Cleavage of Alzheimer's Amyloid Precursor Protein (APP) by Secretases Occurs after O-Glycosylation of APP in the Protein Secretory Pathway
IDENTIFICATION OF INTRACELLULAR COMPARTMENTS IN WHICH APP CLEAVAGE OCCURS WITHOUT USING TOXIC AGENTS THAT INTERFERE WITH PROTEIN METABOLISM*

Susumu TomitaDagger , Yutaka Kirino, and Toshiharu Suzuki§

From the Laboratory of Neurobiophysics, School of Pharmaceutical Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan

    ABSTRACT
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Abstract
Introduction
Procedures
Results
Discussion
References

beta -Amyloid peptide (Abeta ) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Abeta 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 Abeta generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defective O-glycosylation, we demonstrated that the majority of APP cleavage by alpha -, beta -, and gamma -secretases occurs after O-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (alpha APPCOOH), a product of alpha -secretase, and both Abeta 40 and Abeta 42 in medium, a product of beta - and gamma -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 alpha -, beta -, and gamma -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.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Alzheimer's disease (AD)1 is characterized by the presence of parenchymal and cerebrovascular beta -amyloid (Abeta ) deposits (1, 2). Abeta is a 39-43-amino acid peptide that is derived from Alzheimer's amyloid precursor protein (APP). The generation of Abeta is thought to be one of the major events of AD pathogenesis (reviewed in Refs. 3 and 4). 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 (5-12). APP is subject to post-translational modification such as glycosylation, sulfation, and phosphorylation during transit through the intracellular protein secretory pathway (13-22). 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 Abeta 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 (23-28) suggest that APP cleavage by alpha -secretase occurs in a secretory step in late Golgi. Although recent reports indicate that the ER is the site for generation of Abeta 42 but not Abeta 40 in the neuron (29, 30), Abeta 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 (31-33). 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 (alpha APPCOOH), a product of cleavage by alpha -secretase, and also failed to generate Abeta 40 and Abeta 42, products of cleavage by both beta - and gamma -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 (34). Generation of Abeta 42 in the ER (29, 30) may be a neuron-specific and/or a minor event.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Introduction of Random Mutation on APP cDNA and Construction of Plasmid-- cDNA encoding human APP770 was cloned from lambda ZAP HeLa cell cDNA library2 by immunoscreening with anti-APP antibody, G-369 (35). The cDNA was subcloned into pcDNA3 (Invitrogen) at HindIII/XbaI sites (Fig. 1a). 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 (13, 15, 20), was deleted by exclusion of XhoI/BglII fragment. The 3' recessed termini were filled with dNTP and ligated in frame (pDelta APP770wt) (Fig. 1a (i)). To produce EcoO65I site in the cytoplasmic domain of pDelta APP770wt, 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 pDelta APP770wt. 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 pDelta APP770wt. Both PCR products were digested with EcoO65I, ligated, and then inserted into pcDNA3 at HindIII/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. 1a (i).

The pDelta APP770wt 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 (36). 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. 1a (ii)). The constructs for mutant pDelta APP770, pDelta APP770mut, were subcloned and transfected into 293 cells (human transfected primary embryonal kidney) with Lipofectin, and cell lines that expressed stably-transfected pDelta APP770mut 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 pDelta APP770mut, and the resulting amino acid substitution was listed in Table I. The mutation was also introduced into APP695cDNA to construct pAPP695mut by exchanging the HindIII/XcmI fragment from APP695cDNA with that from pDelta APP770mut which carries the mutation (Fig. 1b (ii)). Cell lines that express stably-transfected pAPP695mut were also isolated and analyzed for APP metabolism. APP695mut contains all the N-glycosylation sites and the complete amino acid sequence of APP695 except for the mutated site(s).

Detection of APP-- Intracellular APP and the truncated cytoplasmic domain, alpha APPCOOH, derived from APP cleaved by alpha -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 APLP2.3

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 alpha APPCOOH were recovered through immunoprecipitation as described (18, 21). Immunoprecipitants were analyzed by SDS-PAGE (7.5% (w/v) polyacrylamide for Delta APP770 and APP695 and 15% (w/v) polyacrylamide for alpha APPCOOH) 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 (18, 21). The radioactivity of the immunoblot was quantitated using a Fuji BAS 2000 Imaging Analyzer (Tokyo, Japan) or by autoradiography.

Enzymatic Deglycosylation-- Deglycosylation of APP was performed with a procedure described previously (19). 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 Study-- 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 Delta APP770wt and Delta APP770mut 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. Delta APP770 was immunoprecipitated using UT-421 and analyzed with Fuji BAS 2000 Imaging Analyzer or autoradiography following SDS-PAGE (7.5% (w/v) polyacrylamide).

Immunocytochemistry-- Cultured cells were fixed for 20 min with 4% (w/v) paraformaldehyde in PBS (pH 7.4) containing 0.12 M 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 plus cis-Golgi and with rhodamine-conjugated WGA (Vector Laboratories) which binds with high affinity to glycoproteins in medial- plus trans-Golgi (37, 38). 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.

ELISA Analysis-- Three monoclonal antibodies that recognize distinct portions of Abeta were used for quantification of Abeta species in medium. 2D1, raised against Abeta 1-27, recognizes a human-specific epitope FRH600-602 between the beta - and alpha -secretase sites. 4D1, raised against Cys + Abeta 32-40, recognizes APP derivatives truncated at Abeta 40 but not Abeta 42. 4D8, raised against Gly-Gly + Abeta 37-42, recognizes APP derivatives truncated at Abeta 42 but not Abeta 40.

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 Abeta 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 Abeta 1-40 or Abeta 1-42 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 Abeta 40 and Abeta 42 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 Abeta 40 and Abeta 42 (fmol/100 µl of medium) was divided by the relative APP695 ratio and was indicated as an Abeta /APP ratio.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

Analysis of APPmut-- To differentiate exogenous transfected APP from endogenous APP in 293 cells, a cDNA (pDelta APP770wt) was constructed, encoding APP770 lacking 287 amino acids (APP770379-666: numbering for APP770 isoform) of the extracellular domain (Fig. 1a) as described under "Experimental Procedures." An immunoblot with UT-421 showed that 293 cells, expressing pDelta APP770wt, presented two isoforms (Fig. 2a). The deleted region contains two potential N-glycosylation sites (Fig. 1 and Refs. 13, 15, and 20), and it is well-characterized that endoglycosidase H removes the N-glycan portion of glycoproteins (reviewed in Refs. 39 and 40). Treatment of Delta APP770wt with endoglycosidase H, isolated from cells which expressed it stably, did not alter the mobility of Delta APP770 on SDS-PAGE (Fig. 2a). This confirms that N-glycosylation sites are deleted from the pDelta APP770 cDNA, and the resulting Delta APP770wt is not subject to N-glycosylation in 293 cells. On the other hand, we found that Delta APP770wt is modified by O-glycosylation with a terminal neuraminic acid of O-glycan because the treatment of Delta APP770wt, isolated from the cell, with neuraminidase and a combination of neuraminidase and O-glycanase increased the mobility of Delta APP770 on SDS-PAGE (Fig. 2a). The treatment of Delta APP770wt 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 Delta APP770 species as follows: a high molecular weight O-glycosylation form is ogDelta APP770, and a low molecular weight non-glycosylated form is nonDelta APP770. The Delta APP770wt treated with a combination of neuraminidase and O-glycanase does not show identical mobility with nonDelta APP770 on SDS-PAGE (compare Neu. + O-gly. with Control in Fig. 2a). The Delta APP770 may be subject to further unidentified modification. We also define, in a broad sense, this Delta APP770, which may be carrying only the unidentified modification, as nonDelta APP770.


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Fig. 1.   Schematic model for construction of Delta APP770 and APP695 mutants. a, construction of pDelta APP770mut. (i) To construct pDelta APP770wt, the N-glycosylation site was excluded by deletion of 0.9 kilobase pairs of XhoI/BglII fragment (APP379-666; numbering for amino acid). Primers 1 and 2 were used to produce EcoO65I site. The pDelta APP770wt was amplified with Taq DNA polymerase with EcoO65I (#3) and HindIII (#4) primers. (ii) The PCR product was ligated with pAPP770 lacking HindIII/EcoO65I fragment. The resulting pDelta APP770mut contains 2-4 substitution mutations. Delta APP770mut1 contains mutations at the sites of Ser-124 and Leu-172 (denoted as × and see Table I). b, construction of pAPP695mut1. (i) pAPP695 cDNA. (ii) HindIII/XcmI fragment containing mutation at the sites of Ser-124 and Leu-172 was dissected from pDelta APP770mut1 and exchanged to a HindIII/XcmI fragment from pAPP695 to construct pAPP695mut1. alpha , beta , and gamma  indicate the cleaving site by secretases. TM, transmembrane domain; N, amino-terminal; C, carboxyl-terminal of APP.


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Fig. 2.   Characterization of Delta APP770wt and Delta APP770mut1. Delta APP770wt (a) and Delta APP770mut1 (b) were recovered by immunoprecipitation from 293 cells that express corresponding cDNA and were treated with the enzymes indicated. ogDelta APP770, O-glycosylated Delta APP770; nonDelta APP770, naked Delta APP770 without glycosylation; Control, sample treated without enzymes; Endo H, sample treated with endoglycosidase H; Neu, sample treated with neuraminidase; Neu + O-Gly, samples treated with a combination of neuraminidase and O-glycanase. 116 and 76 are standard molecular mass (kDa) of protein.

When O-glycosylation and degradation of Delta APP770wt were compared with those of endogenous APP in a pulse-chase study (Fig. 3a), we found that the respective metabolic rate of nonDelta APP770 and endogenous imAPP and that of ogDelta APP770 and endogenous mAPP were identical (Fig. 4, a and b). These results indicate that the intracellular metabolism of Delta APP770wt is normal.


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Fig. 3.   Autoradiogram of pulse-chase study of Delta APP770wt and Delta APP770mut1. 293 cells expressing Delta APP770wt (a) and Delta APP770mut1 (b) were pulse-labeled with [35S]methionine for 30 min and chased for periods (0-3 h) as indicated. mAPP, mature (N- and O-glycosylated) endogenous APP; imAPP, immature (N-glycosylated) endogenous APP; ogDelta APP770, O-glycosylated Delta APP770wt and Delta APP770mut1; nonDelta APP770, naked Delta APP770wt and Delta APP770mut1 without glycosylation.


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Fig. 4.   Intracellular metabolism of Delta APP770wt and Delta APP770mut1. The relative ratios of mature endogenous APP (mAPP), immature endogenous APP (imAPP), O-glycosylated Delta APP (ogDelta APP770), and naked Delta APP770 (nonDelta APP770) are indicated relative to maximum levels, which were assigned a reference value of 1.0. a, metabolism of imAPP and nonDelta APP770wt. b, metabolism of mAPP and ogDelta APP770wt. c, metabolism of imAPP and nonDelta APP770mut1. d, metabolism of mAPP and ogDelta APP770mut1. Results are averages of duplicate pulse-chase studies, and the error bars are indicated.

To introduce a mutation into its extracellular domain, pDelta APP770 was amplified with primers 3 and 4 using Taq DNA polymerase as shown in Fig. 1a (i). The PCR fragments were substituted for a fragment from HindIII/EcoO65I digestion of pDelta APP770wt and subcloned into pcDNA3 vector as described under "Experimental Procedures." The plasmid carrying a potential mutation (denoted as × in Fig. 1a (ii)), pDelta APP770mut, was transfected into 293 cells, and approximately 100 independent clones of cells expressing Delta APP770mut stably were tested for intracellular APP metabolism with immunoblot using UT-421 antibody. A cloned cell line that expresses pDelta APP770mut1 presented with abnormal APP metabolism (Fig. 2b). The cells contained large amounts of nonDelta APP770 and relatively little ogDelta APP770. 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. 2b). The mobility is identical to that of Delta APP770wt treated with a combination of neuraminidase and O-glycanase (compare Neu. + O-gly in Fig. 2a with Control in Fig. 2b). These results indicate that Delta APP770mut1 is not subject to O-glycosylation. DNA sequence analysis of pDelta APP770mut1 revealed that Ser-124 (all numbering for amino acid positions is for the APP695 isoform) was substituted for cysteine (Ser-124 right-arrow Cys), and Leu-172 was substituted for proline (Leu-172 right-arrow 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 Delta APP770mut1 (Fig. 3b). Very small amounts of APP770mut1 were O-glycosylated, and the majority of nonAPP770mut1 accumulated intracellularly without O-glycosylation (Figs. 3b and 4c). However, once Delta APP770mut1 is O-glycosylated, ogDelta APP770mut1 is degraded in a process similar to that for endogenous mAPP (Fig. 4d). The results indicate that Delta APP770mut 1 is metabolized normally if it is modified with O-glycan, although the cellular content of ogDelta APP770mut1 is extremely low (Figs. 2b and 3b).

                              
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Table I
Lists of mutant and the position of mutation
Changes introduced into the wild-type (wt) amino acid sequence are listed (numbering of amino acid position for APP695 isoform).

Identical results were obtained when the mutation was carried on the APP695 isoform. To construct pAPP695mut1, a fragment containing the mutations, Ser-124 right-arrow Cys and Leu-172 right-arrow Pro, which was derived from HindIII/XcmI digestion of pDelta APP770mut1, was substituted for a fragment from HindIII/XcmI of pAPP695 wild type (pAPP695wt) and subcloned (Fig. 1b). pAPP695mut1 encodes the entire amino acid sequence including the N-glycosylation sites, except for the two amino acid mutations (Fig. 1b (ii)). When 293 cells stably expressing pAPP695mut1 were selected and analyzed for APP metabolism with immunoblot, a result identical to that for pDelta APP770mut1 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, inhibits O-glycosylation of APP.


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Fig. 5.   Characterization of APP695wt and APP695mut1. APP695wt and APP695mut1 were recovered by immunoprecipitation from 293 cells that express corresponding cDNA, and endogenous APP was also recovered from non-transfected 293 cells. Arrows indicate mAPP695 and imAPP695. Arrowhead indicates endogenous imAPP. mock, non-transfected 293 cells; APP695wt, 293 cells expressing APP695wt; APP695mut1, 293 cells expressing APP695mut1. 170 and 116 are standard molecular mass (kDa) of protein.

Determination of Mutation Site Inhibiting O-Glycosylation-- Delta APP770mut1 and APP695mut1 contain two amino acid substitutions, Ser-124 right-arrow Cys and Leu-172 right-arrow Pro. To determine which mutation inhibits O-glycosylation, we constructed plasmids (pAPP695mut) carrying several mutations including a single amino acid substitution for Ser-124 right-arrow Cys and Leu-172 right-arrow Pro as follows: pAPP695mut1a carries Ser-124 right-arrow Cys, pAPP695mut1b carries Leu-172 right-arrow Pro, pAPP695mut2 carries a mutation of leucine at position 172 changed to alanine (Leu-172 right-arrow Ala), pAPP695mut3 carries a mutation of leucine at position 171 changed to proline (Leu-171 right-arrow Pro), pAPP695mut4 carries a double mutation of Leu-172 right-arrow Pro and a mutation of proline at position 173 changed to leucine (Pro-173 right-arrow Leu), and pAPP695mut5 carries a mutation of leucine 127 changed to proline (Leu-127 right-arrow Pro) (Table I). These mutant APP plasmids were stably expressed in 293 cells, and APP metabolism was examined (Fig. 6a). Among the mutants, APP695mut1b, APP695mut3, and APP695mut4 presented with abnormal imAPP695 accumulation. The ratio of mAPP695 to total APP695 was estimated (Fig. 6b). Generally the ratio of mAPP695 to total APP695 in pAPP695wt was 0.2-0.3 (mAPP695/total APP695). The ratio of mAPP695/total APP695 in cells expressing APP695mut1b, APP695mut3, and APP695mut4 was approximately 0.05, which is identical to that for APP695mut1. The ratio of mAPP695/total APP695 for APPmut1a, APPmut2, and APPmut5 is identical to, or slightly lower, that of APPwt but is significantly higher than that of APPmut1 (the ratio of mAPP695/total APP695 is >0.15). These results indicate that it is the Leu-172 right-arrow Pro substitution that affects O-glycosylation (or maturation) of APP, although a Ser-124 right-arrow Cys substitution in APPmut1 may contribute to the aberrant metabolism of APP. However, a Leu-172 right-arrow Ala substitution (mut2) does not appear to inhibit O-glycosylation. When an unrelated leucyl residue at position 127 was changed to proline, Leu-127 right-arrow Pro (mut5), O-glycosylation was observed in the same manner as APP695wt.


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Fig. 6.   Determination of mutation site inhibiting O-glycosylation. a, APP695mut containing various sites of mutation was recovered by immunoprecipitation from 293 cells that express corresponding cDNA. Arrows indicate mAPP695 and imAPP695. b, relative ratio of mature APP695. Results are the average of three independent studies (n = 3), and the error bar indicates standard deviation. mAPP695 and imAPP695 in a were quantified using a Fuji BAS 2000 Imaging Analyzer. The ratio of mAPP/total APP was determined and compared to quantify the inhibitory level of O-glycosylation. wt, APP695wt; mut1, APP695mut1 (Ser-124 right-arrow Cys/Leu-172 right-arrow Pro double mutant); mut1a, APP695mut1a (Ser-124 right-arrow Cys mutant); mut1b, APP695mut1b (Leu-172 right-arrow Pro mutant); mut2, APP695mut2 (Leu-172 right-arrow Ala mutant); mut3, APPmut3 (Leu-171 right-arrow Pro mutant); mut4, APPmut4 (Leu-172 right-arrow Pro/Pro173 right-arrow Leu double mutant); mut5, APPmut5 (Leu-127 right-arrow Pro mutant). See also Table I for the positions of mutation.

Intracellular Distribution of APPmut1-- To study the intracellular localization of APPmut1, 293 cells expressing APP695mut1 and APP695wt were double-stained with UT-421 and ConA (ER plus cis-Golgi marker) or WGA (medial- plus trans-Golgi marker) and then observed under a confocal laser scanning microscope (Fig. 7). We confirmed that the APP695wt was distributed in ER and Golgi apparatus (Fig. 7, a and b) as described previously (19, 41-44). The APP695wt co-localized with the staining of the ER plus cis-Glogi with ConA (Fig. 7a) and of medial- plus trans-Golgi with WGA (Fig. 7b). However, APP695mut1 seemed to be distributed in cytoplasm, including ER, but not in late Golgi because the distribution of APP695mut1 was identical with the staining using ConA (Fig. 7c) but not using WGA (Fig. 7d). When non-transfected 293 cells were stained, only a background level of fluorescence was observed (Fig. 7e) because the level of expression of endogenous APP is very low in 293 cells (Fig. 5). Therefore, the immunostaining observed in this study is thought to be due to the result of transfected exogenous APP. The stainings of ER plus cis-Glogi with ConA and of medial- plus trans-Golgi with WGA in the transfected cells (Fig. 7, a-d) showed an identical pattern to that in non-transfected 293 cells (Fig. 7e). These results clearly indicate that the intracellular distribution of APP695mut1 is abnormal and that the majority of APP695mut1 is distributed in the ER, in contrast to APP695wt, which is distributed in both the ER and the Golgi equally.


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Fig. 7.   Localization of APP695wt and APP695mut1. Intracellular localization of APP695wt and ER plus cis-Golgi (a), APP695wt and medial-plus trans-Golgi (b), APP695mut1 and ER plus cis-Golgi (c), APP695mut1 and medial-plus trans-Golgi (d), and endogenous APP, ER plus cis-Golgi and medial-plus trans-Golgi in non-transfected 293 cells (e). Cells expressing APP695wt (a and b) and APP695mut1 (c and d) and non-transfected 293 cells (e) were stained with anti-APP cytoplasmic domain antibody, UT-421 and observed under a confocal laser scanning microscope. The cells were also double-stained with rhodamine-conjugated ConA to identify the location of ER plus cis-Golgi and rhodamine-conjugated WGA to identify the location of medial-plus trans-Golgi, respectively. Scale bar, 20 µm in (a-d) and 50 µm in (e).

Cleavage of APP Occurs after O-Glycosylation-- It has been well characterized that APP is cleaved preferentially at the alpha -site compared with the beta -site and that the carboxyl-terminal fragment of APP, alpha APPCOOH, is generated intracellularly. The generation of alpha APPCOOH from APP695mut1 was examined. alpha APPCOOH was recovered by immunoprecipitation with UT-421 from the lysates of 293 cells expressing APP695wt and APP695mut1, separated by SDS-PAGE (15% (w/v) polyacrylamide gel), and analyzed by immunoblot using UT-421 (Fig. 8). APP695wt generates a 14-15-kDa alpha APPCOOH (alpha APPCOOH presents a higher molecular weight on the SDS-PAGE than its actual molecular weight) that has been fully characterized (18, 45, 46). Because expression of endogenous APP in 293 cells is extremely low (Figs. 5 and 7e) and production of endogenous alpha APPCOOH was under the detectable level (data not shown), it is clear that the detected alpha APPCOOH in Fig. 8a is derived from transfected exogenous APP695wt. Only extremely low levels of alpha APPCOOH were detected in cells expressing APP695mut1 (Fig. 8a), although the level of APP695mut1 expression was almost identical to that of APP695wt (Figs. 2, 5, 6, and 7). The lower production of alpha APPCOOH was observed in several independently cloned cells (mut1-1-3) that stably express APP695mut1. The production of alpha APPCOOH was quantified and indicated as a ratio of alpha APPCOOH to total APP (alpha APPCOOH/total APP) in Fig. 8b. The results indicate that APP cleavage by alpha -secretase occurs after, or possibly during, O-glycosylation of APP.


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Fig. 8.   Detection of intracellular alpha APPCOOH, a product by alpha -secretase. a, autoradiogram of alpha APPCOOH derived from APP695. alpha APPCOOH was recovered by immunoprecipitation with UT-421 antibody from two independent clones of 293 cells expressing APP695wt and three independent clones of 293 cells expressing APP695mut1. Arrow indicates alpha APPCOOH. wt-1 and wt-2, independent clones of 293 cells expressing APP695wt; mut1-1, mut1-2, and mut1-3, independent clones of 293 cells expressing APP695mut1. 20.1 and 14.4 are standard molecular mass (kDa) of protein. b, quantification of alpha APPCOOH. APP and alpha APPCOOH were quantified using a Fuji BAS 2000 Imaging Analyzer. The level of alpha APPCOOH production was normalized to the amount of APP. Quantity of alpha APPCOOH was divided by the relative APP ratio and indicated as alpha APPCOOH/total APP ratio. Results are the average of five independent studies (n = 5), and the error bar indicates standard deviation. wt, 293 cells expressing APP695wt; mut1, 293 cells expressing APP695mut1.

We also quantified the amount of Abeta 40 and Abeta 42, the major Abeta isoforms, in the medium of 293 cells expressing APP695mut1 by sandwich ELISA using an Abeta -specific monoclonal antibody 2D1 (epitope is FRH600-602), and 4D1 or 4D8, the Abeta carboxyl-terminal end-specific monoclonal antibodies that recognize Abeta 40 and Abeta 42, respectively. The level of APP expression was quantified with immunoblot as described under "Experimental Procedures." We indicated the level of Abeta production as a ratio of the amount of Abeta to APP expression level (Abeta /APP) in Fig. 9. Production of both Abeta 40 (Fig. 9a) and Abeta 42 (Fig. 9b) from APP695mut1 was found to be very low. Identical results were also obtained from the study with 293 cells expressing Delta APP770wt and Delta APP770mut1 (data not shown). These results suggest that the majority of APP cleavage at beta - and gamma -sites also occurs after, or possibly during, O-glycosylation of APP.


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Fig. 9.   Analysis of Abeta in the medium. Abeta 40 (a) and Abeta 42 (b) were quantified as described under "Experimental Procedures." Quantity of Abeta 40 (a) and Abeta 42 (b) (fmol/100 µl of medium) was divided by the relative level of total APP and indicated as the ratio of Abeta /APP. Results are the average of eight independent clones (n = 8 for APP695wt) and that of two independent clones (n = 4 for APP695 mut1). The error bar indicates standard deviation (***, p < 0.001; **, p < 0.05).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

APP is thought to be cleaved by alpha -, beta -, and gamma -secretases in the protein secretory pathway. Previous studies using protein metabolic inhibitors (chloroquine, brefeldin A, bafilomycin A1, etc.) suggest that APP cleavage by alpha -secretase occurs in the trans-Golgi network or other late compartments of the protein secretory pathway (23, 24, 26-28), and that cleavage by beta -secretase occurs in acidic compartments such as endosome and/or late Golgi (31-33). These results are plausible, but one must consider the fundamental problem of drugs, which may affect protein metabolism nonspecifically, that were used in the previous studies. The results obtained from such studies may have been due to indirect or generic effects on APP metabolism. Furthermore, recent reports suggest that the production of Abeta 42 but not Abeta 40 occurs in the ER (29, 30). However, the biochemical quantification of Abeta 42 production in ER without toxic drugs has not been performed. Therefore, we conducted further biochemical studies to confirm the previous results that had been obtained using metabolic inhibitors.

In the present study, we found that APPmut1 is defective for O-glycosylation and is metabolized aberrantly in normal cells. APPmut1 contains two sites of substitution mutation, Ser-124 right-arrow Cys and Leu-172 right-arrow Pro, and Delta APP770 also has a deleted sequence within the extracellular domain, including N-glycosylation sites. Present results demonstrate that only the Leu-172 right-arrow Pro mutation (mut1b) is effective in inhibiting O-glycosylation of APP. Two explanations for altered metabolism of APP by the mutation are possible. One is that the amino acid sequence around Leu-172 is essential for O-glycosylation itself and/or for recognition by the enzyme(s) which is responsible for O-glycosylation. There are several seryl or threonyl residues, Ser-162, Thr-163, Ser-193, Ser-198, and Ser-206, around the position of Leu-172. Generally, seryl and threonyl residues are candidates for modification by O-linked carbohydrates. However, the amino acid sequences around those seryl and threonyl residues do not appear to contain the recognition motif for GalNAC-transferase, although the peptide motifs that allow for O-glycosylation have not been identified (47). Another possibility is that the mutation of Leu-172 right-arrow Pro (mut1b) may inhibit O-glycosylation by causing a partial mis-folding of the APP amino-terminal which in turn may cause failure of APP transportation within the Golgi complex. Because the Leu-172 right-arrow Ala mutation (mut2) is not effective but the Leu-171 right-arrow Pro mutation (mut3) and a double mutation, Leu-172 right-arrow Pro/Pro-173 right-arrow Leu (mut4), showed an effect identical to the Leu-172 right-arrow Pro mutation (mut1b), it is thought that the prolinyl residue at position 171 or 172 may induce a conformational change and then mis-folding of the APP amino-terminal. This may be critical for O-glycosylation or the passing of APP into the Golgi complex. APPmut1 does not exhibit a conformational change in its beta -amyloid and carboxyl-terminal domains because APPmut1 is metabolized normally if it is modified with O-glycosylation, but this modification is very rare in APPmut1. This result indicates that the mutation does not alter the substrate specificity to the secretases.

The amino acid sequence of the deleted region, APP770379-666, does not contain any known functional domains for APP metabolism. Delta APP770wt matures and degrades identically to APP770. Furthermore, the Leu-172 right-arrow Pro mutation does not affect N-glycosylation, as imAPP695mut1 showed identical mobility to imAPP695wt on SDS-PAGE. The cells expressing APPmut1 present a reliable system to analyze whether APP is cleaved after O-glycosylation, without using drugs which inhibit intracellular protein metabolism indiscriminately. The present results clearly demonstrate that the majority of APPmut1 is not cleaved by alpha -secretase and alpha APPCOOH is not generated. The results indicate, without using cytotoxic drugs, that APP cleavage at the alpha -site occurs in a metabolic step following trans-Golgi after proteins have completed O-glycosylation, although we cannot rule out the possibility that the cleavage occurs during a metabolic step of O-glycosylation. Our results agree with previous observations using monensin and brefeldin A (25, 42, 48).

Quantification, using sandwich ELISA, of Abeta 40 and Abeta 42 in the medium of 293 cells expressing APP695mut1, indicates that a majority of APP cleavage at the beta - and gamma -sites also occurs after O-glycosylation, although we cannot rule out the possibility that a small quantity of APP is subject to cleavage by secretases at an earlier step during O-glycosylation modification. Previous reports using ammonium chloride and chloroquine suggest that Abeta may be generated in acidic compartments following medial-Golgi (31-33). However, the present study suggests that both Abeta 40 and Abeta 42 are generated subsequent to trans-Golgi, as in the case of alpha -cleavage in 293 cells. However, it is not known whether the molecular mechanism of APP processing in 293 cells is identical to that in neurons. Furthermore, recent reports suggested that Abeta 42 but not Abeta 40 is able to accumulate intracellularly (29, 30, 48, 49). Although we do not rule out a possibility of a very minor ratio of intracellular Abeta 42 accumulation in 293 cells expressing APP695mut1, the intracellular accumulation of Abeta , which is not secreted, may not be critical for the pathogenesis of AD because it has been well analyzed that the Abeta accumulation is an extracellular event in the brain of AD patient.

In previous studies, to identify the intracellular site of APP cleavage by beta -secretase, APP carrying the Swedish double mutation was often utilized (50). We have not used such an FAD mutant APP because we feel that the mechanisms of cleavage at the beta -site of APP carrying an FAD mutation differ from those of non-FAD patients.4 Therefore, our approach may be more useful in understanding the molecular mechanism of the pathogenesis of non-FAD.

    ACKNOWLEDGEMENTS

We thank Dr. K. Yamamoto (University of Tokyo) and T. Ozaki (Chiba Cancer Center, Chiba, Japan) for valuable technical advice; Dr. M. Oishi (Montefiore Medical Center, CT) for the critical reading of this manuscript; Dr. P. Greengard (Rockefeller University, NY) for supplying the antibody, G369; Drs. S. Takeda and Y. Yagi (University of Tokyo) for critical comments and helpful discussions; and S. Oguchi for technical assistance.

    FOOTNOTES

* This research was supported in part by a grant from Program for Promotion of Basic Research Activity for Innovative Bioscience.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of Japan Society for the Promotion of Science Research Fellowships for Young Scientists.

§ To whom correspondence should be addressed. Tel./Fax: 81-3-3814-6937; E-mail: t-suzuki{at}mayqueen.f.u-tokyo.ac.jp.

1 The abbreviations used are: AD, Alzheimer's disease; Abeta , beta -amyloid; APP, amyloid precursor protein; alpha APPCOOH, alpha -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.

2 T. Suzuki, unpublished observations.

3 Y. Satoh, unpublished observations.

4 S. Tomita, Y. Kivino, and T. Suzuki, manuscript in preparation.

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