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• W1983996266 abstract "β-Amyloid peptide (Aβ) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Aβ is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Aβ generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defectiveO-glycosylation, we demonstrated that the majority of APP cleavage by α-, β-, and γ-secretases occurs afterO-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (αAPPCOOH), a product of α-secretase, and both Aβ40 and Aβ42 in medium, a product of β- and γ-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by α-, β-, and γ-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway. β-Amyloid peptide (Aβ) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Aβ is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Aβ generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defectiveO-glycosylation, we demonstrated that the majority of APP cleavage by α-, β-, and γ-secretases occurs afterO-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (αAPPCOOH), a product of α-secretase, and both Aβ40 and Aβ42 in medium, a product of β- and γ-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by α-, β-, and γ-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway. Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, β-amyloid; APP, amyloid precursor protein; αAPPCOOH, α-secretase cleaved intracellular APP carboxyl-terminal fragment; ConA, concanavalin A; ELISA, enzyme-linked immunosorbent assay; ER, endoplasmic reticulum; FAD, familial Alzheimer's disease; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; WGA, wheat germ agglutinin; wt, wild type; imAPP, immature APP; mAPP, mature APP; APPmut, mutant APP; PBS, phosphate-buffered saline. is characterized by the presence of parenchymal and cerebrovascular β-amyloid (Aβ) deposits (1Glenner G. Wong C. Biochem. Biophys. Res. Commun. 1984; 122: 885-890Crossref Scopus (4238) Google Scholar, 2Masters C.L. Simms G. Weinmann N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3679) Google Scholar). Aβ is a 39–43-amino acid peptide that is derived from Alzheimer's amyloid precursor protein (APP). The generation of Aβ is thought to be one of the major events of AD pathogenesis (reviewed in Refs. 3Codell B. Annu. Rev. Pharmacol. Toxicol. 1994; 34: 69-89Crossref PubMed Google Scholar and 4Selkoe D.J. Annu. Rev. Neurosci. 1994; 17: 489-517Crossref PubMed Scopus (829) Google Scholar). APP is an integral membrane protein with a receptor-like structure, existing in several isoforms which, in many tissues, arise by alternative splicing of a single gene (5Goldgaber D. Lerman M.I. McBride O.W. Saffiotti U. Gajdusek D.C. Science. 1987; 235: 877-880Crossref PubMed Scopus (1026) Google Scholar, 6Kang J. Lemaire H.G. Unterbeck A.J. Salbaum J.M. Master C.L. Grzeschik K.H. Multhaup G. Beyreuther K. Muller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3956) Google Scholar, 7Robakis N.K. Ramakrishna N. Wolfe G. Wisniewski H.M. Proc. Natl. Acad. Sci. U. S. 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APP is subject to post-translational modification such as glycosylation, sulfation, and phosphorylation during transit through the intracellular protein secretory pathway (13Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Master C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar, 14Oltersdorf T. Ward P.J. Henriksson T. Beattie E.C. Neve R. Lieberburg I. Fritz L.C. J. Biol. Chem. 1990; 265: 4492-4497Abstract Full Text PDF PubMed Google Scholar, 15Påhlsson P. Shakin-Eshleman S.H. Spitalnik S.L. Biochem. Biophys. Res. Commun. 1992; 189: 1667-1673Crossref PubMed Scopus (71) Google Scholar, 16Knops J. Gandy S. Greengard P. Lieberburg I. Sinha S. Biochem. Biophys. Res. Commun. 1993; 197: 380-385Crossref PubMed Scopus (22) Google Scholar, 17Hung A.Y. Selkoe D.J. EMBO J. 1994; 13: 534-542Crossref PubMed Scopus (112) Google Scholar, 18Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 19Graebert K.S. Popp G.M. Kehlw T. Herzog V. Eur. J. Cell Biol. 1995; 66: 39-46PubMed Google Scholar, 20Påhlsson P. Spitalnik S.L. Arch. Biochem. Biophys. 1996; 331: 177-186Crossref PubMed Scopus (55) Google Scholar, 21Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar, 22Walter J. Capell A. Hung A.Y. Langen H. Schnölzer M. Thinakaran G. Sisodia S.S. Selkoe D.J. Haass C. J. Biol. Chem. 1997; 272: 1896-1903Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). APP isoforms exist as immature (imAPP, N-glycosylated) and mature (mAPP, N- and O-glycosylated, tyrosyl-sulfated) species. The imAPP localizes in the ER and cis-Golgi, and the mAPP localizes in compartments following trans-Golgi and on the plasma membrane. The molecular mechanism(s) and cellular compartment(s) involved in APP cleavage and Aβ production have yet to be fully resolved. Studies using agents (i.e. brefeldin A and monensin) or studies with treatments (i.e. cell culture at low temperature) that interfere with secretory metabolic steps (23Sambamurti K. Shioi J.P. Pappolla M.A. Robakis N.K. J. Neurosci. Res. 1992; 33: 319-329Crossref PubMed Scopus (134) Google Scholar, 24De Strooper B. Umans L. Van Leuven F. Van Den Berghe H. J. Cell Biol. 1993; 121: 295-304Crossref PubMed Scopus (140) Google Scholar, 25Haass C. Hung A.Y. Schlossmacher M.G. Teplow D.B. Selkoe D.J. J. Biol. Chem. 1993; 268: 3021-3024Abstract Full Text PDF PubMed Google Scholar, 26Kuentzel S.L. Ali S.M. Altman R.A. Greenberg B.D. Raub T.J. Biochem. J. 1993; 295: 367-378Crossref PubMed Scopus (119) Google Scholar, 27Refolo L.M. Sambamurti K. Efthimiopoulos S. Pappolla M.A. Robakis N.K. J. Neurosci. Res. 1995; 40: 694-706Crossref PubMed Scopus (52) Google Scholar, 28Thinakaran G. Teplow D.B. Siman R. Greenberg B. Sisodia S.S. J. Biol. Chem. 1996; 271: 9390-9397Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar) suggest that APP cleavage by α-secretase occurs in a secretory step in late Golgi. Although recent reports indicate that the ER is the site for generation of Aβ42 but not Aβ40 in the neuron (29Hartmann T. Bieger S.C. Brühl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 30Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar), Aβ in studies using agents that interfere with pH gradients (i.e.chloroquine and ammonium chloride) is believed to be generated in acidic compartments such as endosomes and/or late Golgi (31Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1765) Google Scholar, 32Shoji M. Golde T.E. Ghiso J. Cheung T.T. Estus S. Shaffer L.M. Cai X.-D. McKay D.M. Tintner R. Frangione B. Younkin S.G. Science. 1992; 258: 126-129Crossref PubMed Scopus (1327) Google Scholar, 33Koo E.H. Squazzo S.L. J. Biol. Chem. 1994; 269: 17386-17389Abstract Full Text PDF PubMed Google Scholar). However, these procedures are toxic, and it is possible that these agents interfere with intracellular protein metabolism through nonspecific and unpredictable mechanisms. To identify potential intracellular compartments involved in the cleavage of APP by secretases without utilizing toxic metabolic inhibitors, we prepared cells expressing mutant APP (APPmut) which is not subject to O-glycosylation. In such cells, all other intracellular protein metabolism is thought to be normal. Taking advantage of the property of the cells expressing APPmut, we examined the processing of APP in healthy cells. Cells expressing the APPmut noticeably decreased the generation of the carboxyl-terminal fragment of APP (αAPPCOOH), a product of cleavage by α-secretase, and also failed to generate Aβ40 and Aβ42, products of cleavage by both β- and γ-secretases. The present study shows that, without utilizing metabolic agents which nonspecifically interfere with protein degradation and secretion, APP is cleaved after, or possibly during, maturation (O-glycosylation). These results indicate that APP cleavage occurs in compartment(s) subsequent to trans-Golgi of the protein secretory pathway or possibly during the transport of APP through Golgi complex, where O-glycosylation occurs (34Danphy W.G. Rothman J.E. Cell. 1985; 42: 13-21Abstract Full Text PDF PubMed Scopus (281) Google Scholar). Generation of Aβ42 in the ER (29Hartmann T. Bieger S.C. Brühl B. Tienari P.J. Ida N. Allsop D. Roberts G.W. Masters C.L. Dotti C.G. Unsicker K. Beyreuther K. Nat. Med. 1997; 3: 1016-1020Crossref PubMed Scopus (646) Google Scholar, 30Cook D.G. Forman M.S. Sung J.C. Leight S. Kolson D.L. Iwatsubo T. Lee V.M.-Y. Doms R.W. Nat. Med. 1997; 3: 1021-1023Crossref PubMed Scopus (430) Google Scholar) may be a neuron-specific and/or a minor event. cDNA encoding human APP770 was cloned from λZAP HeLa cell cDNA library 2T. Suzuki, unpublished observations. by immunoscreening with anti-APP antibody, G-369 (35Buxbaum J.D. Gandy S.E. Cicchetti P. Ehrlich M.E. Czernik A.J. Fracasso R.P. Ramabhadran T, V. Unterbeck A.J. Greengard P. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 6003-6006Crossref PubMed Scopus (427) Google Scholar). The cDNA was subcloned into pcDNA3 (Invitrogen) atHindIII/XbaI sites (Fig. 1 a). A sequence of APP770 extracellular domain, 379–666 (the numbering for APP770 and also 304–591 for APP695 isoforms) which includes two potential N-glycosylation sites (13Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Master C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar, 15Påhlsson P. Shakin-Eshleman S.H. Spitalnik S.L. Biochem. Biophys. Res. Commun. 1992; 189: 1667-1673Crossref PubMed Scopus (71) Google Scholar, 20Påhlsson P. Spitalnik S.L. Arch. Biochem. Biophys. 1996; 331: 177-186Crossref PubMed Scopus (55) Google Scholar), was deleted by exclusion of XhoI/BglII fragment. The 3′ recessed termini were filled with dNTP and ligated in frame (pΔAPP770wt) (Fig. 1 a (i)). To produce EcoO65I site in the cytoplasmic domain of pΔAPP770wt, site-directed mutagenesis was introduced with PCR as follows: primer 1, 5′-GCCGCGGTCACCCCAGAGGAGCGCCACACCTGTCC-3′ (the nucleotide underlined were changed to produce EcoO65I site (T to G)), and primer 2, 5′-ATTTAGGTGACACTATAGAATAG-3′ (SP6 promoter primer), were used in PCR with PWO DNA polymerase (Boehringer Mannheim) in the presence of plasmid pΔAPP770wt. Primer 3, 5′-TCTGGGTGACCGCGGCGTCAACCTCCACC-3′ (the nucleotide underlined was changed to produce EcoO65I site (A to C)), and primer 4, 5′-TAATACGACTCACTATAGGG-3′ (T7 promoter primer), were used in PCR with PWO DNA polymerase in the presence of plasmid pΔAPP770wt. Both PCR products were digested with EcoO65I, ligated, and then inserted into pcDNA3 atHindIII/XbaI sites. Production of the EcoO65I site does not change the amino acid sequence in the APP protein, and this PCR procedure with PWO DNA polymerase did not induce nucleotide mutations. The position and direction of primers is indicated in Fig. 1 a (i). The pΔAPP770wt that introduced EcoO65I site was further amplified between primers 3 and 4 with Taq DNA polymerase (Takara Co., Kyoto, Japan). The Taq DNA polymerase introduces nucleotide mutations on newly synthesized DNA strands with a frequency of one base per approximately 400 bases (36Fromant M.B. Blanquest S. Plateau P. Anal. Biochem. 1995; 224: 347-353Crossref PubMed Scopus (236) Google Scholar). The resulting PCR products were ligated with pAPP770COOH, in which the 3′ downstream sequence from EcoO65I site of APP770 has been inserted into pcDNA3, at HindIII and EcoO65I sites (Fig. 1 a (ii)). The constructs for mutant pΔAPP770, pΔAPP770mut, were subcloned and transfected into 293 cells (human transfected primary embryonal kidney) with Lipofectin, and cell lines that expressed stably-transfected pΔAPP770mut were isolated. Among the cell lines isolated, cells displaying aberrant APP metabolism were further characterized. The site of mutation was detected by sequencing the DNA inserted in pΔAPP770mut, and the resulting amino acid substitution was listed in Table I. The mutation was also introduced into APP695cDNA to construct pAPP695mutby exchanging the HindIII/XcmI fragment from APP695cDNA with that from pΔAPP770mutwhich carries the mutation (Fig. 1 b (ii)). Cell lines that express stably-transfected pAPP695mut were also isolated and analyzed for APP metabolism. APP695mutcontains all the N-glycosylation sites and the complete amino acid sequence of APP695 except for the mutated site(s).Table ILists of mutant and the position of mutationAPPAmino acid position (APP695)124127171172173wtSerLeuLeuLeuPromut 1CysPromut 1aCysmut 1bPromut 2Alamut 3Promut 4ProLeumut 5Pro Open table in a new tab Intracellular APP and the truncated cytoplasmic domain, αAPPCOOH, derived from APP cleaved by α-secretase were detected by a combination of immunoprecipitation and immunoblot with anti-APP cytoplasmic domain antibody, UT-421, which is raised against a peptide (Cys)APP676–695 (the numbering for APP695 isoform). UT-421 is specific to APP, and does not react with amyloid precursor-like proteins, APLP1 and APLP2. 3Y. Satoh, unpublished observations. 293 cells (2–3 × 106 cells) were grown in Dulbecco's modified Eagle's medium containing 10% (v/v) heat-inactivated fetal bovine serum. APP and αAPPCOOHwere recovered through immunoprecipitation as described (18Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 21Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar). Immunoprecipitants were analyzed by SDS-PAGE (7.5% (w/v) polyacrylamide for ΔAPP770 and APP695 and 15% (w/v) polyacrylamide for αAPPCOOH) and transferred electrophoretically to a nitrocellulose membrane. The membrane was probed with UT-421 antibody followed by 125I-protein A (Amersham Corp., IM144). Specificity and identification of the immunoprecipitants were examined by a competition study with antigen peptide as described previously (18Suzuki T. Oishi M. Marshak D.R. Czernik A.J. Nairn A.C. Greengard P. EMBO J. 1994; 13: 1114-1122Crossref PubMed Scopus (212) Google Scholar, 21Oishi M. Nairn A.C. Czernik A.J. Lim G.S. Isohara T. Gandy S.E. Greengard P. Suzuki T. Mol. Med. 1997; 3: 111-123Crossref PubMed Google Scholar). The radioactivity of the immunoblot was quantitated using a Fuji BAS 2000 Imaging Analyzer (Tokyo, Japan) or by autoradiography. Deglycosylation of APP was performed with a procedure described previously (19Graebert K.S. Popp G.M. Kehlw T. Herzog V. Eur. J. Cell Biol. 1995; 66: 39-46PubMed Google Scholar). Antibody (UT-421)·APP complex was recovered from cell lysates following addition of protein A-Sepharose (Pharmacia Biotech Inc.). The beads were washed twice with reaction buffer, 40 mm Tris maleate (pH 6.0), 2.25 mm CaCl2, and then incubated with 1 milliunit of O-glycanase and/or 10 milliunits of neuraminidase (Seikagaku Co., Tokyo, Japan) in the same reaction buffer containing protease inhibitors as follows: 200 μg/ml (w/v) pepstatin A, 200 μg/ml (w/v) chymostatin, and 200 μg/ml (w/v) leupeptin. In a separate study, the beads were washed twice with reaction buffer, 50 mm citrate buffer (pH 5.5), and then incubated with 4 milliunits of endoglycosidase H (Seikagaku Co.) in the same reaction buffer containing protease inhibitors as follows: 200 μg/ml (w/v) pepstatin A, 200 μg/ml (w/v) chymostatin, and 200 μg/ml (w/v) leupeptin. After overnight digestion at 37 °C, the samples were subject to SDS-PAGE (7.5% (w/v) polyacrylamide) and analyzed by immunoblot using UT-421. Pulse-chase labeling of cells was carried out with [35S]methionine (1 mCi/ml; NEN Life Science Products, NEG-072). 293 cell lines that express stably-transfected ΔAPP770wt and ΔAPP770mut were labeled metabolically for 30 min, followed by a chase period as indicated. The chase was initiated by replacing the labeling medium with medium containing excess unlabeled methionine. ΔAPP770 was immunoprecipitated using UT-421 and analyzed with Fuji BAS 2000 Imaging Analyzer or autoradiography following SDS-PAGE (7.5% (w/v) polyacrylamide). Cultured cells were fixed for 20 min with 4% (w/v) paraformaldehyde in PBS (pH 7.4) containing 0.12m sucrose, permeabilized with 0.3% (v/v) Triton X-100 for 5 min, and blocked in 10% (w/v) solution of bovine serum albumin. The cells were incubated with the affinity purified primary antibody, UT-421, and then with fluorescein isothiocyanate-conjugated secondary antibody (Zymed, San Francisco, CA). The same cells were double-stained with rhodamine-conjugated ConA (Vector Laboratories, Burlingame, CA) which binds with high affinity to glycoproteins in the ER pluscis-Golgi and with rhodamine-conjugated WGA (Vector Laboratories) which binds with high affinity to glycoproteins in medial- plus trans-Golgi (37Virtanen I. Ekblom P. Laurila P. J. Cell Biol. 1980; 85: 429-434Crossref PubMed Scopus (163) Google Scholar, 38Tartakoff A.M. Vassalli P. J. Cell Biol. 1983; 97: 1243-1248Crossref PubMed Scopus (149) Google Scholar). The coverslips were mounted in Immersion oil type B (R. P. Cargille Laboratory Inc., Cedar Grove, NJ), and cells were viewed using a confocal laser scanning microscope, Bio-Rad MRC 600. Three monoclonal antibodies that recognize distinct portions of Aβ were used for quantification of Aβ species in medium. 2D1, raised against Aβ1–27, recognizes a human-specific epitope FRH600–602 between the β- and α-secretase sites. 4D1, raised against Cys + Aβ32–40, recognizes APP derivatives truncated at Aβ40 but not Aβ42. 4D8, raised against Gly-Gly + Aβ37–42, recognizes APP derivatives truncated at Aβ42 but not Aβ40. All monoclonal antibodies were purified with protein G-Sepharose (Pharmacia) from the ascites. Purified 2D1 was biotinylated with ECL protein biotinylation module (Amersham, RPN 2202). Conditioned media from cells (2 × 106 cells) were collected 18–20 h after medium change. Wells were coated with the monoclonal Aβ end-specific antibody, 4D1 or 4D8 (0.3 μg of antibody in a phosphate-buffered saline (PBS, 140 mm NaCl, 10 mm sodium phosphate (pH 7.2))), washed with PBS containing 0.05% (v/v) Tween 20 (washing buffer, WB), blocked with bovine serum albumin (3% (w/v) in PBS), washed with WB, and then a sample (100 μl) diluted suitably with WB containing 1% (w/v) bovine serum albumin (dilution buffer, DB) was incubated together with a standard of synthetic Aβ1–40 or Aβ1–42 peptides (synthesized at the W. M. Keck Foundation Biotechnology Resource Laboratory, Yale University). After washing, wells were treated with biotinized 2D1 (12.5 ng in DB), washed, and incubated with 100 μl of a streptavidin-horseradish peroxidase complex (1:2000 dilution: Amersham RPN1051). The plate was further washed, and 100 μl of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) peroxidase substrate solution (KPL 5062-01, Kirkegaard & Perry Laboratories Inc., Gaithersburg, MD) was added to wells and then incubated at room temperature. Reaction was stopped by addition of 100 μl of 1% (w/v) SDS, and the absorbance at 405 nm was determined. This procedure can quantify >0.4 ng of Aβ40 and Aβ42 in 100 μl of medium. To estimate the level of APP695 expression, APP from cells that expressed stably-transfected plasmids was immunoprecipitated from the same amount of protein lysate, detected by immunoblot with UT-421 following SDS-PAGE, and quantified using a Fuji BAS 2000 Imaging Analyzer. The level of APP695mut expression was normalized to the level of APP695wt expression, which was assigned a reference value of 1.0 and was indicated as a relative ratio. Quantity of Aβ40 and Aβ42 (fmol/100 μl of medium) was divided by the relative APP695 ratio and was indicated as an Aβ/APP ratio. To differentiate exogenous transfected APP from endogenous APP in 293 cells, a cDNA (pΔAPP770wt) was constructed, encoding APP770 lacking 287 amino acids (APP770379–666: numbering for APP770 isoform) of the extracellular domain (Fig. 1 a) as described under “Experimental Procedures.” An immunoblot with UT-421 showed that 293 cells, expressing pΔAPP770wt, presented two isoforms (Fig. 2 a). The deleted region contains two potential N-glycosylation sites (Fig. 1 and Refs. 13Weidemann A. Konig G. Bunke D. Fischer P. Salbaum J.M. Master C.L. Beyreuther K. Cell. 1989; 57: 115-126Abstract Full Text PDF PubMed Scopus (1038) Google Scholar, 15Påhlsson P. Shakin-Eshleman S.H. Spitalnik S.L. Biochem. Biophys. Res. Commun. 1992; 189: 1667-1673Crossref PubMed Scopus (71) Google Scholar, and 20Påhlsson P. Spitalnik S.L. Arch. Biochem. Biophys. 1996; 331: 177-186Crossref PubMed Scopus (55) Google Scholar), and it is well-characterized that endoglycosidase H removes the N-glycan portion of glycoproteins (reviewed in Refs. 39Ashwell G. Morell A.G. Adv. Enzymol. Relat. Areas Mol. Biol. 1974; 41: 99-128PubMed Google Scholar and 40Muramatsu T. Methods Enzymol. 1978; 50: 555-559Crossref PubMed Scopus (25) Google Scholar). Treatment of ΔAPP770wt with endoglycosidase H, isolated from cells which expressed it stably, did not alter the mobility of ΔAPP770 on SDS-PAGE (Fig. 2 a). This confirms thatN-glycosylation sites are deleted from the pΔAPP770 cDNA, and the resulting ΔAPP770wt is not subject to N-glycosylation in 293 cells. On the other hand, we found that ΔAPP770wt is modified by O-glycosylation with a terminal neuraminic acid of O-glycan because the treatment of ΔAPP770wt, isolated from the cell, with neuraminidase and a combination of neuraminidase and O-glycanase increased the mobility of ΔAPP770 on SDS-PAGE (Fig. 2 a). The treatment of ΔAPP770wt with O-glycanase alone had no effect because the sialic acid first needs to be removed to release O-glycan from the protein (data not shown). We tentatively assigned different ΔAPP770 species as follows: a high molecular weight O-glycosylation form is ogΔAPP770, and a low molecular weight non-glycosylated form is nonΔAPP770. The ΔAPP770wt treated with a combination of neuraminidase and O-glycanase does not show identical mobility with nonΔAPP770 on SDS-PAGE (compare Neu. +O-gly. with Control in Fig. 2 a). The ΔAPP770 may be subject to further unidentified modification. We also define, in a broad sense, this ΔAPP770, which may be carrying only the unidentified modification, as nonΔAPP770. When O-glycosylation and degradation of ΔAPP770wt were compared with those of endogenous APP in a pulse-chase study (Fig. 3 a), we found that the respective metabolic rate of nonΔAPP770 and endogenous imAPP and that of ogΔAPP770 and endogenous mAPP were identical (Fig. 4, a and b). These results indicate that the intracellular metabolism of ΔAPP770wt is normal.Figure 4Intracellular metabolism of ΔAPP770wt and ΔAPP770mut1. The relative ratios of mature endogenous APP (mAPP), immature endogenous APP (imAPP), O-glycosylated ΔAPP (ogΔAPP770), and naked ΔAPP770 (nonΔAPP770) are indicated relative to maximum levels, which were assigned a reference value of 1.0. a, metabolism of imAPP and nonΔAPP770wt. b, metabolism of mAPP and ogΔAPP770wt. c, metabolism of imAPP and nonΔAPP770mut1.d, metabolism of mAPP and ogΔAPP770mut1. Results are averages of duplicate pulse-chase studies, and the error bars are indicated.View Large Image Figure ViewerDownload (PPT) To introduce a mutation into its extracellular domain, pΔAPP770 was amplified with primers 3 and 4 using Taq DNA polymerase as shown in Fig. 1 a (i). The PCR fragments were substituted for a fragment from HindIII/EcoO65I digestion of pΔAPP770wt and subcloned into pcDNA3 vector as described under “Experimental Procedures.” The plasmid carrying a potential mutation (denoted as × in Fig. 1 a(ii)), pΔAPP770mut, was transfected into 293 cells, and approximately 100 independent clones of cells expressing ΔAPP770mut stably were tested for intracellular APP metabolism with immunoblot using UT-421 antibody. A cloned cell line that expresses pΔAPP770mut1 presented with abnormal APP metabolism (Fig. 2 b). The cells contained large amounts of nonΔAPP770 and relatively little ogΔAPP770. Treatment with glycosidases of APP recovered from the cells using UT-421 does not affect its mobility on SDS-PAGE when detected by immunoblot using UT-421 (Fig. 2 b). The mobility is identical to that of ΔAPP770wt treated with a combination of neuraminidase and O-glycanase (compare Neu. + O-gly in Fig. 2 a with Control in Fig. 2 b). These results indicate that ΔAPP770mut1 is not subject to O-glycosylation. DNA sequence analysis of pΔAPP770mut1 revealed that Ser-124 (all numbering for amino acid positions is for the APP695 isoform) was substituted for cysteine (Ser-124 → Cys), and Leu-172 was substituted for proline (Leu-172 → Pro) (Table I). It is reasonable to assume that either or perhaps both mutations interfere with the O-glycosylation of APP. Pulse-chase studies also confirmed aberrant metabolism of ΔAPP770mut1 (Fig. 3 b). Very small amounts of APP770mut1 wereO-glycosylated, and the majority of nonAPP770mut1accumulated intracellularly without O-glycosylation (Figs.3 b and 4 c). However, once ΔAPP770mut1 is O-glycosylated, ogΔAPP770mut1 is degraded in a process similar to that for endogenous mAPP (Fig. 4 d). The results indicate that ΔAPP770mut 1 is metabolized normally if it is modified with O-glycan, although the cellular content of ogΔAPP770mut1 is extremely low (Figs. 2 b and3 b). Identical results were obtained when the mutation was carried on the APP695 isoform. To construct pAPP695mut1, a fragment containing the mutations, Ser-124 → Cys and Leu-172 → Pro, which was derived from HindIII/XcmI digestion of pΔAPP770mut1, was substituted for a fragment from HindIII/XcmI of pAPP695 wild type (pAPP695wt) and subcloned (Fig. 1 b). pAPP695mut1 encodes the entire amino acid sequence including the N-glycosylation sites, except for the two amino acid mutations (Fig. 1 b (ii)). When 293 cells stably expressing pAPP695mut1 were selected and analyzed for APP metabolism with immunoblot, a result identical to that for pΔAPP770mut1 was observed (Fig. 5). Because 293 cells do not endogenously express APP695, a neuron-specific APP isoform, it is easy to identify exogenous APP695mut1. In the cells expressing APP695mut1, imAPP695 accumulated in large quantities, whereas only very small amounts of mAPP695 were detected (Fig. 5). The results confirm that the mutation, mut1, inhibitsO-glycosylation of APP. ΔAPP770mut1 and APP695mut1 contain two amino acid substitutions, Ser-124 → Cys and Leu-172 → Pro. To determine whic" @default.
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• W1983996266 title "Cleavage of Alzheimer's Amyloid Precursor Protein (APP) by Secretases Occurs after O-Glycosylation of APP in the Protein Secretory Pathway" @default.
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