From the Institute for Medical Biology and Human
Genetics, University of Innsbruck, Schoepfstrasse 41, A-6020 Innsbruck, Austria and the § Center of Molecular
Biology, University of Heidelberg, D-69120 Heidelberg, Germany
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ABSTRACT |
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Variation at the APOE gene locus has
been shown to affect the risk for Alzheimer's disease. To gain deeper
insight into the postulated apoE-mediated amyloid formation, we have
characterized the three common apoE isoforms (apoE2, apoE3, and apoE4)
regarding their binding to amyloid precursor protein (APP). We employed the yeast two-hybrid system and co-immunoprecipitation experiments in
cell culture supernatants of COS-1 cells, ectopically expressing apoE
isoforms and APP751 holoprotein or a COOH-terminal A
deletion mutant protein, designated APPtrunc. We found that
all three apoE isoforms were able to bind APP751
holoprotein in an A
-independent fashion. The interacting domains
could be mapped to the NH2 termini of APP (amino acids
1-207) and apoE (amino acids 1-191). As a functional consequence of
this novel APP751 ectodomain-mediated apoE binding, the
secretion of soluble APP751 is differentially affected by
distinct apoE isoforms in vitro, suggesting a new "chaperon-like" mechanism by which apoE isoforms may modulate APP
metabolism and consequently the risk for Alzheimer's disease.
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INTRODUCTION |
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Human apolipoprotein E (apoE),1 which is coded by a gene on chromosome 19q13.2, is a polymorphic protein and is known to play a major role in lipoprotein metabolism and cholesterol homeostasis in the brain (1-5). ApoE exists in three common genetic forms, designated apoE2, apoE3, and apoE4, which differ by single amino acid substitutions at one of two positions of the proteins 299-amino acids long primary structure (6, 7). Variation at the APOE gene locus has long been known to affect plasma cholesterol concentrations and subsequently the risk for atherosclerosis and coronary heart disease (1, 8). Recently it was realized that the apoE4 isoform is also associated with late onset sporadic and familial Alzheimer's disease (AD), the cause of dementia, effecting up to 5% of the population over age 65. Continuing gains in life expectancy have made AD by far the most common form of dementia worldwide (9-16). The effect of APOE4 appears to be dependent on gene dosage, i.e. homozygotes for the APOE4 allele tend to develop AD at an earlier age then heterozygotes (11, 17). Presence of an APOE4 allele is, however, neither necessary nor sufficient to develop AD (18). ApoE4 is therefore considered a susceptibility factor for AD. Interestingly, the apoE2 isoform seems to demonstrate a protective effect in AD, emphasizing the significant influence of the APOE genotype as a modifier of AD progression (17).
Although the genetic link between APOE and AD has been
confirmed by many laboratories, the pathophysiological mechanism(s) by
which the apoE4 isoform confers increased susceptibility and the apoE2
isoform confers decreased susceptibility to the disease is not yet
understood. Quantitative neuropathological assessment reveals that
amyloid deposits (19, 20) but not neurofibrillary tangles (19) are
elevated in association with the apoE4 isoform, suggesting an influence
of apoE primarily on amyloid formation in AD. Additionally, AD patients
with the apoE4 isoform have more but not larger plaques than AD
patients with the apoE3 isoform (21). Because amyloid A peptide, a
proteolytic product of the APP (22) and main component of the senile
plaques, is hydrophobic, apoE molecules have been implicated in its
aggregation and/or clearance (10, 23, 24). Differential binding of apoE
isoforms to the amyloid A
peptide has been suggested as a mechanism.
In our study the three common apoE isoforms, apoE2, apoE3, and apoE4, have been biochemically characterized regarding their differential binding to APP holoprotein in vitro.
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EXPERIMENTAL PROCEDURES |
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Plasmids--
pAS2-1/apoE2, pAS2-1/apoE3, pAS2-1/apoE4, and
pAS2-1/apoE3 NH2 (amino acids 1-191) were constructed by
cloning the full-length or partial apoE cDNA (lacking the leader
sequence) into the EcoRI/SalI sites of GAL4-DBD
vector pAS2-1 using PCR and recombinant PCR primers apoE-SENSE: 5'-GGA
TGC GAA TTC AAG GTG GAG CAA GCG GTG GA-3' and apoE-ANTISENSE: 5'-AGG
CTT GTC GAC TCA GTG ATT GTC GCT GGG CAC-3' or
apoE-NH2/1-191 ANTISENSE: 5'-GCC CAC GTC GAC CTA CCG CAC
GCG GCC CTG TTC CA-3', respectively, engineered to create proper
compatible ends and the correct reading frame for expression as fusion
proteins in the context of the GAL4 DNA binding domain. Similarly,
pACT2/APP751 were cloned into SmaI/SalI of pACT2,
respectively, using APP-SENSE: 5'-GAC GGT CCC GGG GCT GGA GGT ACC CAC
TGA T-3' and APP-ANTISENSE: 5'- GAT GAC GTC GAC TTC AGC TAT GAC AAC ACC GCC C-3'. pACT2/APPtrunc (amino acids 1-635) was
constructed by COOH-terminal deletion using the endogenous
BglII site, 5' of the A sequence in the APP cDNA.
pACT2/APP COOH-terminal deletion mutants, amino acids 1-342 and
1-207, have been constructed employing the endogenous restrictions
sites XhoI and BbsI, respectively. The plasmids
pEF-apoE2, apoE3, and apoE4 were constructed by shuffling the
full-length coding regions (including the leader sequence) from
pUC-apoE3 (obtained from J. Smith, Rockefeller University, NY),
pCTV-apoE2, pCTV-apoE4 (obtained from K. Weisgraber, University of
California San Francisco, CA), and apoE3-NH2 cDNA
(amino acids 1-191, generated by PCR using recombinant PCR primers
apoE-LEADER: 5'-CCA ATC TCT AGA GTC GAC ATG AAG GTT CTG TGG GCT GCG
TTG-3' and apoE-NH2/1-191 ANTISENSE: 5'-GCC CAC ACT AGT
CTA CCG CAC GCG GCC CTG TTC CA-3') into expression plasmid pEF-neo (a
kind gift from Y. Liu, LIAI, CA) downstream and under the control of
the elongation factor-1
promoter. Human APP full-length cDNA was loned into the HindIII/SalI sites of the CMV
expression vector pTAG-CMVneo (25) employing PCR primers APP-Leader:
5'-GCC CCG AAG CTT GTC GCG ATG CTG CCC GGT TT-3' and APP-751-TAG-COOH:
5'-GCG GGG GTC GAC GTT CTG CAT CTG CTC AAA GAA CTT GT-3' to engineer a
COOH-terminal fusion-TAG. Similarly, APP-trunc was cloned into pTAG-CMV
using PCR primers APP-Leader (see above) and
APPtrunc-TAG-COOH: 5'-CTT CAC GTC GAC GAT CTC CTC CGT CTT
GAT ATT TG-3'. Correct constructs have been identified and confirmed by
restriction analysis and partial DNA sequencing using vector-specific
primers (5'-GAL4-DBD: 5'-CAT CGG AAG AGA GTA G-3', 3'-GAL4-DBD: 5'-CCT
AAG AGT CAC TTT AAA A-3', 5'-GAL4-AD: 5'- TAC CAC TAC AAT GGA TG-3',
3'-GAL4-AD: 5'- ATA AAT GAA AGA AAT TGA GAT-3', 5'-EF-1
: 5'-TGG ATC
TTG GTT CAT TCT CAA GCC-3', 5'-T7: 5'- TAA TAC GAC TCA CTA TA-3' and
3'-SP6: 5'- ATT TAG GTG ACA CTA TA-3').
Yeast Two-hybrid Screen--
The genotype of the
Saccharomyces cerevisiae reporter strain HF7c used for the
two-hybrid screening, is MATa, ura3-52, his3-200, ade2-101, lys2-801,
trp1-901, leu2-3, 112, gal4-542, gal80-538, LYS2::GAL1-HIS3,
URA3:: (GAL4, 17-mers)3-CYC1-lacZ
(CLONTECH). Stains were grown under standard
conditions in rich or synthetic medium with appropriate supplements at
30 °C. For the yeast two-hybrid screening, DBD-apoE baits were
co-transformed with the APP-AD fusion cDNA in the pACT vector
(CLONTECH) into the HF7c yeast strain as described
by the manufacturer, and the transformants were assayed for
-galactosidase activity by transferring individual colonies on
filters placed on selection medium. The plates were incubated for 2 days at 30 °C, the filters lifted and immersed in liquid nitrogen
for 10 s. After thawing at room temperature, the filters were
placed on filter circles saturated with X-gal solution in a Petri dish
(permeabilized cells up) and incubated overnight. For quantitative
analysis,
-galactosidase reporter protein was measured in the cell
extracts using a
-galactosidase ELISA (5 Prime
3 Prime, Inc.,
Boulder, CO). The results shown are obtained with different
preparations of expression plasmids and represent the mean ± S.E.
from four representative experiments done in triplicate.
Immunoblotting of GAL4-DBD Fusion Baits in Yeast Extracts--
5
ml of transformed yeast cells grown overnight in selective medium
lacking tryptophan were used to inoculate 15 ml of YPD medium. At an
optical density (600 nm) of 0.5, the cells were pelleted, washed,
resuspended at 5 × 108 cells/ml in ice-cold
radioimmune precipitation buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 µg/ml aprotinin/leupeptin, and 1 mM phenylmethylsulfonyl fluoride) and frozen at 20 °C.
Samples were analyzed by SDS-polyacrylamide gel electrophoresis (10%),
transferred to polyvinylidene difluoride membrane (Millipore, Vienna),
and fusion proteins were detected using a GAL4-DBD (Santa Cruz
Biotechnology, Inc.) or GAL4-AD (Upstate Biotechnology) specific
antibodies, followed by a rabbit anti-mouse IgG-peroxidase conjugate
and a chemiluminescence detection kit (Pierce).
Transient Transfection of COS-1 Cells-- The SV40-transformed African green monkey kidney cell line COS-1 was obtained from the American Type Culture Collection (ATCC, Rockville, MD) and cultured as recommended by ATCC. For transient transfections, cells were seeded at a density of 2 × 106 cells/well in 10-cm Petri plates in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum at 37 °C. One day later, the cells were transfected with 20 µg of circular plasmid DNA/dish by electroporation (BTX, San Diego, CA) with the following settings: 100 V, 48 Ohm and 1275 µF, or by LipofectAMINETM in Opti-MEM medium (Life Technologies, Inc.) as described by the manufacturer. 24 h post-transfection, cells were lysed in lysis buffer (5 mM NaP2P, 5 mM NaF, 5 mM EDTA, 50 mM NaCl, 50 mM Tris, pH 7.3, 2% Triton X-100, 50 µg/ml each aprotinin and leupeptin). Similarly, cell culture media were harvested by precipitation in 5% trichloroacetic acid.
Co-immunoprecipitation-- Cell culture media were precleared by incubating with 50 µl of protein G-Sepharose beads for 2 h at 4 °C and then immunoprecipitated overnight at 4 °C with the indicated antibodies at 5 µg/ml final antibody concentration followed by addition of 50 µl of protein G-Sepharose beads for the last 2 h. Immunoprecipitates were collected by centrifugation for 1 min at 13,000 rpm and 4 °C in an Eppendorf microfuge and washed six times with phosphate-buffered saline, 0.02% Tween buffer. Immuno-precipitates were resuspended in SDS-polyacrylamide gel electrophoresis sample buffer, boiled for 5 min at 95 °C and separated on 10% Tris-glycine gels (Novex). Immunoblot analysis using apoE (Calbiochem), APP (Boehringer Mannheim 22C11 or WO-2, obtained from K. Beyreuther, Heidelberg) and TAG-specific (H902, Ref. 25) antibodies was performed.
Metabolic Labeling and Immunoprecipitation--
COS-1 cells
transfected with apoE and APP expression plasmids were treated
with minimum essential medium lacking methionine, supplemented
with 100 µCi of [35S]methionine (Amersham
Pharmacia Biotech, Braunschweig, Germany) for 1 h. For
pulse-chase analysis, the chase was performed with 1 mM L-methionine in minimum essential medium for
various periods of time as indicated before standard cell
lysis and immunoprecipitation analysis of APP holoprotein employing mAb
WO-2 (anti-A1-16).
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RESULTS AND DISCUSSION |
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In our study the three common apoE isoforms, apoE2, apoE3, and
apoE4, have been biochemically characterized regarding their binding to
APP in vitro. In contrast to previous studies employing synthetic A peptides (10, 23, 24), we investigated the role of the
A
region in a potential direct interaction of apoE and APP
holoprotein, initially employing the GAL4 two-hybrid system. As result,
a specific and direct physical association of apoE and APP holoprotein
could be demonstrated (Fig.
1A). Additionally and to our
surprise, an A
-independent interaction of apoE and APPtrunc, a COOH-terminal deletion mutant of APP (amino
acids 1-635) devoid of the A
region, was observed (Fig.
1B). Interaction results were strictly dependent on the
combined presence of one of the GAL4-apoE isoform baits plus one of the
GAL4-APP ligands. Specificity controls employing GAL4-p53 and GAL4-SV40
ligands or empty vector in exchange of either apoE or APP and
APPtrunc did not activate the
-galactosidase expression
(data not shown).
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To biochemically confirm the relevance of this putative apoE and
APP751 interaction, we used COS-1 cells, which do not
express apoE, for transient transfection with expression plasmids
encoding distinct apoE isoforms and APP751 holoprotein or
APPtrunc, the COOH-terminal A deletion mutant,
respectively (Fig. 2) First, overexpressed and, due to endoproteolytic processing, soluble (s)APP751 was immunoprecipitated from cell culture media,
and the resultant precipitates, e.g. apoE-APP complexes,
were detected by immunoblotting with antisera specific to apoE or APP.
As shown in Fig. 2A, apoE specifically co-immunoprecipitates
with sAPP751, indicating an interaction of these two
proteins. In another set of experiments, ectopically expressed apoE
isoforms (and apoE-associated APPtrunc) were
immunoprecipitated from cell culture media, and the resultant
precipitates were analyzed by immunoblotting with either mAb
TAG,
specific for the epitope-TAG engineered into the recombinant
sAPPtrunc (see cartoon in Fig. 2C) or anti-apoE antibodies. Consistently with the apoE-APP interaction detected in the
yeast two-hybrid results, a specific and A
-independent interaction
of apoE and sAPP could be demonstrated in these co-immunoprecipitation assays (Fig. 2B). Again, all three apoE isoforms were able
to form complexes with sAPP751. Since the
APPtrunc used for transfections lacks the A
region, the
findings of Fig. 2B demonstrate that the observed apoE-APP
interaction requires domains of APP upstream of the A
region.
Recently, a physical interaction of sAPP and baculovirus-expressed
apoE3 and apoE4 has been reported (26), an observation consistent with
our co-expression studies reported here.
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To define the critical domains in the apoE-APP interaction, APP and apoE3 COOH-terminal deletion mutants have been employed in the yeast two-hybrid system. As shown in Fig. 3A, the initial definition of the binding pockets could be assigned to the NH2 terminus (amino acids 1-207) of APP and the NH2 terminus (amino acids 1-191) of apoE. This has been independently demonstrated in co-immunoprecipitation assays of ectopically expressed apoE and APP COOH-terminal deletion mutants (Fig. 3B), confirming the binding of sAPPtrunc to the NH2 terminus (amino acids 1-191) of apoE3.
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To investigate whether this novel interaction may affect APP
processing, we used COS-1 cells for transient co-transfection with
apoE2, apoE3, apoE4, and APP holoprotein. 24 h post-transfection, COS-1 cell culture media were investigated for apoE and sAPP protein concentrations. As shown in Fig.
4A, a significant apoE
isoform-dependent inhibition of sAPP751
secretion could be observed. ApoE2 demonstrated the strongest effect on
sAPP followed by apoE3 and apoE4. Reduced concentrations of the soluble
form of APP in the cellular supernatant may be explained by
intracellular retention of APP. This was indeed demonstrated by the
apoE-mediated accumulation of mature APP in the Triton X-100 soluble
cellular fraction (Fig. 4B). No significant amount of APP
could be detected in the detergent-resistant fraction (not shown).
Similar effects could be seen with APP695 (data not shown).
ApoE isoforms therefore influence secretion and/or retro-endocytosis of
APP from transfected COS-1 cells in an isoform-specific manner. Again,
this apoE isoform-specific effect on sAPP751 was
A-independent since they were also observed with
APPtrunc (Fig. 4C).
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To further distinguish between apoE-mediated retention of sAPP or retro-endocytosis of sAPP, pulse-chase experiments have been performed over chase periods between 30 and 150 min. A representative result is shown in Fig. 5 demonstrating, only in the presence of apoE2, a significant intracellular accumulation of the APP holoprotein up to 90 min. Similar albeit reduced retention effects on APP holoprotein could be observed with apoE3 and apoE4 (data not shown). Due to the time frame of intracellular APP accumulation, apoE isoforms seem to mediate retention of APP mainly by reducing its secretion rate, an observation consistent with the postulated chaperon-like function of intracellular apoE. Additionally, however, apoE-mediated APP retro-endocytosis may also contribute to the observed intracellular APP accumulation. Undoubtedly, more work is necessary to determine the precise mechanism utilized by apoE isoforms, but our data represent an important step toward the identification and characterization of this novel apoE function involved in modulation of APP metabolism.
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Consistently, we found that the effect of apoE/APP751
interaction is bilateral, since APP751 prevents apoE
secretion to the culture medium (Fig.
6A) and furthermore leads to
an apparent reduction of intracellular apoE (Fig. 6B). This
appears to be mediated in part by the ubiquitin-proteasome pathway,
since N-acetyl-leucyl-leucyl-norleucinal, a potent inhibitor
of this degradation pathway, can block apoE degradation by 30%
(data not shown). The effects shown in Figs. 4-6 are not due to
different expression levels of apoE2, apoE3, and apoE4 in transfected
COS-1 cells (Fig. 6, see for control apoE the lanes lacking APP (
)).
Additionally, no similar apoE effect was observed with other
glycoproteins besides APP, e.g.
-2 glycoprotein I
secretion was not inhibited by apoE2 co-expression (data not shown).
This further indicates a physiological association of apoE isoforms
with APP, suggesting a potential chaperon-like function of apoE
isoforms in APP processing in the order apoE2
apoE3
apoE4.
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In conclusion, apoE was demonstrated to directly bind via its
NH2 terminus to APP holoprotein within the ectodomain,
independent of the A region in vitro. This was
independently shown by the yeast two-hybrid system and by
co-immunoprecipitation assays from ectopically transfected COS-1 cell
culture media. Co-expressed apoE affects sAPP secretion in an apoE
isoform-specific way in vitro. This novel apoE-APP
ectodomain interaction may reflect a new potential physiological role
of distinct apoE isoforms in APP metabolism, e.g. in
astrocytes and microglia cells, endogenously co-expressing apoE (27,
28) and APP (29, 30). This may affect either A
formation or
alternatively may interfere with APP-mediated signal transduction
pathways (31, 32). The relevance for the in vivo situation,
however, remains to be shown.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. V. Zannis (Boston) for helpful discussions and to Dr. E. Preuss (Innsbruck), PDL, for the art work.
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FOOTNOTES |
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* This work was supported by Grants from the Austrian Ministry of Science and the European Communities/Biomed 2 Program (BMH4-CT96-0898).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.
¶ To whom correspondence should be addressed. Tel.: 43-512-507-3451; Fax: 43-512-507-2861; E-mail: Gottfried.Baier{at}uibk.ac.at.
1
The abbreviations used are: apoE, apolipoprotein
E; AD, Alzheimer's disease; PCR, polymerase chain reaction; X-gal,
5-bromo-4-chloro-3-indoyl -D-galactopyranoside; ELISA,
enzyme-linked immunosorbent assay; mAb, monoclonal antibody.
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REFERENCES |
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