From the
Apolipoprotein E (apoE), particularly the e4 allele, is
genetically linked to the incidence of Alzheimer's disease.
In vitro, apoE has been shown to bind
Apolipoprotein E (apoE),
A major neuropathological feature
of AD is the presence of extracellular senile plaques composed
predominantly of aggregated
Genetic data have defined the presence of the e4 allele of
apoE as a major risk factor for the occurrence of sporadic
(4) and late-onset familial
(5) Alzheimer's
disease. However, the physiological mechanism by which apoE isoform
specificity contributes to the pathogenesis of the disease is unknown.
Previously, using unpurified apoE from conditioned medium, we showed
that the amount of the apoE3
We first examined the
effect of purification of apoE from conditioned medium on the formation
of apoE
There are several possibilities that could account for
the loss of isoform specificity in the binding of purified apoE to
A
Another more likely explanation is
that apoE acquires a conformation when lipid-associated that confers
isoform-specific binding to A
The data presented here are
consistent with a function for apoE in the pathogenesis of AD by
sequestering A
In summary, using unpurified apoE from tissue culture medium
and intact VLDL particles, the apoE3
We thank Warren Wade and Richard Perner for synthesis
of A
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-amyloid (A
),
an amyloidogenic peptide that aggregates to form the primary component
of senile plaques. In previous work, we demonstrated that apoE3 from
tissue culture medium binds to A
with greater avidity than apoE4
(LaDu, M. J., Falduto, M. T., Manelli, A. M., Reardon, C. A., Getz, G.
S., and Frail, D. E. (1994) J. Biol. Chem. 269,
23403-23406). This is in contrast to data using purified apoE
isoforms as substrate for A
(Strittmatter, W. J., Weisgraber, K.
H., Huang, D. Y., Dong, L.-M., Salvesen, G. S., Pericak-Vance, M.,
Schmechel, D., Saunders, A. M., Goldgaber, D., and Roses, A. D. (1993)
Proc. Natl. Acad. Sci. U. S. A. 90, 8098-8102). Here we
resolve this apparent discrepancy by demonstrating that the
preferential binding of A
to apoE3 is attenuated and even
abolished with purification, a process that includes delipidation and
denaturation. We compared the A
binding capacity of unpurified
apoE isoforms from both tissue culture medium and intact human very low
density lipoproteins with that of apoE purified from these two sources.
The interaction of human
A
-
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) -peptide
and apoE was analyzed by nonreducing SDS-polyacrylamide gel
electrophoresis followed by Western immunoblotting for either A
or
apoE immunoreactivity. While the level of the apoE3
A
complex
was
20-fold greater compared with the apoE4
A
complex in
unpurified conditioned medium, apoE3 and apoE4 purified from this
medium bound to A
with comparable avidity. Moreover, using
endogenous apoE on very low density lipoproteins from plasma of apoE3/3
and apoE4/4 homozygotes, apoE3 was again a better substrate for A
than apoE4. However, apoE purified from these plasma lipoproteins
exhibited little isoform specificity in binding to A
. These
results suggest that native preparations of apoE may be a more
physiologically relevant substrate for A
binding than purified
apoE and further underscore the importance of subtle differences in
apoE conformation to its biological activity.
(
)
a component of
several classes of lipoproteins, acts as a ligand for lipoprotein
receptors, thus regulating lipid transport and clearance. ApoE is also
expressed in the brain
(1) and in response to injury in both
the peripheral
(2) and central nervous
(3) systems. In
humans, apoE has three major isoforms, E2 (Cys
,
Cys
), E3 (Cys
, Arg
), and E4
(Arg
, Arg
), which are products of three
alleles at a single gene locus. The presence of cysteine in apoE2 and
apoE3 allow these isoforms to form disulfide-linked dimers. Recently,
it has been demonstrated that the apoE e4 allele is present with
increased frequency in patients with sporadic
(4) and
late-onset familial
(5) Alzheimer's disease (AD). Due
primarily to this genetic linkage, the role of apoE in the pathogenesis
of AD is being actively pursued.
-amyloid peptide (A
). The
physiological mechanism by which apoE contributes to AD pathology may
be by means of an isoform-specific interaction with A
. ApoE and
A
colocalize in senile plaques
(6) , and synthetic A
peptides bind in vitro to apoE from tissue culture medium
(7) and cerebrospinal fluid
(5, 8, 9) as
well as to purified apoE
(9, 10) . However, studies
involving apoE isoform specificity in binding to A
have been
limited and contradictory. In previous work, we demonstrated that apoE3
from tissue culture medium binds to A
with greater avidity than
apoE4
(7) . This is in contrast to data from Strittmatter et
al. (10) , who used apoE isoforms purified from human
plasma as substrate for A
. Here we resolve the apparent
discrepancy between these studies by demonstrating that the previously
observed preferential association of A
with apoE3 is attenuated or
abolished by purification, a process that includes delipidation and
denaturation.
Expression of Human ApoE in Cultured Cells
Human
apoE3 and apoE4 were expressed in HEK-293 cells stably transfected with
human apoE3 or apoE4 (products of the e3 and e4 alleles, respectively)
cDNA as described previously
(7) . Conditioned medium containing
apoE was concentrated (Centriprep, Amicon, Inc.) 50-fold prior to
binding reactions or purification.
Purification of ApoE
Human plasma was screened for
apoE genotype using a modification of the method of Hixson and Vernier
(11) . Intermediate and very low density lipoprotein (VLDL)
particles ( d < 1.02 g/ml) were isolated
(12) from
the plasma of individuals homozygous for apoE3 and apoE4. Unpurified
preparations of VLDL were used within 2 weeks of isolation.
Purification of apoE from this lipoprotein fraction and from
concentrated conditioned medium was carried out according to standard
procedures
(13) . Briefly, the conditioned medium and
lipoproteins were dialyzed against 0.01% EDTA, lyophilized, and
delipidated in CHCl:MeOH (2:1). Delipidated proteins were
pelleted in MeOH and solubilized in 6
M guanidine, 0.1
M Tris, 0.01% EDTA (pH 7.4), and 1% 2-mercaptoethanol.
Proteins were fractionated on a Sephacryl S-300 column (Pharmacia
Biotech Inc.) equilibrated in 4
M guanidine, 0.1
M
Tris, 0.01% EDTA (pH 7.4), and 0.1% 2-mercaptoethanol. Fractions
containing apoE were dialyzed in 5 m
M
NH
HCO
, lyophilized, and resuspended in 0.1
M NH
HCO
. Unpurified and purified apoE
proteins were quantified by SDS-polyacrylamide gel electrophoresis,
protein staining, and densitometry (Molecular Dynamics, Inc.) of serial
dilutions of apoE-containing samples using a purified apoE standard.
ApoE
For binding reactions, synthetic human
AA
Complex Formation and
Detection
-
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40) -peptide,
purified by high performance liquid chromatography, was resuspended to
5 m
M in 100% Me
SO. ApoE (25 µg/ml,
700
n
M) was incubated for 2 h (except as noted in the legend to
Fig. 4
) at room temperature with 250 µ
M A
at pH
7.4 as described previously
(7) . Control reactions without
A
contained 5% Me
SO. Reactions were stopped by the
addition of 2
nonreducing Laemmli buffer
(14) (4% SDS,
no 2-mercaptoethanol) and frozen at -20 °C. Samples were
boiled for 5 min, electrophoresed on 10-20% SDS-Tricine gels,
transferred to Immobilon-P membranes (Millipore Corp.), and probed with
antibodies to A
or apoE (1:1000 dilution). Monoclonal antibody 4G8
to amino acids 17-24 of A
was provided by Drs. H. M.
Wisniewski and K. S. Kim
(15) . ApoE antiserum was obtained by
immunizing rabbits with apoE purified from human serum. Proteins on
Western blots were visualized by enhanced chemiluminescence (Amersham
Corp.) and quantified by densitometry.
Figure 4:
Time course of A binding to apoE
isoforms purified from human plasma. Shown are Western blots of
apoE/A
binding reactions assayed under the conditions described in
the legend to Fig. 1 and probed with 4G8 antibody ( A) or apoE
antiserum ( B). ApoE was purified from human plasma of apoE3/3
( lanes 1-6) and apoE4/4 ( lanes 7-12)
homozygotes. Lanes 1 and 7, apoE alone;
lanes 2-6 and 8-12, apoE + A
for 2 min, 10 min, 2 h, 8 h, and 24 h,
respectively.
A
complex was much greater
compared with the apoE4
A
complex
(7) . These results
are in contrast to data published by Strittmatter et al. (10) . Using apoE purified from human plasma, they reported
that apoE4 binds A
more rapidly than apoE3. However, the level of
A
binding to apoE3 and apoE4 after several hours of incubation was
comparable. The major differences between these two studies are the
source of apoE (plasma versus secreted by cultured cells) and
whether the apoE protein was purified prior to use in the binding
assays. In this study, we examined whether these differences could
account for the discrepancy between these two reports regarding apoE
isoform-specific binding to A
. We compared the A
binding
capacity of unpurified apoE from both conditioned medium and intact
human VLDL from plasma of apoE3/3 and apoE4/4 homozygotes with the
A
binding capacity of apoE purified from these two sources. The
purification procedure
(13) used was the same as that used by
Strittmatter et al. (10) .
A
complexes. ApoE3 and apoE4 were purified from the
medium of transfected HEK-293 cells, and the binding of this material
to A
was compared with the binding of unpurified apoE from medium
(Fig. 1). Prior to purification, the amount of the
apoE3
A
complex (Fig. 1, A and B,
lane 2) was
20-fold greater compared with the
apoE4
A
complex ( lane 4). These results are
consistent with our previous data that demonstrated that this
preferential binding of A
to apoE3 is maintained over time, pH
range, and concentration of apoE and A
(7) . Purification
of the apoE isoforms from this medium resulted in a decrease in A
binding to apoE3 (Fig. 1, A and B, lane
6) and an increase in A
binding to apoE4 ( lane 8).
To determine whether an auxiliary component that influences the isoform
specificity of apoE binding to A
is present in the medium of
HEK-293 cells, we reconstituted purified apoE with mock-transfected
conditioned medium. The isoform specificity of apoE binding to A
was not restored to pre-purification levels, indicating that
purification does not remove a component present in the medium that
confers the preferential binding of A
to apoE3 (compare Figs. 2
and 1 A, lanes 5-8).
Figure 1:
ApoE/A binding: effect of
purifying apoE isoforms from conditioned medium. Shown are Western
blots of binding reactions using 25 µg/ml apoE (
700
n
M) incubated with ( lanes 2, 4,
6, and 8) or without ( lanes 1,
3, 5, and 7) 250 µ
M
A
-(1-40)-peptide for 2 h at room temperature. Sources of
apoE were as follows: conditioned medium of transfected cells (apoE3,
lanes 1 and 2; apoE4, lanes 3 and 4) and purified from conditioned medium (apoE3,
lanes 5 and 6; apoE4, lanes 7 and 8). Samples were run in nonreducing Laemmli buffer on
10-20% SDS-Tricine gels, transferred to Immobilon-P membrane, and
probed with 4G8 antibody ( A) or apoE antiserum
( B).
To examine the binding
of A to unpurified plasma apoE isoforms, VLDL was isolated from
the plasma of apoE3/3 and apoE4/4 homozygotes and used for binding
reactions with A
. Similar to the results using unpurified apoE
from conditioned medium, we observed an
20-fold difference in the
amount of the apoE3
A
complex (Fig. 3, A and
B, lane 2) compared with the apoE4
A
complex
( lane 4) when using intact VLDL particles as the source of
apoE. As with purification of apoE from medium, purification of apoE
from the VLDL fraction of human plasma attenuated the preferential
binding of apoE3 to A
(Fig. 3 A and B, lanes
6 and 8). The level of A
binding to both apoE4 and
apoE3 dimer was increased after purification of apoE from plasma. In
apoE3/3 plasma, the apoE immunoreactive species migrating slightly
higher than the apoE
A
complex (
55 kDa) is the
apoE3-apolipoprotein A-II heterodimer (Fig. 3 B,
lanes 1 and 2).
(
)
The
autoradiographs shown in Figs. 1 and 3 are representative of several
experiments with similar results using different apoE-containing
preparations. While some variation in the purification-mediated loss of
preferential apoE3 binding to A
was observed, in general,
purification of apoE from both medium and plasma abolished
isoform-specific binding to A
.
Figure 3:
ApoE/A binding: effect of purifying
apoE isoforms from human plasma. Shown are Western blots of apoE/A
binding reactions assayed under the conditions described in the legend
to Fig. 1. Sources of apoE were as follows: intact VLDL from apoE3/3
( lanes 1 and 2) and apoE4/4 ( lanes 3 and 4) homozygotes and purified from VLDL of
apoE3/3 ( lanes 5 and 6) and apoE4/4
( lanes 7 and 8) homozygotes. Samples were
run as described in the legend to Fig. 1 and probed with 4G8 antibody
( A) or apoE antiserum ( B). For apoE3 VLDL, total
protein was 95 ng/µl, and triglyceride was 600 ng/µl. For apoE4
VLDL, total protein was 110 ng/µl, and triglyceride was 400
ng/µl.
Strittmatter et al. (10) used apoE purified from plasma to show that A binds to
apoE4 monomer at a faster rate than to apoE3 monomer. However, the
contribution of the apoE3-A
complex in their time course data is
difficult to discern since the blots were probed for apoE and A
immunoreactivity simultaneously
(10) . Therefore, we repeated
the time course study using purified plasma apoE isoforms and probed
for A
(Fig. 4 A) and apoE (Fig. 4 B) immunoreactivity individually. Similar to the results obtained by
Strittmatter et al. (10) , apoE4 monomer did form a
complex with A
at a faster rate compared with apoE3 monomer.
However, when the apoE3 dimer-A
complex was taken into account,
the total binding of A
to apoE3 and apoE4 remained comparable
across time.
. Purification may result in a change in the oxidative state of
apoE that influences its binding to A
. Although the apoE isoforms
are subject to the same conditions while undergoing purification,
oxidation could be specific to the Cys
in apoE3. This
could affect conformation-dependent binding of apoE to A
, either
directly or via interaction of other amino acids with this residue.
However, apoE3 dimer, particularly in its purified form, readily binds
A
, indicating that at least oxidation of cysteine does not seem to
adversely affect apoE3
A
complex formation. In addition,
oxidation provides no obvious explanation for the increase in A
binding to purified apoE4 monomer.
. Purification, which includes
delipidation, may increase the binding of A
to apoE3 dimer and
apoE4 monomer by disrupting the endogenous conformation of these apoE
species. In each of the unpurified preparations used here, apoE was
lipid-associated. ApoE from human plasma was in the VLDL fraction,
while the majority of apoE in conditioned medium was in a high density
lipid fraction.
(
)
Purified apoE has been shown to
require lipid to restore its biological activity in other assay
systems, presumably by allowing the denatured protein to refold to its
functional conformation. These apoE activity assays include such
diverse functions as receptor binding and modulation of neuritic
growth. While purified apoE is unable to displace
I-labeled low density lipoprotein (LDL) from LDL
receptors
(16) , high affinity binding is restored when apoE is
added in the presence of a variety of lipid particles, including
phospholipid vesicles
(17) . Similarly, the binding of purified
apoE to LDL receptor-related protein is enhanced by the addition of
-migrating VLDL
(18) . In primary dorsal root ganglia
cultures, purified apoE3 and apoE4 have no effect on neuritic growth.
However, when added in the presence of
-migrating VLDL, both apoE
isoforms affect neuritic growth, with apoE3 increasing neuritic
extension and decreasing branching and apoE4 decreasing both branching
and extension
(19) . Experiments are in progress using the assay
system described here to determine the nature of the interaction of
A
with lipid-associated apoE using both endogenous lipoproteins
and reconstituted lipid vesicles.
. Avid binding of apoE3 to soluble A
in the
neuropil could lead to enhanced clearance or altered fibril formation,
both of which could prevent the conversion of A
into a neurotoxic
species. Since lipid-associated apoE3 and apoE4 bind the LDL receptor
and LDL receptor-related protein with equal affinity
(18) ,
efficient uptake and clearance of A
may depend on its preferential
binding to apoE3. Alternatively, purified apoE3 and apoE4 are known to
differ with respect to their effect on A
fibril formation
(20, 21, 22) , suggesting that native apoE may
play an even greater isoform-specific role in the extracellular
aggregation of A
due to differences in A
binding
characteristics. Finally, there is evidence that A
-induced
toxicity in hippocampal neurons is attenuated by the addition of rabbit
apoE
(23) , leading to the intriguing possibility that human
apoE may contribute to A
-induced toxicity in an isoform-specific
manner.
A
complex was
20-fold more abundant than the apoE4
A
complex. This
isoform specificity was attenuated or abolished when apoE purified from
these two sources was used in binding reactions with A
. ApoE is an
apolipoprotein, and as such, its endogenous conformation requires
lipid. It is therefore not surprising that the type of apoE preparation
used can affect the results. The avidity of A
binding to apoE3
compared with apoE4 demonstrated here may be involved in the
isoform-specific effect underlying the genetic correlation between the
apoE e4 allele and AD. The physiological relevance of this complex to
plaque formation or neurodegeneration awaits further investigation.
,
-amyloid; VLDL, very low
density lipoprotein; Tricine,
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
LDL, low density lipoprotein.
peptides, Randy Metzger for genotyping of plasma sources, and
John Lukens for lipoprotein isolation. We also thank Brad Hyman and
Bill Rebeck for helpful discussions and providing apoE4/4 plasma.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.