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Single Chain Variable Fragment against Nicastrin Inhibits the�-Secretase Activity*□S

Received for publication, August 11, 2009 Published, JBC Papers in Press, August 14, 2009, DOI 10.1074/jbc.M109.055061

Ikuo Hayashi‡1, Sho Takatori‡1, Yasuomi Urano§, Hiroko Iwanari§¶, Noriko Isoo‡, Satoko Osawa‡, Maiko A. Fukuda‡,Tatsuhiko Kodama§, Takao Hamakubo§, Tong Li�, Philip C. Wong�, Taisuke Tomita‡**2, and Takeshi Iwatsubo‡**‡‡

From the ‡Department of Neuropathology and Neuroscience, Graduate School of Pharmaceutical Sciences, The University ofTokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan, the §Laboratory for Systems Biology and Medicine, Research Center forAdvanced Science and Technology, The University of Tokyo, 4-6-1 Komaba, Meguro-ku, Tokyo 153-8904, Japan, ¶PerseusProteomics, Inc., 4-7-6 Komaba, Meguro-ku, Tokyo 153-0041, Japan, the �Department of Pathology, The Johns Hopkins UniversitySchool of Medicine, Baltimore, Maryland 21205, and **Core Research for Evolutional Science and Technology, Japan Science andTechnology Corporation, and the ‡‡Department of Neuropathology, Graduate School of Medicine, The University of Tokyo,7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

�-Secretase is a membrane protein complex that catalyzesintramembrane proteolysis of a variety of substrates includingthe amyloid� precursor protein of Alzheimer disease. Nicastrin(NCT), a single-pass membrane glycoprotein that harbors alarge extracellular domain, is an essential component of the�-secretase complex. Here we report that overexpression of asingle chain variable fragment (scFv) against NCT as an intra-body suppressed the �-secretase activity. Biochemical analy-ses revealed that the scFv disrupted the proper folding andthe appropriate glycosyl maturation of the endogenous NCT,which are required for the stability of the �-secretase com-plex and the intrinsic proteolytic activity, respectively, impli-cating the dual role of NCT in the �-secretase complex. Ourresults also highlight the importance of the calnexin cycle inthe functional maturation of the �-secretase complex. Theengineered intrabodies may serve as rationally designed,molecular targeting tools for the discovery of novel actions ofthe membrane proteins.

�-Secretase catalyzes intramembrane proteolysis of a varietyof substrates including amyloid � precursor protein (APP)3 togenerate amyloid � peptide (A�), the latter being a major com-

ponent of senile plaques in the brains of Alzheimer diseasepatients. Thus, agents that inhibit �-secretase activity couldserve as an effective therapeutics for Alzheimer disease,whereas the �-secretase activity plays important roles in cellsignaling pathways including Notch signaling (1, 2). �-Secre-tase consists of at least four integral membrane proteins, i.e.presenilin (PS), nicastrin (NCT), APH-1, and PEN-2, all ofwhich are essential to the proteolytic activity (3–5). Molecularcellular and chemical biological analyses have revealed that PSforms a hydrophilic pore involving the transmembrane domain6 and 7, where conserved catalytic aspartates reside to functionas catalytic residues of �-secretase complex (6, 7). APH-1 is amultipass membrane protein that plays a role in stabilizationand trafficking of the �-secretase complex (8), and PEN-2 is acofactor for the activation and the regulation of the �-secretaseactivity (3, 9).NCT, which was identified as a PS-binding protein (10), is a

single-pass membrane protein that harbors an extracellulardomain (ECD) with a number of N-glycosylation sites. Inmammalian cells NCT undergoes Endo H-resistant complexglycosylation and acquires trypsin resistance during theassembly process of the �-secretase complex (11–17).Molecular and cellular analyses revealed that the trypsinresistance, presumably indicating the proper structural fold-ing of NCT, might be directly linked to the enzymatic activ-ity, whereas the complex glycosylation is dispensable. More-over, multiple sequence alignment analyses revealed thatNCT ECD have a similarity to an aminopeptidase (18),whereas certain catalytic residues are not conserved.Recently one study has suggested that NCT plays a criticalrole in substrate recognition (19). During the proteolyticprocess, NCT ECD captures the most N terminus of thesubstrate as a primary substrate receptor (i.e. exosite) for the�-secretase via the aminopeptidase-like domain. However,this view has been recently challenged (20). Nevertheless, asstructural information of NCT ECD is totally lacking, thefunctional role of the structural maturation of NCT in theformation and activity of the �-secretase remains unclear.Molecular engineering of monoclonal antibodies opens a

venue for the functional analyses of targeted molecule and thetherapeutic intervention for several diseases (21). A single-

* This work was supported in part by grants-in-aid for Young Scientists (S)from the Japan Society for the Promotion of Science, Scientific Researchon Priority Areas Research on Pathomechanisms of Brain Disordersfrom the Ministry of Education, Culture, Sports, Science, and Technol-ogy, Japan, by the Program for Promotion of Fundamental Studies inHealth Sciences of the National Institute of Biomedical Innovation, byTargeted Proteins Research Program of the Japan Science and Technol-ogy Corporation (JST), and by Core Research for Evolutional Scienceand Technology of JST, Japan.

□S The on-line version of this article (available at http://www.jbc.org) containssupplemental Figs. 1–3.

1 Research Fellows of the Japan Society for the Promotion of Science.2 To whom correspondence should be addressed: Dept. of Neuropathology

and Neuroscience, Graduate School of Pharmaceutical Sciences, The Uni-versity of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan. Tel.:81-3-5841-4868; Fax: 81-3-5841-4708; E-mail: [email protected].

3 The abbreviations used are: APP, amyloid-� precursor protein; A�, amy-loid-� peptide; CHAPSO, 3-[(3-cholamidopropyl)dimethylammonio]-2-hy-droxy-1-propanesulfonate; CNX, calnexin; CST, castanospermine; CTF,C-terminal fragment; NCT, nicastrin; PS, presenilin; Endo H, endoglycosi-dase H; ECD, extracellular domain; ER, endoplasmic reticulum; scFv, singlechain variable fragment.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 284, NO. 41, pp. 27838 –27847, October 9, 2009© 2009 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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chain antibody fragment (scFv) is comprised of heavy- andlight-chain sequences of an antibody linked by a short linkerand preserves binding abilities of its parental antibody. scFv canbe expressed intracellularly as an intrabody (22, 23), which pro-vides a powerfulmethod for phenotypic knock-out of the genes.Intrabodies have been investigated as treatments for a variety ofpathological conditions, including neurodegenerative diseasessuch as Parkinson disease and Huntington disease. Moreover,several recent publications have highlighted the therapeuticpotential of intrabodies targeting intra- as well as extracellularepitopes (24–29). Here, we generated scFv against NCT froman anti-NCT monoclonal antibody. Unexpectedly, the overex-pression of the anti-NCT scFv as an intrabody abolished theproteolytic activity by the destabilization of the �-secretasecomplex and the inappropriate glycosylation of NCT. This isthe first example showing that engineered antibody would be auseful tool for the directmodulation of the�-secretase complexand its activity.

EXPERIMENTAL PROCEDURES

Plasmids—C-terminal V5-His-tagged human NCT ECDinserted in pBlueBac4.5 (Invitrogen) was generated from NCT/V5-His in pBlueBac4.5 (30) by long PCR. Cytoplasmic RNA wasprepared from 1 � 107 hybridoma cells by using Isogen reagent(Nippongene, Tokyo, Japan). The cells were lysed by mixing withIsogen and incubated at room temperature for 5 min. After cen-trifugationof the lysate, theRNAwasprecipitated anddissolved indistilled water. This RNA was used as a template for first-strandcDNA synthesis with 3� primers specific for the mouse IgG genes(Novagen,Darmstadt,Germany).ThecDNAfragmentswere thenamplified by PCRwith LATaq (Takara, Shiga, Japan) using 3� and5�primers from themouse Ig primer set as per themanufacturer’sinstructions (Ig-Primekit protocols;Novagen).ThePCRproductswere subcloned into the pEF6/V5-His-TOPO vector (Invitrogen)by the TOPO cloning method. scFv cDNAs inserted intopSecTag2C (Invitrogen) were constructed as follows. The PCR-derivedDNA fragments in pEF6/V5-HisTOPOwere subjected tosplice overlapping extension PCR to connect heavy- and light-chain genes to give a single fusion protein gene. In the first roundPCR, heavy- and light-chain genes were amplified by using thefollowing primers: the variable region heavy-chain gene, 5�-gggg-aattcGAAGTGAAGCTGGTGGAG-3� (VHF#1) and 5�-caccacc-tccggaaccaccaccaccggaaccaccacctccGGCTGAGGAGACTGTG-AGAGT-3� (VHR#1); the variable region light-chain genes, 5�-ggtggttccggtggtggtggttccggaggtggtggttcaGACATTGTGCTGA-CACAGTCT-3� (VLF#1) and 5�-cccgcggccgcTTTTATTTCCA-GCTTGGT-3� (VLR#1) or 5�-ggtggttccggtggtggtggttccggaggtggt-ggttcaGATATCCAGATGACACAGACT-3� (VLF#2) and 5�-cccgcggccgcTTTGATTTCCAGCTTGGT-3� (VLR#2). In thesecond round PCR, the amplified heavy- and light-chain frag-ments were linked by using VHF#1 and VLR#1 or #2. The ampli-fied scFv cDNAs were digested with EcoRI and NotI to subcloneinto the EcoRI-NotI-digested pSecTag2C vector. Wild-type aswell as mutant (i.e.�312, 648ATAA) humanNCT inserted in thepEF6/V5-His-TOPO was generated as previously described (14).All cDNAs were sequenced by automated sequencer (LI-COR,Lincoln, NE). cDNAs encoding deletion mutants of human NCTfusedwithV5 tagwere kindly gifted fromDrs. Keiro Shirotani and

Christian Haass (Ludwig-Maximilians-University, Munich, Ger-many) (17).Cell Culture and Transfection—Maintenance of Sf9 cells,

transfection, and recombinant baculovirus preparation weredone as previously described (30, 31). Hybridoma cells weremaintained in RPMI 1640medium supplemented with L-glu-tamine (Nikken Bio Medical Laboratory, Kyoto, Japan) con-taining 15% (v/v) fetal bovine serum, 100 internationalunits/ml of penicillin, 100 �g/ml of streptomycin, and 1 mM

sodium pyruvate (Sigma) at 37 °C in 5% CO2. All transfec-tions were achieved by FuGENE 6 (Roche Applied Science)according to the manufacturer’s instructions. HEK293 celllines stably expressing scFv were selected by Zeocin (Invitro-gen). Ncstn knock-out fibroblasts (NKO cells) (32) stablyexpressing wild-type andmutant NCTwere selected by Blas-ticidin (Calbiochem).Purification of NCT ECD and Secreted scFv—For NCT ECD

production, Sf9 cells were infected with recombinant virusencodingNCTECD atmultiplicity of infection 2 and incubatedfor 72 h. For scFv, 5201F-expressing cells (clone 2) were incu-bated in regular media for 72 h. NCT ECD or scFv was recov-ered from the culturemedia by using a nickel-chelating column(GEHealthcare). Bound proteins were eluted by a stepwise gra-dient of imidazole (5–300 mM) in phosphate-buffered saline.

FIGURE 1. Binding of A5201A to recombinant NCT ECD. Sequentiallydiluted A5201A, anti-V5 monoclonal antibody, or mouse IgG fraction wasapplied to NCT ECD- or bovine serum albumin-coated plates. Subsequently,anti-mouse IgG antibody conjugated with horseradish peroxidase was incu-bated, and monoclonal antibody binding was quantitated by measuring A450using peroxidase substrate. AU, absorbance units.

Intrabody against Nicastrin

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Eluted fractions were analyzed by Coomassie or silver stainingas well as immunoblotting.Analysis of A5201A Binding Ability by Enzyme-linked Immu-

nosorbent Assay—Purified NCT ECD or bovine serum albuminwas coated on 96-well plates at the concentration of 2 �g/ml ina coating buffer (0.1 M sodium bicarbonate, pH 8.6), and theplates were incubated overnight at 4 °C. After the coating, the

plates were blocked by a blockingbuffer (phosphate-buffered salinecontaining 1� BlockAce (Dainip-pon Sumitomo Pharma, Osaka,Japan) and 0.02% (w/v) sodiumazide) and stored at 4 °C until used.A5201A, anti-V5 antibody (Invitro-gen), as a positive control or mouseIgG fraction (SIGMA) as a negativecontrol was added at various con-centrations to the wells, and theplates were incubated overnight at4 °C. Binding of antibody wasdetected by anti-mouse IgG anti-body conjugated with horseradishperoxidase (GE Healthcare) andtobaccomosaic virus substrate.A450was measured and quantitated bySpectraMax M2 microplate reader(Molecular Devices, Sunnyvale,CA).Antibodies, Immunological Anal-

yses, and in Vitro �-Secretase As-say—Anti-G1Nr3, G1L3, and PNT3polyclonal antibodies against gluta-thione S-transferase-fused humanPS1 N terminus, cytoplasmic loopregion, or synthetic peptide corre-sponding to the N-terminal 26amino acids of human/mousePEN-2, respectively, were previ-ously described (30, 33–35). Anti-PS1NT polyclonal antibody waskindly gifted from Drs. Gopal Thi-nakaran and Sangram Sisodia (TheUniversity of Chicago, Chicago, IL).Other antibodies were purchasedfrom Cell Signaling Technology(Danvers,MA) (anti- c-myc (9B11)),Covance (Princeton, NJ) (anti-APH-1aL (O2C2)), Santa Cruz Bio-technology (Santa Cruz, CA) (anti-NCT N terminus (N19)), Sigma(anti-NCT C terminus (N1660),anti-�-tubulin (DM1A)), or Stress-gen (Ann Arbor, MI) (anti-Cal-nexin). Cells were solubilized withHEPES buffer (10 mM HEPES, pH7.4, 150 mM NaCl) containing 1%(w/v) CHAPSO. Immunoprecipita-tion, immunoblot analysis, meta-

bolic labeling, and enzymatic digestion experiments were pre-viously described (14, 33–36). For detection of the �-secretaseactivity in vitro, solubilized HeLa cell membranes were co-in-cubated with the APP-based recombinant substrates in thepresence (�) or absence (�) of 100 nM DBZ (YO01027), whichwas kindly provided fromDr. Haruhiko Fuwa (TohokuUniver-sity, Miyagi, Japan) (3, 37–39). A� was quantified by human �

FIGURE 2. Effects of overexpression of 5201F on the expression levels of the �-secretase componentsand proteolytic activity. A, the sequence alignment of the obtained scFvs, 5201C, and 5201F. B, immu-noblot analysis of HEK293 cells stably expressing intrabodies with each antibody, indicated below thepanel. mNCT and imNCT represent mature and immature NCT, respectively. FL, full-length. Mature NCTwith faster migration in 5201F-expressing cells (NCT*) was indicated by an asterisk. C, specific �-secretaseactivity of the intrabody-expressing cells measured by in vitro assay. Solubilized cell membranes werecoincubated with the substrates in the presence (�) or absence (�) of 100 nM DBZ. De novo generationlevels of A�40 (open columns) or A�42 (filled columns) peptides were normalized by the �-secretase levels,which were assessed by densitometric analysis of PS1 CTF on the immunoblotting (n � 3, *, p � 0.01,Student’s t test).




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amyloid enzyme-linked immunosorbent assay kit (WAKO,Osaka, Japan). Cell surface biotinylation was performed usingPierce cell surface protein isolation kit (Pierce) according to themanufacturer’s instruction.

RESULTS

Anti-NCT Intrabody Decreases the Expression Levels of NCTand Suppresses the �-Secretase Activity—Budded baculovirusfrom Sf9 cells infected with recombinant virus displays therecombinant proteins on its virion membrane (30, 40). Thus,budded baculovirus can be used as an optimal immunogen togenerate monoclonal antibodies against the membrane pro-teins. Using this technology, we have generated a monoclonalantibody A5201A that specifically binds to NCT ECD. A5201Ashowed specific binding ability to V5-tagged NCT ECD in asimilar manner to anti-V5 antibody, whereas an irrelevant IgGexhibited no reactivity (Fig. 1). Next, we generated two intra-bodies based on A5201A, named 5201C and 5201F. Both intra-bodies consist of light and heavy chain variable regions thatwere cloned from hybridoma cells producing A5201A, conju-gated with three GGGGS pentapeptide repeats as a linker byPCR. Each cDNA was cloned into a pSecTag2C vector, whichenables the targeting of the intrabodies into the lumen by theIg� leader sequence and detection with c-myc tag attached tothe C terminus (Fig. 2A). 5201C and 5201F harbored a differ-ence only in the light chain variable region sequences, whereasthe heavy chain variable region sequenceswere totally identical.As NS-1 cells, themousemyeloma cells used for the generationof the hybridoma (40) endogenously express � light chain gene,one of the two light chain sequences might be derived fromNS-1 cells.We then generated HEK293 cell lines stably expressing

5201C or 5201F (Fig. 2B, supplemental Fig. S1). Immunoblotanalysis revealed that both intrabodies were expressed intra-cellularly as a �36-kDa protein and secreted into culturemedia. Intriguingly, the expression levels of NCT, especiallythat of mature NCT, were markedly reduced in 5201F-ex-pressing cell lines, and the remaining “mature-like” NCTshowed slightly longer migration on SDS-PAGE than that ofmock- or 5201C-expressing cells. Hereafter, we refer to thismature-like NCT of�115 kDa observed in 5201F-expressingcells as NCT*. Moreover, the protein levels of other compo-nents of the �-secretase complex, i.e. PS1, APH-1aL, andPEN-2, were also decreased in 5201F-expressing cells. Incontrast, none of the �-secretase components was affected in5201C-expressing cells. Next, we examined whether theintrinsic �-secretase activity was affected in the intrabody-expressing cells by in vitro assay using an APP-based recom-binant substrate (3, 38). We then normalized the activityagainst the levels of PS1 CTF to measure the specific activityper active complex (20). 5201F-expressing cells showed sig-nificant reduction in the A�-generating activities (for A�40,34.5% (#2) and 23.9% (#4) compared with that of mock cells;for A�42, 21.3% (#2) and 39.4% (#4)) (Fig. 2C). These resultssuggest that the overexpression of intrabody 5201F, but not5201C, reduces the steady-state expression levels as well asthe intrinsic activity of the �-secretase complex.

5201F, but Not 5201C, Binds to NCT ECD in the �-SecretaseAssembly Process—We then examined the interactions of theintrabodies with the �-secretase complex by immunoprecipita-tion analysis. NCT* as well as immature NCT were co-precipi-tated with intrabody only from 5201F-expressing cells (Fig. 3).

FIGURE 3. Incorporation of 5201F into the �-secretase complex. Co-immu-noprecipitation analysis of 1% CHAPSO-solubilized fractions from intrabody-expressing stable HEK293 cells with control IgG (c) or anti-c-myc antibody9B11 (m). Immunoprecipitates were analyzed by immunoblotting with eachantibody indicated below the panel. The white circle indicates the immuno-globulin chain. imNCT immature NCT.

FIGURE 4. Secreted 5201F failed to bind NCT. A, culture media of 5201F-expressing cells were applied to a nickel chelating column, and the boundproteins were eluted with stepwise increased concentrations of imidazoleand EDTA. Eluates were dialyzed against phosphate-buffered saline and thenanalyzed with silver staining (upper panel) and immunoblotting with anti-c-myc antibody 9B11 antibody (lower panel). B, partially purified secreted scFvfrom the 100 mM imidazole fraction in A was mixed with 1% CHAPSO-solubi-lized HEK293 cell lysates and immunoprecipitated with control IgG or anti-c-myc 9B11 antibody. Immunoprecipitates were analyzed by immunoblottingusing each antibody indicated below the panels. Note that secreted scFv failedto bind with endogenous NCT. mNCT and imNCT represent mature and imma-ture NCT, respectively.




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All �-secretase components were also detected in the immuno-precipitates with 5201F. These data suggest that 5201F directlyinteractswithNCTand that 5201F-boundNCT is incorporatedinto the �-secretase complex. Unexpectedly, however, 5201Fpurified from conditionedmedia failed to pull downNCT fromHEK cell lysates (Fig. 4). Then, we transiently expressed intra-body in the presence or absence of human NCT in NKO cells(32) and performed the immunoprecipitation analysis usingmixed lysates. The coexpressed 5201F precipitated NCTpolypeptides, whereas the intrabody in NKO cells failed tointeract with the independently expressed NCT (Fig. 5, Aand B), suggesting that the intrabody 5201F is incorporatedinto the �-secretase complex during its assembly process.Moreover, significant reduction of mature NCT wasobserved in stable NKO cells coexpressing 5201F and humanNCT in a similar manner to that in HEK293 cells expressing5201F, suggesting that 5201F was able to formNCT* in NKOcells (supplementary Fig. S2). Next, we analyzed the locationof the epitope of the intrabody 5201F using systematicallydeleted constructs (NCT/�1-�5) of NCT ECD (17) in NKOcells. Previous results have suggested that these deletionconstructs encode loss-of-function mutant forms of NCT.Although 5201F bound to all deletion NCT mutants, theimmunoreactivities against NCT/�2, �3, �4, and �5 weresignificantly reduced, suggesting that 5201F directly recog-nizes a broad region in NCT ECD irrespective of the forma-tion of functional �-secretase complex (Fig. 5, C and D).Collectively, these results suggest that the intracellularlyexpressed scFv 5201F directly targets the nascent or newlysynthesized NCT polypeptides during the biosynthetic path-way and is incorporated into the �-secretase complex.

Binding of 5201F Prevents theGlycosyl and the Structural Matu-ration of NCT—During the matura-tion process of the �-secretase com-plex, NCT ECD undergoes acomplex glycosylation and a confor-mational change to acquire trypsinresistance along with the traffickingfrom ER to the cell surface (11–17).To investigate the molecular mech-anism by which the binding of5201F decreased the expression lev-els of NCT as well as of the �-secre-tase components, we biochemicallycharacterized NCT*. In the meta-bolic labeling experiment, NCTwassynthesized as �110-kDa core-gly-cosylated intermediate form ofpolypeptides at 0 h of chase (Fig. 6A)(11, 13). The levels of NCT corepolypeptides were comparableamong the stable cell lines, indicat-ing that the overexpression of intra-bodies had no effect on the transla-tion efficiency of NCT. Three hoursafter synthesis, measurable levels of

NCT were converted to the 100 kDa immature form by trim-ming of glucose and mannose in the ER. Then these NCTmatured into the complex glycosylated forms that exhibitedretarded migration at 120 kDa. This mature NCT was long-lived, and significant levels of labeled mature NCT were stillpresent 48 h after labeling as previously reported (11). In 5201F-expressing cells, however, the levels of immature NCT wererelatively low at 3 h of chase and the conversion to NCT* wascompletedwithin 6 h.Moreover, 48 h after synthesis, NCT*wasstill present but clearly lesser in amount compared with that ofmature NCT in mock- or 5201C-expressing cells, suggestingthat the binding of 5201F caused rapid and inappropriate mat-uration of NCT polypeptides and rendered the NCT* unstable.Next we examined the glycosylation state of NCT* by

Endo H digestion. Mature NCT in mock- or 5201C-express-ing cells was Endo H-resistant and migrated at �120 kDa inSDS-PAGE, whereas immature NCT was completely degly-cosylated as previously described (Fig. 6B) (11–17). Unex-pectedly, NCT* showed Endo H resistance, too. Moreover,cell surface biotinylation experiments revealed that matureNCT*was displayed on the plasmamembrane in a similarman-ner to the mature NCT of the control cell lines (Fig. 6C). Thelevels of PS1N-terminal fragment inmock- and 5201F-express-ing cells were almost comparable (supplemental Fig. S3), sug-gesting that the steady-state level of the �-secretase complexcontaining 5201F on the cell surface is not significantly altered.Finally, we examined the trypsin resistance of NCT*. As previ-ously described (17), substantial levels ofmatureNCT inmock-or 5201C-expressing cells remained intact after 30-min of incu-bation with trypsin, whereas immature NCT was completelydigested (Fig. 6D). In contrast, in 5201F-expressing cells, NCT*was completely digested by trypsin in a similar manner to that

FIGURE 5. Direct binding of intracellular 5201F to NCT. A, immunoblotting of the NKO cells overexpressingintrabodies 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 antibody9B11 (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, immu-noblotting of the NKO cells coexpressing the deletion mutant of human NCT together with 5201F. White circlesindicate nonspecific proteins appeared in NKO cells. D, immunoprecipitation of 1% CHAPSO-solubilized NKOcells in C with anti-c-myc antibody 9B11.




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of immature NCT. These results indicate that the binding of5201F partially prevents the conformational as well as glycosylmaturation of NCT.Conformational Maturation of NCT Is Required for the Sta-

bilization of the �-Secretase Complex—Large deletions in NCTECD caused total loss of function of NCT (i.e. NCT/�1-�5)by failure in acquiring conformational maturation (10, 17).However, it is difficult to analyze the effect of the conforma-tional defects in NCT ECD using these mutants because ofthe nature of deletion mutation; in fact, the overexpressionof NCT/�312 in NKO cells completely failed to generatemature NCT (Refs. 10, 14, and 17; see below). To testwhether a “partial” conformational defect of NCT affects theintrinsic �-secretase activity, we introduced amino acid sub-stitutions into the highly conserved residues at the jux-tamembrane region of NCT ECD (i.e. Trp-648, Glu-650, andSer-651 to alanine; 648ATAA), which play an importantfunctional role in the �-secretase complex formation (Fig.7A) (41). The overexpression of NCT/648ATAA in NKO

cells partially rescued the generation of mature NCT and PS1fragments (Fig. 7B). Cycloheximide treatment caused rapiddegradation of PS1 CTF in NCT/648ATAA-overexpressingcells, whereas PS1 CTF in cells expressing wild-type NCTwas stable as previously reported (42), indicating that thereconstituted �-secretase complex by NCT/648ATAAmutant is unstable (Fig. 7C). The trypsin digestion experi-ment revealed that mature NCT/648ATAA was readilydegraded, suggesting that mutant NCT failed to acquire theconformational maturation, thereby causing the instabilityof the �-secretase complex (Fig. 7D). The de novo A� gener-ating activity in NCT/648ATAA-expressing NKO cells wasalso decreased (66.3% of that in wild-type human NCT-ex-pressing cells). Notably, however, the de novo activity nor-malized by PS1 CTF levels in cells expressing NCT/648ATAA was not reduced compared with that in wild-typehuman NCT-expressing cells (Fig. 7E). These data suggestthat the defect in the conformational maturation of NCTECD caused the decrease in the total A� generating activityby loss of stability of the functional �-secretase complexes,whereas the intrinsic activity of the enzyme was unaltered.Glucose Trimming in ER Is Required for the Intrinsic Activ-

ity of the �-Secretase Complex—N-Glycosylated proteins arefolded by ER-resident chaperones (e.g., calnexin (CNX) orcalreticulin) that recognize a monoglucose on unfoldedpolypeptides (43, 44). During the folding process, the trans-fer and the trimming of glucose are executed by the ER-resident glucosyltransferase and glucosidase, respectively.Thus, unfolded, but still glucose-attached proteins are cap-tured by CNX to be folded; this process is called “the CNXcycle.” To test the possibility that the CNX cycle is involvedin the maturation of NCT, the immunoprecipitation analysiswas performed. We confirmed the association of CNX andNCT as previously described (Fig. 8A) (45). Moreover, thisinteraction was significantly reduced by the overexpressionof 5201F (Fig. 8B), suggesting that the glucose trimmingand/or the CNX cycle would be inhibited by the scFv. Cas-tanospermine (CST) is an �1,2-glucosidase inhibitor thatcauses the inhibition of interaction between N-linked glyco-proteins and CNX (46, 47). The CST treatment caused theaccumulation of aberrant NCT polypeptides, which presum-ably represent the glucosylated form of NCT (glucoNCT)(Fig. 9A). Intriguingly, glucoNCT showed a similar molecu-lar weight to that of NCT* and acquired the Endo H resist-ance (Fig. 9B). In contrast, the levels of the �-secretase com-ponents and the trypsin resistance of NCT were unaffected,suggesting that the glucose trimming is dispensable for theproper folding of NCT and the trafficking of the stable�-secretase complex (Fig. 9, A and C). However, specific denovo A� generating activity normalized by PS1 CTF levels inCST-treated cell membrane was significantly decreased to asimilar extent to that in 5201F-expressing cells (for A�40,32.0% compared with that of mock-treated cells; for A�42,33.4%) (Fig. 9D). These data indicate that the proper glucosetrimming of NCT ECD accompanied by the CNX cycle isrequired for the intrinsic �-secretase activity but not for theformation of the stable enzyme complex. Taken together,the binding of scFv 5201F has detrimental effects both on the

FIGURE 6. Characterization of NCT* observed in 5201F-expressingcells. A, metabolic labeling of stable HEK293 cells. Cells were pulse-la-beled with [35S]methionine and cysteine for 30 min and chased for indi-cated time periods. Lysates were immunoprecipitated with anti-NCT Cterminus antibody N1660 and analyzed. NCT* is denoted by asterisks.mat., mature; imm., immature; int., intermediate. B, immunoblotting ofNCT polypeptides digested by Endo H. Immunoprecipitates (IP) with anti-NCT C terminus antibody N1660 or anti-c-myc antibody 9B11 were incu-bated with (�) or without (�) Endo H and analyzed by immunoblottingwith anti-NCT N terminus antibody N19. C, cell surface biotinylation ofHEK293 cells. Cell lysates (input) as well as biotinylated proteins (IP-SA)were analyzed using each antibody indicated below the panels. D, trypsindigestion of NCT polypeptides. Immunoprecipitates with anti-NCT C ter-minus antibody N1660 or anti-c-myc antibody 9B11 were incubated with(�) or without (�) 100 �g/ml trypsin and analyzed as in B.




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conformational maturation andthe glucose trimming of NCTECD, thereby causing the destabi-lization and the loss of the enzy-matic activity of the �-secretasecomplex, respectively.

DISCUSSION

Recently, much attention is beingfocused on the use of scFv frag-ments as intrabodies. Intrabodieshave been used for phenotypicknock-out of endogenous targetproteins by several different strate-gies. In this study we generated twointrabodies by using an anti-NCTECD monoclonal antibody A5201Aas a template. Biochemical analysesrevealed that the specific binding of5201F on NCT ECD inhibited theconformational change and theproper glycosylation of NCT,thereby causing the destabilizationof the �-secretase complex and theloss of proteolytic activity. Ourresults suggest that the functionalmaturation of NCT ECD regulatesthe proper trafficking, stability, andthe specific activity of the �-secre-tase complex.

�-Secretase is an unusually stableprotease that has 24 h of half-lifein mammalian cells (42). Biochemi-cal studies have shown that NCTforms a subcomplex with APH-1within the biosynthetic pathway(48) and functions as a stabilizingcofactor as well as a substrate recep-tor for the �-secretase complex (19,20). The assembly of the �-secretasecomplex occurs in the ER (49, 50),and only “functionally” assembled�-secretase is subsequently sortedout to the Golgi apparatus in Rer1-and COPII-regulated manners (5,51–53). However, the molecularinformation on the quality controlof a prefunctional �-secretase com-plex in the ER remains unknown.Here, we showed that the overex-pression of 5201F caused an inap-propriate glycosylation and pre-vented NCT polypeptides fromacquiring the trypsin resistance,thereby causing the phenotypic“knock-out” of the �-secretase com-ponents. Notably, we found that5201F accelerated the inappropriate

FIGURE 7. The role of conformational maturation of NCT in the �-secretase activity. A, schematic depictionof NCT mutants analyzed in this experiment. The black box indicates highly conserved region containing DYIGSmotif (312–369). The C-terminal V5 tag is indicated by shaded circle. B, immunoblot analysis of wild-type andmutant NCT-expressing NKO cell lysates. mNCT and imNCT represent mature and immature NCT, respectively.C, immunoblot analysis of wild-type and mutant NCT expressing NKO cells treated with cycloheximide (CHX) aspreviously reported. Lysates were prepared after cycloheximide treatment for various incubation times asindicated above the lanes. D, trypsin digestion of wild- type or mutant NCT polypeptides. E, de novo �-secretaseactivity of the mutant NCT-expressing cells measured by in vitro assay. White bars indicate the proteolyticactivity in the solubilized membrane containing equal protein amounts. Black bars denote the relative activitynormalized by the �-secretase levels, which were assessed by densitometric analysis of PS1 CTF onimmunoblotting.

FIGURE 8. Interaction of CNX and NCT. A, immunoprecipitation (IP) analysis of NKO cell lysate transientlycoexpressing human NCT and intrabodies. Immunoreactivities of NCT-bound CNX were quantified and nor-malized by the amount of total NCT in B (n � 7; *, p � 0.05, Student’s t test).




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Endo H-resistant glycosylation of NCT, suggesting that thequality control and the trafficking system for the prefunctional�-secretase in the ER are altered upon 5201F expression. Ingeneral, the folding and maturation states of glycoproteins aremonitored by the CNX cycle. Properly folded glycoproteinsescape this cycle and are sorted out from the ER. Trypsin resist-ance of NCT is tightly correlated with the proteolytic activity ofthe�-secretase complex andmay reflect its structural change inthe ER (17), whereas themolecular basis of this conformationalchange has not been clarified to date. Of note, NCT fused withER retention dilysine signal at the C terminus retained the abil-ity to form the functional �-secretase complex and is sorted outto the cell surface (49, 50). In addition, after trypsin digestion,the immunoreactivity of the C terminus of mature NCT waspreserved, and no molecular weight change was observed (17).These results suggest that the most C terminus of NCT is alsotightly folded in the functional �-secretase complex. Thus, thefolding state of NCT throughout the molecule might be undersurveillance by ER-associated chaperones (i.e. CNX) and/ortrafficking-related molecules (e.g. Rer1, coatmer subunits) as amolecular signature for the functional assembly of the �-secre-tase complex. Incorporation of 5201Fwould interrupt the func-tional folding of NCT and the binding with CNX; the latterpresumably caused the aberrant escape of the �-secretase com-plex from the CNX cycle, thereby leading to the destabilizationof the �-secretase complex.An ineffective CNX cycle is also caused by the inhibition of

glucose trimming. The treatment with CST induced the accu-mulation of the glucosylated NCT polypeptides and reducedthe �-secretase activity. In general, aberrant glucosylated pro-teins generated by CST treatment are rapidly transported outfrom the ER or degraded by ER-associated degradation (46, 54,

55). However, our results showed that biochemical character-istics of the �-secretase were unaffected by CST, whereas theintrinsic proteolytic activity was reduced. In contrast, it hasbeen shown that treatment by kifunensine, which inhibits themannose trimming occurring at the later stage of glycosyl mat-uration, has no effect on the �-secretase activity (16, 17), sug-gesting that the glucose/mannose trimmingwas dispensable forthe proper folding of NCT and the formation of the �-secretasecomplex. Rather, our data indicate that the glucosyl state ofNCT per se is involved in the specific enzymatic activity, sug-gesting the functional significance of the CNX cycle in the bio-genesis of the �-secretase complex. The analysis of 648/ATAAmutant NCT also supported our notion that the conforma-tional maturation of NCT determines the amount, but not theintrinsic activity, of the functional �-secretase complex.Although the molecular mechanism(s) by which the glycosyla-tion of NCT modulates the intramembrane cleavage remainunknown, aberrant glucosylatedNCTmight have lesser activityin the substrate-capturing function.We propose a model for the mode of action of 5201F as

depicted in Fig. 10. NCT would be an essential component inthe �-secretase complex with dual functions, which were dis-rupted by the binding with 5201F. The properly glycosylatedand foldedNCTmight function not only as a substrate receptor(19) but as a gatekeeper for the trafficking and the stability ofthe �-secretase complex (20). Our data presented here alsoexpand the repertoire of antibody-based functional reagentsvaluable for the cell biology research on the �-secretase. Tomodify/engineer their binding profile, scFvs can be applicableto phage display, to which “rational design and directed molec-ular evolution” process with high throughput platform is appli-cable (56). In addition, structural studies using scFv have pro-vided crucial information for the rational development of thesmall compound targeting protein-protein interactions (57).

FIGURE 9. The role of glucose trimming in NCT maturation and the�-secretase activity. A, immunoblot analysis of CST-treated HEK293 celllysates. Note that CST treatment caused the accumulation of aberrant molec-ular weight NCT (glucoNCT). mNCT and imNCT represent mature and imma-ture NCT, respectively. NTF, N-terminal fragment. B, Endo H and C, trypsindigestion of glucoNCT accumulated by CST treatment. D, specific �-secretaseactivity of the CST-treated HEK293 cells analyzed by in vitro assay as in Fig. 1C(n � 3; *, p � 0.01, Student’s t test).

FIGURE 10. A putative model for the mode of action of 5201F. In biosyn-thetic pathway of the �-secretase (black arrows), folding and glycosylationstates of NCT are monitored by CNX in the ER. After the assembly of the�-secretase complex together with PS, APH-1, and PEN-2 (white circle), NCTECD is glycosylated with complex oligosaccharides (black hexagon) in theGolgi. CST treatment yields the stable �-secretase complex containing NCTwith aberrant glycosylation (white hexagon), which reduces the proteolyticactivity. 5201F binding to NCT ECD inhibits the functional folding and glucosetrimming to form the unstable and inactive �-secretase complex with NCT*.mNCT and imNCT represent mature and immature NCT, respectively.




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Notably, amino acid substitutions at certain sites in NCT ECDdifferently affected the A� generation and Notch processing(20). Thus, search for small compounds or peptides targetingNCT ECD might provide a new class of biological tools as wellas therapeutics for �-secretase modulation. Further investiga-tions including structural analyses of NCT ECD complexedwith scFvs would shed light on themolecularmechanism of thefunctional assembly of �-secretase and the role of NCT ECD inthe �-secretase-mediated intramembrane cleavage.

Acknowledgments—We acknowledge Drs. Keiro Shirotani (Fuku-shima Medical University), Harald Steiner, Christian Haass (Lud-wig-Maximilians-University), Gopal Thinakaran, Sangram Sisodia(The University of Chicago), Haruhiko Fuwa (Tohoku University),Satoshi Yokoshima, and Tohru Fukuyama (The University of Tokyo)for valuable reagents. We are also grateful to our current/previouslaboratory members for helpful discussions and technical assistance.

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Wong, Taisuke Tomita and Takeshi IwatsuboOsawa, Maiko A. Fukuda, Tatsuhiko Kodama, Takao Hamakubo, Tong Li, Philip C. Ikuo Hayashi, Sho Takatori, Yasuomi Urano, Hiroko Iwanari, Noriko Isoo, Satoko

-Secretase ActivityγSingle Chain Variable Fragment against Nicastrin Inhibits the

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