). For detection of the γ-secretase activity in vitro, solubilized HeLa cell membranes were co-incubated with the APP-based recombinant substrates in the presence (+) or absence (−) of 100 nm DBZ (YO01027), which was kindly provided from Dr. Haruhiko Fuwa (Tohoku University, Miyagi, Japan) (3Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 37Morohashi Y. Kan T. Tominari Y. Fuwa H. Okamura Y. Watanabe N. Sato C. Natsugari H. Fukuyama T. Iwatsubo T. Tomita T. J. Biol. Chem. 2006; 281: 14670-14676Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 38Takahashi Y. Hayashi I. Tominari Y. Rikimaru K. Morohashi Y. Kan T. Natsugari H. Fukuyama T. Tomita T. Iwatsubo T. J. Biol. Chem. 2003; 278: 18664-18670Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar, 39Fuwa H. Takahashi Y. Konno Y. Watanabe N. Miyashita H. Sasaki M. Natsugari H. Kan T. Fukuyama T. Tomita T. Iwatsubo T. ACS Chem. Biol. 2007; 2: 408-418Crossref PubMed Scopus (86) Google Scholar). Aβ was quantified by human β amyloid enzyme-linked immunosorbent assay kit (WAKO, Osaka, Japan). Cell surface biotinylation was performed using Pierce cell surface protein isolation kit (Pierce) according to the manufacturer's instruction. Budded baculovirus from Sf9 cells infected with recombinant virus displays the recombinant proteins on its virion membrane (30Hayashi I. Urano Y. Fukuda R. Isoo N. Kodama T. Hamakubo T. Tomita T. Iwatsubo T. J. Biol. Chem. 2004; 279: 38040-38046Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 40Saitoh R. Ohtomo T. Yamada Y. Kamada N. Nezu J. Kimura N. Funahashi S. Furugaki K. Yoshino T. Kawase Y. Kato A. Ueda O. Jishage K. Suzuki M. Fukuda R. Arai M. Iwanari H. Takahashi K. Sakihama T. Ohizumi I. Kodama T. Tsuchiya M. Hamakubo T. J. Immunol. Methods. 2007; 322: 104-117Crossref PubMed Scopus (41) Google Scholar). Thus, budded baculovirus can be used as an optimal immunogen to generate monoclonal antibodies against the membrane proteins. Using this technology, we have generated a monoclonal antibody A5201A that specifically binds to NCT ECD. A5201A showed specific binding ability to V5-tagged NCT ECD in a similar manner to anti-V5 antibody, whereas an irrelevant IgG exhibited no reactivity (Fig. 1). Next, we generated two intrabodies based on A5201A, named 5201C and 5201F. Both intrabodies consist of light and heavy chain variable regions that were cloned from hybridoma cells producing A5201A, conjugated with three GGGGS pentapeptide repeats as a linker by PCR. Each cDNA was cloned into a pSecTag2C vector, which enables the targeting of the intrabodies into the lumen by the Igκ leader sequence and detection with c-myc tag attached to the C terminus (Fig. 2A). 5201C and 5201F harbored a difference only in the light chain variable region sequences, whereas the heavy chain variable region sequences were totally identical. As NS-1 cells, the mouse myeloma cells used for the generation of the hybridoma (40Saitoh R. Ohtomo T. Yamada Y. Kamada N. Nezu J. Kimura N. Funahashi S. Furugaki K. Yoshino T. Kawase Y. Kato A. Ueda O. Jishage K. Suzuki M. Fukuda R. Arai M. Iwanari H. Takahashi K. Sakihama T. Ohizumi I. Kodama T. Tsuchiya M. Hamakubo T. J. Immunol. Methods. 2007; 322: 104-117Crossref PubMed Scopus (41) Google Scholar) endogenously express κ light chain gene, one of the two light chain sequences might be derived from NS-1 cells.FIGURE 2Effects of overexpression of 5201F on the expression levels of the γ-secretase components and proteolytic activity. A, the sequence alignment of the obtained scFvs, 5201C, and 5201F. B, immunoblot analysis of HEK293 cells stably expressing intrabodies with each antibody, indicated below the panel. mNCT and imNCT represent mature and immature NCT, respectively. FL, full-length. Mature NCT with faster migration in 5201F-expressing cells (NCT*) was indicated by an asterisk. C, specific γ-secretase activity of the intrabody-expressing cells measured by in vitro assay. Solubilized cell membranes were coincubated with the substrates in the presence (+) or absence (−) of 100 nm DBZ. De novo generation levels of Aβ40 (open columns) or Aβ42 (filled columns) peptides were normalized by the γ-secretase levels, which were assessed by densitometric analysis of PS1 CTF on the immunoblotting (n = 3, *, p < 0.01, Student's t test).View Large Image Figure ViewerDownload Hi-res image Download (PPT) We then generated HEK293 cell lines stably expressing 5201C or 5201F (Fig. 2B, supplemental Fig. S1). Immunoblot analysis revealed that both intrabodies were expressed intracellularly as a ∼36-kDa protein and secreted into culture media. Intriguingly, the expression levels of NCT, especially that of mature NCT, were markedly reduced in 5201F-expressing cell lines, and the remaining “mature-like” NCT showed slightly longer migration on SDS-PAGE than that of mock- or 5201C-expressing cells. Hereafter, we refer to this mature-like NCT of ∼115 kDa observed in 5201F-expressing cells as NCT*. Moreover, the protein levels of other components of the γ-secretase complex, i.e. PS1, APH-1aL, and PEN-2, were also decreased in 5201F-expressing cells. In contrast, none of the γ-secretase components was affected in 5201C-expressing cells. Next, we examined whether the intrinsic γ-secretase activity was affected in the intrabody-expressing cells by in vitro assay using an APP-based recombinant substrate (3Takasugi N. Tomita T. Hayashi I. Tsuruoka M. Niimura M. Takahashi Y. Thinakaran G. Iwatsubo T. Nature. 2003; 422: 438-441Crossref PubMed Scopus (789) Google Scholar, 38Takahashi Y. Hayashi I. Tominari Y. Rikimaru K. Morohashi Y. Kan T. Natsugari H. Fukuyama T. Tomita T. Iwatsubo T. J. Biol. Chem. 2003; 278: 18664-18670Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). We then normalized the activity against the levels of PS1 CTF to measure the specific activity per active complex (20Chávez-Gutiérrez L. Tolia A. Maes E. Li T. Wong P.C. de Strooper B. J. Biol. Chem. 2008; 283: 20096-20105Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). 5201F-expressing cells showed significant reduction in the Aβ-generating activities (for Aβ40, 34.5% (#2) and 23.9% (#4) compared with that of mock cells; for Aβ42, 21.3% (#2) and 39.4% (#4)) (Fig. 2C). These results suggest that the overexpression of intrabody 5201F, but not 5201C, reduces the steady-state expression levels as well as the intrinsic activity of the γ-secretase complex. We then examined the interactions of the intrabodies with the γ-secretase complex by immunoprecipitation analysis. NCT* as well as immature NCT were co-precipitated with intrabody only from 5201F-expressing cells (Fig. 3). All γ-secretase components were also detected in the immunoprecipitates with 5201F. These data suggest that 5201F directly interacts with NCT and that 5201F-bound NCT is incorporated into the γ-secretase complex. Unexpectedly, however, 5201F purified from conditioned media failed to pull down NCT from HEK cell lysates (Fig. 4). Then, we transiently expressed intrabody in the presence or absence of human NCT in NKO cells (32Li T. Ma G. Cai H. Price D.L. Wong P.C. J. Neurosci. 2003; 23: 3272-3277Crossref PubMed Google Scholar) and performed the immunoprecipitation analysis using mixed lysates. The coexpressed 5201F precipitated NCT polypeptides, whereas the intrabody in NKO cells failed to interact with the independently expressed NCT (Fig. 5, A and B), suggesting that the intrabody 5201F is incorporated into the γ-secretase complex during its assembly process. Moreover, significant reduction of mature NCT was observed in stable NKO cells coexpressing 5201F and human NCT in a similar manner to that in HEK293 cells expressing 5201F, suggesting that 5201F was able to form NCT* in NKO cells (supplementary Fig. S2). Next, we analyzed the location of the epitope of the intrabody 5201F using systematically deleted constructs (NCT/Δ1-Δ5) of NCT ECD (17Shirotani K. Edbauer D. Capell A. Schmitz J. Steiner H. Haass C. J. Biol. Chem. 2003; 278: 16474-16477Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar) in NKO cells. Previous results have suggested that these deletion constructs encode loss-of-function mutant forms of NCT. Although 5201F bound to all deletion NCT mutants, the immunoreactivities against NCT/Δ2, Δ3, Δ4, and Δ5 were significantly reduced, suggesting that 5201F directly recognizes a broad region in NCT ECD irrespective of the formation of functional γ-secretase complex (Fig. 5, C and D). Collectively, these results suggest that the intracellularly expressed scFv 5201F directly targets the nascent or newly synthesized NCT polypeptides during the biosynthetic pathway and is incorporated into the γ-secretase complex.FIGURE 4Secreted 5201F failed to bind NCT. A, culture media of 5201F-expressing cells were applied to a nickel chelating column, and the bound proteins were eluted with stepwise increased concentrations of imidazole and EDTA. Eluates were dialyzed against phosphate-buffered saline and then analyzed with silver staining (upper panel) and immunoblotting with anti-c-myc antibody 9B11 antibody (lower panel). B, partially purified secreted scFv from the 100 mm imidazole fraction in A was mixed with 1% CHAPSO-solubilized HEK293 cell lysates and immunoprecipitated with control IgG or anti-c-myc 9B11 antibody. Immunoprecipitates were analyzed by immunoblotting using each antibody indicated below the panels. Note that secreted scFv failed to bind with endogenous NCT. mNCT and imNCT represent mature and immature NCT, respectively.View Large Image Figure ViewerDownload Hi-res image Download (PPT)FIGURE 5Direct binding of intracellular 5201F to NCT. A, immunoblotting of the NKO cells overexpressing intrabodies with or without human NCT. mNCT and imNCT represent mature and immature NCT, respectively. B, immunoprecipitation (IPed) of 1% CHAPSO-solubilized NKO cells with control IgG (c) or anti-c-myc antibody 9B11 (m). Coexpressed samples were the lysates from NKO cells coexpressing intrabodies and human NCT. Mixed samples were the mixture of the lysate from NKO cells either expressing intrabodies or NCT. C, immunoblotting of the NKO cells coexpressing the deletion mutant of human NCT together with 5201F. White circles indicate nonspecific proteins appeared in NKO cells. D, immunoprecipitation of 1% CHAPSO-solubilized NKO cells in C with anti-c-
Referência(s)