From the Laboratory of Neurobiophysics, School of Pharmaceutical
Sciences, The University of Tokyo, Hongo 7-3-1, Bunkyo-ku, Tokyo 113-0033, Japan
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INTRODUCTION |
Alzheimer's disease
(AD)1 is characterized by the
presence of parenchymal and cerebrovascular
-amyloid (A
) deposits
(1, 2). A
is a 39-43-amino acid peptide that is derived from
Alzheimer's amyloid precursor protein (APP). The generation of A
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 A
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
-secretase occurs in a secretory step
in late Golgi. Although recent reports indicate that the ER is the site
for generation of A
42 but not A
40 in the neuron (29, 30), A
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 (
APPCOOH), a product
of cleavage by
-secretase, and also failed to generate A
40 and
A
42, products of cleavage by both
- and
-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 A
42 in the ER (29, 30) may be a neuron-specific and/or
a minor event.
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EXPERIMENTAL PROCEDURES |
Introduction of Random Mutation on APP cDNA and Construction
of Plasmid--
cDNA encoding human APP770 was cloned from
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
(p
APP770wt) (Fig. 1a (i)). To
produce EcoO65I site in the cytoplasmic domain of
p
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 p
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 p
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 p
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 p
APP770, p
APP770mut, were
subcloned and transfected into 293 cells (human transfected primary
embryonal kidney) with Lipofectin, and cell lines that expressed
stably-transfected p
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 p
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 p
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,
APPCOOH, derived from APP cleaved by
-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
APPCOOH
were recovered through immunoprecipitation as described (18, 21).
Immunoprecipitants were analyzed by SDS-PAGE (7.5% (w/v)
polyacrylamide for
APP770 and APP695 and 15% (w/v) polyacrylamide
for
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
APP770wt and
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.
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 A
were used for quantification of A
species
in medium. 2D1, raised against A
1-27, recognizes a human-specific
epitope FRH600-602 between the
- and
-secretase
sites. 4D1, raised against Cys + A
32-40, recognizes APP derivatives
truncated at A
40 but not A
42. 4D8, raised against Gly-Gly + A
37-42, recognizes APP derivatives truncated at A
42 but not
A
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 A
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 A
1-40 or A
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 A
40 and A
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 A
40 and A
42 (fmol/100 µl of medium) was divided by the
relative APP695 ratio and was indicated as an A
/APP ratio.
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RESULTS |
Analysis of APPmut--
To differentiate exogenous transfected APP
from endogenous APP in 293 cells, a cDNA (p
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 p
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
APP770wt with endoglycosidase H, isolated from cells
which expressed it stably, did not alter the mobility of
APP770 on
SDS-PAGE (Fig. 2a). This confirms that
N-glycosylation sites are deleted from the p
APP770
cDNA, and the resulting
APP770wt is not subject to
N-glycosylation in 293 cells. On the other hand, we found
that
APP770wt is modified by O-glycosylation
with a terminal neuraminic acid of O-glycan because the
treatment of
APP770wt, isolated from the cell, with
neuraminidase and a combination of neuraminidase and
O-glycanase increased the mobility of
APP770 on SDS-PAGE
(Fig. 2a). The treatment of
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
APP770
species as follows: a high molecular weight O-glycosylation
form is og
APP770, and a low molecular weight non-glycosylated form
is non
APP770. The
APP770wt treated with a combination
of neuraminidase and O-glycanase does not show identical
mobility with non
APP770 on SDS-PAGE (compare Neu. + O-gly. with Control in Fig. 2a). The
APP770 may be subject to further unidentified modification. We also
define, in a broad sense, this
APP770, which may be carrying only
the unidentified modification, as non
APP770.

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Fig. 1.
Schematic model for construction of APP770
and APP695 mutants. a, construction of
p APP770mut. (i) To construct
p 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 p 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
p APP770mut contains 2-4 substitution mutations.
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 p APP770mut1 and exchanged to a
HindIII/XcmI fragment from pAPP695 to construct
pAPP695mut1. , , and 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 APP770wt
and APP770mut1. APP770wt
(a) and APP770mut1 (b) were
recovered by immunoprecipitation from 293 cells that express
corresponding cDNA and were treated with the enzymes indicated.
og APP770, O-glycosylated APP770; non APP770, naked 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.
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When O-glycosylation and degradation of
APP770wt were compared with those of endogenous APP in a
pulse-chase study (Fig. 3a),
we found that the respective metabolic rate of non
APP770 and
endogenous imAPP and that of og
APP770 and endogenous mAPP were
identical (Fig. 4, a and
b). These results indicate that the intracellular metabolism
of
APP770wt is normal.

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Fig. 3.
Autoradiogram of pulse-chase study of
APP770wt and APP770mut1. 293 cells
expressing APP770wt (a) and
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; og APP770,
O-glycosylated APP770wt and
APP770mut1; non APP770, naked
APP770wt and APP770mut1 without glycosylation.
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Fig. 4.
Intracellular metabolism of
APP770wt and APP770mut1. The
relative ratios of mature endogenous APP (mAPP),
immature endogenous APP (imAPP), O-glycosylated
APP (og APP770), and naked APP770
(non APP770) are indicated relative to maximum
levels, which were assigned a reference value of 1.0. a,
metabolism of imAPP and non APP770wt. b,
metabolism of mAPP and og APP770wt. c,
metabolism of imAPP and non APP770mut1.
d, metabolism of mAPP and
og APP770mut1. Results are averages of duplicate
pulse-chase studies, and the error bars are indicated.
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To introduce a mutation into its extracellular domain, p
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 p
APP770wt and subcloned into pcDNA3
vector as described under "Experimental Procedures." The plasmid
carrying a potential mutation (denoted as × in Fig. 1a
(ii)), p
APP770mut, was transfected into 293 cells, and approximately 100 independent clones of cells expressing
APP770mut stably were tested for intracellular APP
metabolism with immunoblot using UT-421 antibody. A cloned cell line
that expresses p
APP770mut1 presented with abnormal APP
metabolism (Fig. 2b). The cells contained large amounts of
non
APP770 and relatively little og
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
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
APP770mut1 is not subject to
O-glycosylation. DNA sequence analysis of
p
APP770mut1 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
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
APP770mut1 is O-glycosylated,
og
APP770mut1 is degraded in a process similar to that for
endogenous mAPP (Fig. 4d). The results indicate that
APP770mut 1 is metabolized normally if it is modified
with O-glycan, although the cellular content of og
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).
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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
p
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
p
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.
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Determination of Mutation Site Inhibiting
O-Glycosylation--
APP770mut1 and
APP695mut1 contain two amino acid substitutions, Ser-124
Cys and Leu-172
Pro. To determine which mutation inhibits
O-glycosylation, we constructed plasmids
(pAPP695mut) carrying several mutations including a single
amino acid substitution for Ser-124
Cys and Leu-172
Pro as
follows: pAPP695mut1a carries Ser-124
Cys,
pAPP695mut1b carries Leu-172
Pro, pAPP695mut2 carries a mutation of leucine at position 172 changed to alanine (Leu-172
Ala), pAPP695mut3 carries a mutation of leucine
at position 171 changed to proline (Leu-171
Pro),
pAPP695mut4 carries a double mutation of Leu-172
Pro and
a mutation of proline at position 173 changed to leucine (Pro-173
Leu), and pAPP695mut5 carries a mutation of leucine 127 changed to proline (Leu-127
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
Pro substitution that
affects O-glycosylation (or maturation) of APP, although a
Ser-124
Cys substitution in APPmut1 may contribute to
the aberrant metabolism of APP. However, a Leu-172
Ala substitution
(mut2) does not appear to inhibit O-glycosylation. When an unrelated leucyl residue at
position 127 was changed to proline, Leu-127
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
Cys/Leu-172 Pro double mutant); mut1a,
APP695mut1a (Ser-124 Cys mutant); mut1b,
APP695mut1b (Leu-172 Pro mutant); mut2,
APP695mut2 (Leu-172 Ala mutant); mut3,
APPmut3 (Leu-171 Pro mutant); mut4,
APPmut4 (Leu-172 Pro/Pro173 Leu double mutant);
mut5, APPmut5 (Leu-127 Pro mutant). See also
Table I for the positions of mutation.
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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).
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Cleavage of APP Occurs after O-Glycosylation--
It has been well
characterized that APP is cleaved preferentially at the
-site
compared with the
-site and that the carboxyl-terminal fragment of
APP,
APPCOOH, is generated intracellularly. The
generation of
APPCOOH from APP695mut1 was
examined.
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
APPCOOH (
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
APPCOOH was under the detectable level (data not shown),
it is clear that the detected
APPCOOH in Fig.
8a is derived from transfected exogenous APP695wt. Only extremely low levels of
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
APPCOOH was observed in several independently cloned
cells (mut1-1-3) that stably express APP695mut1.
The production of
APPCOOH was quantified and indicated as a ratio of
APPCOOH to total APP
(
APPCOOH/total APP) in Fig. 8b. The results
indicate that APP cleavage by
-secretase occurs after, or possibly
during, O-glycosylation of APP.

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Fig. 8.
Detection of intracellular
APPCOOH, a product by -secretase. a,
autoradiogram of APPCOOH derived from APP695.
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 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 APPCOOH. APP
and APPCOOH were quantified using a Fuji BAS 2000 Imaging Analyzer. The level of APPCOOH production was
normalized to the amount of APP. Quantity of
APPCOOH was divided by the relative APP ratio and
indicated as 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.
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We also quantified the amount of A
40 and A
42, the major A
isoforms, in the medium of 293 cells expressing APP695mut1
by sandwich ELISA using an A
-specific monoclonal antibody 2D1
(epitope is FRH600-602), and 4D1 or 4D8, the A
carboxyl-terminal end-specific monoclonal antibodies that recognize
A
40 and A
42, respectively. The level of APP expression was
quantified with immunoblot as described under "Experimental
Procedures." We indicated the level of A
production as a ratio of
the amount of A
to APP expression level (A
/APP) in Fig.
9. Production of both A
40 (Fig.
9a) and A
42 (Fig. 9b) from
APP695mut1 was found to be very low. Identical results were
also obtained from the study with 293 cells expressing
APP770wt and
APP770mut1 (data not shown).
These results suggest that the majority of APP cleavage at
- and
-sites also occurs after, or possibly during,
O-glycosylation of APP.

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Fig. 9.
Analysis of A in the medium. A 40
(a) and A 42 (b) were quantified as described
under "Experimental Procedures." Quantity of A 40 (a)
and A 42 (b) (fmol/100 µl of medium) was divided by the
relative level of total APP and indicated as the ratio of A /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).
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DISCUSSION |
APP is thought to be cleaved by
-,
-, and
-secretases in
the protein secretory pathway. Previous studies using protein metabolic
inhibitors (chloroquine, brefeldin A, bafilomycin A1, etc.) suggest
that APP cleavage by
-secretase occurs in the
trans-Golgi network or other late compartments of
the protein secretory pathway (23, 24, 26-28), and that cleavage by
-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 A
42 but not A
40 occurs in the ER (29, 30). However,
the biochemical quantification of A
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
Cys and Leu-172
Pro, and
APP770 also has a deleted
sequence within the extracellular domain, including
N-glycosylation sites. Present results demonstrate that only
the Leu-172
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
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
Ala mutation (mut2) is not effective but the
Leu-171
Pro mutation (mut3) and a double mutation,
Leu-172
Pro/Pro-173
Leu (mut4), showed an effect
identical to the Leu-172
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
-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.
APP770wt matures and degrades
identically to APP770. Furthermore, the Leu-172
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
-secretase and
APPCOOH is not generated. The results indicate, without
using cytotoxic drugs, that APP cleavage at the
-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 A
40 and A
42 in the
medium of 293 cells expressing APP695mut1, indicates that a majority of APP cleavage at the
- and
-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 A
may
be generated in acidic compartments following medial-Golgi (31-33). However, the present study suggests that both A
40 and A
42 are generated subsequent to trans-Golgi, as in the
case of
-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 A
42 but
not A
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 A
42 accumulation in 293 cells expressing
APP695mut1, the intracellular accumulation of A
,
which is not secreted, may not be critical for the pathogenesis of AD because it has been well analyzed that the A
accumulation is an
extracellular event in the brain of AD patient.
In previous studies, to identify the intracellular site of APP cleavage
by
-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
-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.
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.