(Received for publication, November 30, 1995; and in revised form, February 21, 1996)
From the
An important feature of Alzheimer's disease (AD) is the
cerebral deposition of amyloid. The main component of the amyloid is a
39-44-amino acid residue protein called amyloid (A
),
which also exists as a normal protein in biological fluids, known as
soluble A
. A major risk factor for late-onset AD is the
inheritance of the apolipoprotein (apo) E4 isotype of apoE. How apoE is
involved in the pathogenesis of AD is unclear; however, evidence exists
for a direct apoE/A
interaction. We and others have shown that
apoE copurifies with A
from AD amyloid plaques and that under
certain in vitro conditions apoE promotes a
-sheet
structure in A
peptides. Currently we document the high affinity
binding of A
peptides to both human recombinant apoE3 and -E4 with
a K
of 20 nM. This interaction
is greatly influenced by the conformational state of the A
peptide
used. Furthermore, we show that the fibril modulating effect of apoE is
also influenced by the initial secondary structure of the A
peptide. The preferential binding of apoE to A
peptides with a
-sheet conformation can in part explain the copurification of
A
and apoE from AD amyloid plaques.
Amyloid deposition is one of the hallmarks of the
Alzheimer's disease (AD). ()The major protein
component of the amyloid, which is deposited in the form of senile
plaques (SP) and cerebrovascular amyloid angiopathy, is a
39-44-residue protein, called amyloid
-peptide
(A
)(1, 2, 3, 4, 5, 6, 7) .
A
is derived by proteolytic processing from a large transmembrane
precursor termed
-amyloid precursor
protein(8, 9, 10, 11) . In both
parenchymal and vascular amyloid deposits, there is heterogeneity at
both the N and C termini of the peptide that is greater for the peptide
isolated from SP amyloid (2, 3, 6, 12) . Immunohistochemical
and biochemical studies suggest that the major initially deposited
A
consists of A
42/43 species (13, 14, 15, 16) . Both A
40
and 42/43, as well as numerous shorter A
fragments, have a
propensity to self-aggregate and form fibrils in
vitro(17, 18, 19, 20) .
However, it has been documented that longer A
pep-tides, extending
beyond Val-40, display a higher propensity for aggregation and
fibrillogenesis(20) . This leads to the hypothesis that A
42/43 is critical for the formation of SP (21) , although this
hypothesis has recently been questioned(22) . In 1992, a
soluble form of A
, termed sA
, was found in cerebrospinal
fluid and plasma of both healthy and AD
patients(23, 24, 25, 26) . The
circulating form of A
contains mostly A
1-40 but also
includes A
1-42/1-28(27) . Thus, the
fibrillogenic A
peptide can exist in both a soluble form and as a
fibril in SP. These data raise the question of what factors determine
the aggregation and fibrillogenesis of A
in vivo.
In vitro studies have shown that amyloid-like fibril
formation by A peptides is dependent on several factors such as
the length of A
(20) , pH(28) , and metal
ions(29, 30) . Recent data disclosed that a group of
compounds, collectively termed as amyloid-associated proteins, could
also significantly modulate the process of A
fibrillogenesis.
Apolipoprotein E (apoE) is one of the proteins shown in vivo to be associated with A
amyloid and other cerebral and
systemic amyloids(31, 32) . It was proposed that apoE
could play a role of ``pathological chaperone''-promoting
aggregation/fibrillogenesis of A
peptide, possibly through
propagation of a
-sheet
conformation(31, 33, 34, 35) .
ApoE in humans is expressed in three common isoforms of E2, E3, E4
that differ by the presence or absence of cysteine at two amino acid
residues, 112 and 158(36) . Genetic studies have shown that
inheritance of apoE4 is a significant risk factor for late onset AD,
decreasing the age of onset and the duration of
disease(37, 38) . Although apoE4 is neither necessary
nor sufficient to cause AD, apoE4 carriers appear to have a higher
amyloid load(39) . There are a number of ways by which apoE may
be involved in the pathogenesis of AD. For example, a differential
effect on neurite outgrowth of apoE3 and -E4 has been
demonstrated(40, 41, 42) . However, several
pieces of evidence also suggest a direct apoE/A interaction. Under in vitro conditions, apoE hydrophobically binds A
,
forming complexes resistant to SDS, as well as organic and chaotropic
solvents(38, 43, 44) . Initially, a striking
difference between apoE3 and apoE4 binding to A
was found, showing
that apoE4 binds A
peptide more avidly(45) , but a recent
study has demonstrated that this is dependent on whether apoE is
associated with lipids(46, 47) . In addition to
binding A
, apoE promotes fibril formation by
A
(33, 34, 35) , and other synthetic
amyloidogenic peptides(48) , under the conditions used in
several studies; however, some investigators (49) have not
found apoE to promote A
fibrillogenesis. Recent biochemical
studies have indicated that apoE is complexed in vivo to the
amyloid in both AD senile plaques (44, 50) and
systemic amyloid deposits of serum amyloid A and immunoglobulin light
chain(51) . These in vivo and in vitro data
suggest a potentially important role of apoE in amyloidogenesis in
vivo. However, the nature of apoE interaction with A
remains
to be clarified. The aim of this study was to analyze the sequential
and structural determinants of the A
/apoE interaction.
To
quantitate the relative affinity of different A analogs and its
different lots, competitive inhibition studies were performed. Plates
were coated with A
C1-40 in the same manner as for the
saturation curve for apoE. Following blocking, a saturating amount of
apoE was added (150 nM) along with serial dilutions of
different A
peptides at a concentration of 0-50
µM, in a volume of 100 µl. This allowed for
competition between the peptide immobilized and peptides in solution
for apoE binding and the calculation of IC
values.
A peptides were dissolved at 250-1000 µM, with or
without 2.5 µM apoE (peptide:apoE molar ratio
100-400:1), in 100 mM Tris, pH 7.4, in 30-µl
aliquots. After 3 days of incubation, samples were diluted to 2 ml in
50 mM glycine, 2 µM Thioflavin T (Sigma), pH 9.2,
and were read on Hitachii F-2000 spectrofluorometer at excitation and
emission wavelengths 435 and 485 nm, respectively. Time scans were
performed, and values at 280, 290, and 300 s were averaged to give the
final reading.
Incubation of serial dilutions of recombinant apoE3 with
wells coated with A 1-40 revealed a dose-dependent binding
that reached saturation at 150 nM (Fig. 1.). Based on
our previous data of 1:1 stoichiometry of apoE to A
binding (58, 59) , K
was calculated as 20
nM ± 2 (S.D.). The affinity of recombinant apoE4,
apoE3, and apoE2 to A
under these conditions was in a similar
range (data not shown). Therefore, all the following experiments were
performed on the most frequent apoE isoform in humans, apoE3. These
experiments were performed on delipidized apoE; however, different
states of lipid association may affect the K
of
the binding of apoE with A
.
Figure 1:
Saturation curve for apoE
binding to immobilized A. A
C1-40 was immobilized at
400 ng/well and incubated in the presence of serial dilutions of apoE
(0-150 nM). Bound apoE was revealed by using monoclonal
antibody 3D12. Using nonlinear regression fit, the K
value of this interaction was calculated as 20 nM.
Plotted data represent mean values (±S.D.) from three
independent experiments performed in
duplicate.
To characterize the binding site of
apoE on A 1-40, we used a competitive inhibition assay using
different A
fragments, which were allowed to interact with apoE in
solution in the presence of immobilized full-length peptide. The
ability of A
1-16, 10-20, 12-28, and 25-35
to inhibit binding of apoE to A
1-40 was tested. None of
these short A
peptides, which together span almost the entire
A
molecule, were able to significantly inhibit this interaction (Fig. 2A). Since we found that sequence determinants
are not decisive for A
-apoE binding, we have looked for
conformational aspects of this interaction.
Figure 2:
Inhibition of apoE and A 1-40
interaction by using different shortened and full-length A
peptides. A, inhibition by peptides A
12-28
(
), A
25-35 (
), and A
C1-40
(
). B, comparison of the inhibitory effect of some
full-length A
peptides, A
Y1-40 (
), A
C1-40 (
), A
1-42 (
), and A
B1-40
(
). IC
values for these inhibitions are presented
in Table 1. Plotted data represent mean values (±S.D.)
from three independent experiments done in
duplicate.
To evaluate the
influence of the secondary structure of A on the affinity to apoE,
we did inhibition studies using A
1-42, A
1-40Q,
and A
1-40G, as well as different lots of A
1-40.
The greater propensity for aggregation of A
1-42 (20) as well as peptides with a substitution of Gln for Glu at
residue 22 of A
(found in a familial variant of AD) (60, 61, 62) and A
1-40G (Gly for
Glu substitution also at residue 22 found in another familial type of
AD) (63) has been previously demonstrated and appears to result
from their higher initial
-sheet content(64) . The
lot-to-lot variability in fibrillogenesis of different synthetic A
preparations may also be related to their initial secondary
structure(65) . We also have examined the affinity to apoE of
two major A
conformers, A
and
A
(52) . Varying lots of A
and A
analogs, as well as isolated A
conformers, had quite different
abilities to inhibit the binding of apoE to A
immobilized to the
plate (Fig. 2). Calculated IC
values for this
inhibition varied from 315 nM to above 10 µM.
Hence, a severalfold difference in the relative affinity to apoE was
found for A
peptides sharing identical or very similar chemical
structure (Table 1). The highest relative affinities for apoE was
shown by A
, followed by A
1-40Q and A
1-42. A
, A
S1-40, and A
Y1-40 showed the lowest affinities for apoE. In addition, the
affinity of some of these peptides to apoE3 was directly measured by
immobilizing them on the plate and obtaining saturation curves. This
also revealed severalfold differences in the calculated K
value (data not shown). To evaluate the
influence of the secondary structure of these different A
peptides
on their relative affinity to apoE, we have performed CD analyses of
these peptides. A
B1-40, 1-40Q, 1-40G, and
1-42 displayed predominantly
-pleated sheet spectra with a
minimum at 215 nm and a maximum at 195 nm, whereas A
S1-40,
C1-40, and Y1-40 showed predominantly random coil structure
with a minimum at 190 nm (as shown partially in Fig. 3). By gel
filtration all these A
peptides were >90% monomeric or dimeric
(data not shown). As presented in Table 1, the affinity to apoE
of the peptides used appears to correlate with their secondary
structure; peptides with the highest
-pleated sheet content, such
as A
and A
1-40Q, displayed the greatest
relative affinities for apoE3, whereas A
S1-40, Y1-40,
and A
, with the lower content of
-sheet, were
the weaker inhibitors of the A
/apoE interaction.
Figure 3:
CD analysis of full-length A
peptides. Peptides were dissolved at 0.15-0.20 mg/ml in 10 mM sodium phosphate buffer, centrifuged, and their spectra recorded
on Jasco 720 spectropolarimeter. The CD spectra are labeled 1-4; corresponding to peptides A
1-42, A
1-40Q, A
Y1-40, and A
C1-40, respectively.
Obtained spectra were fitted by using the Lincomb
algorithm(54) . Resulting estimations of
-sheet content
are shown in Table 1.
Once having
established the secondary structure of the A peptides used and
their binding affinities to apoE, we correlated these data with the
known modulatory effect of apoE on the fibrillogenesis of A
peptides(33, 34, 35, 58) . We
performed incubations of these A
peptides in the presence or
absence of apoE3 and measured the amount of fibril formation using a
Thioflavin T assay(56, 57) . ApoE3 enhanced the
fibrillogenesis of all full-length A
peptides, although to a
variable extent. Fibrillogenesis of the peptides that showed an initial
random coil structure was stimulated the most, whereas fibrillogenesis
of the peptides with an initial high
-sheet content was stimulated
the least (Fig. 4). The highest modulatory effect of apoE was
observed for A
12-28, which suggests that this peptide
constitutes a target sequence for apoE effect in A
. None of the
other shortened A
peptides showed a significant increase in
fibrillogenicity upon coincubation with apoE.
Figure 4:
Thioflavin T fluorescence of A
peptides incubated in the presence and absence of apoE. Peptides A
1-16, 12-28, 10-20, and 25-35 were incubated at
2 mg/ml and full-length peptides at 1 mg/ml with or without 2.5
µM of apoE (apoE:A
molar ratio 1:100-400) in
30-µl aliquots for 3 days. The fluorescence of Thioflavin T (2
µM) was measured at an excitation of 435 nm and and an
emission of 485 nm(56, 57) . The mean value
(±S.D.) from three independent experiments is
plotted.
By using solid-phase assay we have shown that delipidated,
recombinant apoE saturably binds to A to give a final K
value of 20 nM, indicating a high
affinity binding. A similar K
value has recently
been reported between human purified, delipidated apoE3 and
A
1-40, using surface plasmon resonance(72) .
However, in normal physiological fluids apparently little sA
exists in complex with apoE(59) . ApoE under normal conditions
exists mainly in lipid particles, and under these conditions the
affinity of binding of apoE to A
may be lower. Furthermore, the
calculated affinity of A
1-40 to another apolipoprotein-apoJ
is 10 times higher than the value we found for apoE/A
binding(55) . This is in agreement with the major binding
protein of sA
under normal conditions being
apoJ(59, 66) . Additionally, we were able to correlate
the affinity of apoE binding to A
with the secondary structure of
the peptide. We show that apoE has a greater affinity for A
peptides with a high
-sheet content, whereas binding to the mainly
random coil peptides is low. Consistent with these in vitro findings, it has recently been shown that A
in senile
plaques, which is predominantly
-sheet, is complexed to apoE and
apoE carboxyl fragments(44, 50) .
Furthermore, we
show that the modulatory effect of apoE on A fibrillogenesis is
dependent on the initial secondary structure of the peptide.
Fibrillogenesis of A
peptides with a high content of
-sheet
is only slightly stimulated, whereas fibrillogenesis of the peptides
with a random coil initial secondary structure is stimulated
severalfold. The distinct behavior of these A
peptides with the
same amino acid sequence may be one explanation for different
laboratories obtaining varying results in A
/ApoE fibrillogenesis
experiments. We have identified residues 12-28 of A
as a
most probable target for this stimulatory effect; fibrillogenesis of
this peptide was stimulated by apoE about 10-fold. This portion of
A
has already been shown to be important for the adoption of a
-pleated sheet structure(28, 67) , and a recent
NMR-based study has shown that A
residues 10-35 are required
for the binding of A
peptides to authentic amyloid
plaques(68) . By interacting with this sequence, apoE could
mimic the effect of known mutations associated with FAD that are found
within this region and which tend to increase A
fibrillogenicity(60, 61, 62, 63) .
Additionally, other factors that have been shown to promote A
fibril formation, such as metal cations (Zn
,
Al
), as well as heparan sulfate proteoglycans, also
bind in this region(29, 30) . This portion of the
A
protein may serve as a potential target for the designed
compounds to interfere with the initial random coil to
-sheet
conformational change that occurs in amyloidogenesis.
Late-onset AD
has been linked to the presence of the apoE4 isotype, with individuals
with the 4 allele having a greater A
load (37, 38, 39) . Under the conditions studied,
we did not find major differences in the binding between the three apoE
isotypes and A
1-40. When we used human-purified,
delipidated apoE3 for these same binding experiments to A
1-40, a slightly different K
of 15 nM was observed (data not shown). Therefore, we cannot rule out that
some variations in the binding of three apoE isotypes to A
peptides will be found when other apoE preparations are used.
Alternatively, distinct binding affinities of apoE isotypes to A
peptides may not be related to the linkage of apoE4 to late-onset AD.
To explain the conformational requirements of A for apoE
binding and its modulatory effect, we propose that apoE in the process
of A
fibrillogenesis displays chaperone-like activity, modulating
A
molecules to adopt or stabilize a pathological
-sheet
secondary structure. Secondary structure predictions and direct
measurements show that apoE contains about 30%
-sheet, with the
-sheet content being slightly increased in the absence of
lipids(69, 70) . Some of these
-sheet stretches
could form hydrophobic pockets, easily accessible for small hydrophobic
peptides. We propose that a
-sheet motif of apoE acts as an
instructor for the random coil A
peptides. In vitro we
have recently shown that apoE can act to increase the
-sheet
content of A
peptides(73) . Although
-sheet-structured A
binds apoE with high affinity, this
interaction has little effect on fibrillogenesis. The critical
interaction in AD is the low affinity binding between apoE and the
random coil sA
that leads to the greatest modulation of fibril
formation.
Our previous (58) and current data show that the
aggregation of poorly fibrillogenic A peptides, even in the
presence of apoE, do not proceed to the level observed for more
amyloidogenic peptides. Fibrillogenesis of the initially
-sheet-rich peptides studied here is not significantly affected by
the presence of apoE. This can be explained on the basis of the molar
ratio of
-sheet and random coil peptide. An excess of
-sheet
conformers will change the equilibrium of the bound and free apoE,
sequestering most of the templates. Fibrillogenesis of these peptides,
if not inhibited by other factors, proceeds until it reaches a high
level, predetermined by their specific initial secondary structure.
This model incorporates the concepts of pathological chaperone (31, 33) and conformational mimicry previously
proposed by us(71) .
In summary, we show the preferential
binding of apoE to A peptides with a high
-sheet content.
This could in part explain the reported in vivo complexing of
A
with apoE within senile plaques, while at the same time little
sA
is found complexed to apoE in biological fluids. Our findings
underscore the importance of the conformation of A
peptides in the
pathogenesis of AD.