(Received for publication, February 21, 1995; and in revised form, May 1, 1995)
From the
Apolipoprotein E (ApoE) immunoreactivity is consistently present
in the senile plaques and neurofibrillary tangles of Alzheimer's
disease (AD) brain. In vitro, apoE, and in particular its
apoE4 isoform, can bind to and promote fibrillogenesis of the amyloid
A
Amyloidosis is a disorder of protein conformation in which low
molecular weight proteins that are soluble under physiological
conditions become deposited and accumulate either intact or partially
digested in diverse tissues and organs as insoluble amyloid fibrils. In
spite of their biochemical diversity, amyloid proteins adopt a common
secondary structure, the
ApoE is a 34-kDa exchangeable
apolipoprotein, present in all types of lipoprotein particles, that is
involved in cholesterol transport as well as other less defined
functions such as nerve regeneration after
injury(9, 10, 11, 12) . The recently
reported association between certain apoE genetic isoforms and
Alzheimer's disease (AD), a condition characterized by the
massive deposition in the brain of the 39-44-residue amyloid
This widespread
association of apoE with biochemically and clinically diverse types of
amyloidoses suggests that apoE may participate in a general manner in
the process of amyloid formation. In order to gain insight into the
biochemistry of the association between apoE and amyloid proteins, we
have characterized by Western blot and amino-terminal sequence analysis
apoE fragments that co-purified with amyloid subunits from cases of
systemic amyloidosis AA and AL. We also studied the in vitro binding of human apoE isolated from plasma to these amyloid
proteins.
Crude amyloid fibrils and fractions obtained
by gel filtration and HPLC were subjected to 12.5% Tricine-sodium
dodecyl sulfate-polyacrylamide gel electrophoresis
(SDS-PAGE)(28) , and the proteins were electrophoretically
transferred (1 h, 400 mA, 4 °C) to polyvinylidene difluoride
membranes (Immobilon, Millipore) using 10 mM CAPS buffer, pH
11, containing 10% methanol. The membranes were blocked with 5% nonfat
dry milk in 10 mM Tris-HCl, 150 mM sodium chloride,
0.1% Tween 20, pH 7.6, for 2 h at room temperature and then incubated
overnight at 4 °C with the following antibodies: rabbit polyclonal
antibodies to amyloid A(29) ,
Immunohistochemistry of spleen sections from patients COH and
RAM showed that polyclonal anti-human apoE labeled amyloid deposits as
described previously(7) . This immunoreactivity was completely
abolished after adsorption with purified human apoE, indicating its
specificity (Fig. 1).
Figure 1:
Immunohistochemistry on spleen sections
of patient COH with amyloid A. a, anti-apoE antibody labels
amyloid-laden vessels. The bar represents 20 µm. b, an adjacent section as in a after adsorption of
anti-apoE with human purified apoE.
Figure 3:
Microsequence Analysis. Amino-terminal
sequence of amyloid L (
Figure 2:
Purification of RAM Amyloid L. a,
reverse phase HPLC of RAM amyloid L on a Deltapak C
Purification of
amyloid A subunits from COH was performed by saline-water extraction
followed by separation on HPLC (Fig. 4a). This
procedure yielded broad ill defined peaks that eluted at 60-78%
solvent B, which were pooled into four fractions, A, B, C, and D, that
were rechromatographed and analyzed by SDS-PAGE. A major protein band
of 6-8 kDa was present in all the fractions, and a minor 16-kDa
component, which may be a polymer of the 6-8-kDa peptide, was
detected in fractions B and C (Fig. 4b). Amino-terminal
sequence analysis of the HPLC fractions revealed only one residue per
cycle. The sequence was identical to serum amyloid A protein (Fig. 3). No additional sequences were identified. When the same
fractions were subjected to Western blot using anti-apoE antibody, the
fluorogram revealed a broad band at 8-9 kDa in fraction D (Fig. 4c). Immunoreactivity of this band was completely
suppressed after adsorption of the antiserum with purified human apoE,
reflecting the specificity of the reaction. There was no band
corresponding to intact apoE (34 kDa), suggesting that only small
fragments of apoE were associated with the amyloid A subunit.
Figure 4:
Purification of COH amyloid A proteins. a, reverse phase HPLC of COH amyloid A on a Deltapak C
When
0.5 micrograms of purified human apoE were incubated with 10 µg of
purified COH amyloid A for 6 h at room temperature in 0.1 M Tris, pH 7.4, a novel component of approximately 44 kDa was
detected by Western immunoblot analysis using anti-apoE (Fig. 5a) and anti-AA antibodies (not shown),
indicating that an apoE-amyloid complex partially resistant to SDS was
formed. Under nondenaturing conditions in 7.5% polyacrylamide gels,
this complex resulted in a shift of the electrophoretic mobility of
apoE (Fig. 5b). No complex formation was detected when
apoE was incubated with 20 µg of ubiquitin. The influence of apoE
upon AA polymerization was evaluated by detection of the apoE-AA
incubation mixture with an anti-AA antibody on Western blots. AA alone
incubated for 6 h at room temperature in 0.1 M Tris, pH 7.4,
showed mainly monomeric forms and minor polymeric components after
SDS-PAGE. In contrast, AA co-incubated with apoE at a 1:85 molar ratio
(apoE:amyloid) under the same conditions as above resulted in the
appearance on the gel of a higher amount of AA dimers, trimers,
tetramers, and higher molecular weight components (Fig. 5c). AA co-incubated with BSA showed no increment
in polymerization when compared with AA alone (not shown). When the
same experiment was done using RAM AL, no additional bands were
present. However, most of the amyloid-apoE incubation mixture remained
on top of the stacking and running gels as very high molecular weight
components (not shown). This result is consistent with a higher degree
of aggregation of amyloid AL subunits in the presence of apoE that
resisted denaturation by SDS treatment.
Figure 5:
In vitro co-incubation of amyloid
A with apoE. a, ApoE (0.5 µg) and amyloid A (10 µg)
were incubated in 0.1 M Tris, pH 7.4, for 6 h at room
temperature. The incubation was stopped by the addition of 2
Apo E is a two-domain protein, modeled by its two major
fragments after thrombin digestion. The 22-kDa amino-terminal domain
(residues 1-191) is a stable globular structure containing the
sequence that mediates low density lipoprotein-receptor
binding(9, 30) . In contrast, the 10-kDa
carboxyl-terminal fragment is less stable, binds to lipoproteins, and
mediates lipid-free apoE tetramerization in aqueous
solutions(31) . Previously, we published the finding of a
tryptic fragment of apolipoprotein E corresponding to residues
270-278 in association with two amyloid A proteins(29) .
Our present data extend that observation. Three fragments of apoE,
located within the thrombolytic carboxyl-fragment of apoE, co-purified
with RAM amyloid L. A slightly larger fragment of apoE starting in the
region connecting the thrombolytic amino-terminal and carboxyl-terminal
domains of apoE co-purified with COH amyloid A (Fig. 6). Similar
apoE fragments have been recently obtained from AD senile plaques (32
Figure 6:
Schematic representation of the structural
domains of apolipoprotein E (a) modeled by thrombin
proteolytic cleavage(31) . Alignment of the apoE
carboxyl-terminal peptides associated with AL and AA proteins is shown
as well as a representation of the purported apoE ``binding
domain'' for A
It
is likely that the initial formation of stable apoE-amyloid complexes
requires the interaction of the amino and carboxyl-terminal domains of
apoE. Binding to the low density lipoprotein receptor through the
amino-terminal domain of apoE requires lipid association, largely
mediated by the carboxyl end of the molecule(31) . The
interdependence of the domains of apoE is further reflected by the
effect that amino acid substitutions within the amino-terminal region
have on the association of apoE with diverse lipoprotein classes. Such
association is known to be mediated by the carboxyl terminus of apoE (9) . The apoE3 isoform with a Cys at position 112
preferentially binds high density lipoproteins, whereas the presence of
Arg at the same position favors binding to very low density and
intermediate density lipoproteins(33, 34) . A
cooperation between domains unique to each apoE isoform has been
postulated to explain this selective lipoprotein
distribution(35) . Whether a similar mechanism can account for
the higher avidity of the apoE4 isoform for A
ApoE is capable of binding to synthetic A
Our finding of a higher degree of polymerization of AA and AL native
peptides upon incubation with apoE together with previous work using
synthetic A
ApoE may act as one of a group of pathological chaperones that
promote the aggregation of amyloidogenic precursors of diverse primary
structure into the
Recently it has
been reported that apoE can inhibit A
The abnormal amyloid response in each
individual will be determined finally by a complex process involving
critical concentrations of amyloid precursors and a set of
amyloid-associated proteins such as apoE, other apolipoproteins, AP,
peptide, the main constituent of senile plaques. These findings,
together with the strong genetic association between late onset AD and
the E4 allele of apoE, have strengthened the hypothesis that apoE may
have a central role in the pathogenesis of AD by modulating A
cerebral accumulation. However, apoE immunoreactivity is present in all
cerebral and systemic amyloidoses tested, and tryptic apoE fragments
have been identified in association with amyloid A (AA). In order to
further elucidate the interaction between apoE and amyloids, we
purified AA and amyloid L (AL) fibrils from patients with familial
Mediterranean fever and primary amyloidosis, respectively, and studied
the association of apoE with AA and AL proteins. In each case, apoE
fragments, detected by Western blot, co-purified with the amyloid
fibrils. Microsequencing analysis identified COOH-terminal fragments of
apoE, similar to the 10-kDa fragment produced by thrombin digestion
that contains the purported binding region to A
. In vitro co-incubation of AA with purified human apoE resulted in the
formation of an SDS-resistant AA
apoE complex and a higher degree
of polymerization of the AA peptide. These findings and similar results
obtained from AD senile plaques suggest that 1) the carboxyl-terminal
fragment of apoE is complexed to amyloid fibrils and resists
proteolysis in vivo and 2) apoE may promote amyloidogenesis
through a conformation-dependent interaction regardless of the primary
structure of the amyloid precursors.
-pleated sheet, and form fibrils of
similar morphology. These fibrils are characteristically long straight
filaments 5-12 nm wide that share the tinctorial properties of
green birefringence after Congo red staining and specific affinity for
fluorescent dyes such as Thioflavine S or T. In addition to these
features shared by most types of amyloids, there are several factors
consistently associated with this pathological condition that can be
viewed as part of the biochemical setting in which amyloid deposition
arises and develops. These include elevated concentration of amyloid
precursors in fluids and tissues and the invariable presence of certain
amyloid-associated proteins of which amyloid P-component
(AP)
(
)(1, 2) , sulfated
proteoglycans (3, 4, 5) , apolipoprotein E
(apoE) (6, 7) and apolipoprotein J (8) are the
most notorious. Whether these amyloid-associated proteins play an
active role by promoting or inhibiting amyloidogenesis or are inert
bystanders is at present unknown.
-protein
(A
)(13, 14, 15, 16, 17, 18) ,
has strengthened the hypothesis that this apolipoprotein may be
directly involved in the pathogenesis of this cerebral
amyloidosis(19, 20) . Moreover, in vitro binding experiments using human apoE isolated from plasma or
recombinant human apoE with synthetic peptides homologous to A
have shown that these two proteins can form an SDS-resistant complex
with A
and promote A
fibril formation in
vitro(21, 22, 23, 24) . These
lines of evidence, together with the immunohistochemical colocalization
of apoE and A
in the senile plaques and its presence in the
intraneuronal amyloid of neurofibrillary tangles(6) , suggest
that apoE may play an active role in the pathogenesis of AD by
promoting fibrillogenesis. However, immunohistochemical studies have
shown that apoE is tightly associated with other types of cerebral
amyloidosis, including Down syndrome related to A
, hereditary
cerebral amyloid angiopathy of Icelandic type, related to a cystatin C
variant, and spongiform encephalopathies such as Creutzfeldt-Jakob,
kuru, and Gerstmann-Sträussler-Scheinker disease that are
associated with the prion amyloid(6, 7, 25) .
Furthermore, apoE has been immunohistochemically identified within
amyloid deposits in systemic forms of the disease such as secondary
amyloidosis and familial Mediterranean fever related to amyloid A,
immunoglobulin-related primary amyloidosis (AL), and familial
amyloidotic polyneuropathy due to deposition of transthyretin genetic
variants(7) . Noteworthy, apoE is rarely detected in the
nonfibrillar monoclonal deposits of light chain deposition disease and
light and heavy chain deposition disease, which may be considered
preamyloid forms of AL disease(26) .
Protein Purification
Amyloid A and AL fibrils
were isolated by the method of Pras et al.(27) from
spleen tissue of patients with familial Mediterranean fever (COH) and
primary amyloidosis (RAM). Briefly, 20 g of spleen tissue were
homogenized in 0.15 M sodium chloride and centrifuged at 8,000
g for 30 min at 4 °C, and the supernatant was
discarded. This procedure was repeated until the supernatant had an
absorbance of less than 0.075 at 280 nm. Then the insoluble residue was
homogenized in distilled water and centrifuged at 8,000
g at 4 °C for 1 h. The amyloid fibrils that appear as a mucoid
mass in the upper layer were dialyzed against water and lyophilized.
After lyophilization, AA protein was purified on high performance
liquid chromatography (HPLC) (see below). AL fibrils were solubilized
in 3 ml of 6 M guanidine hydrochloride, 0.1 M Tris,
0.17 M dithiothreitol, pH 10.2 and stirred for 48 h at room
temperature. Then 1 ml of 2 M guanidine hydrochloride, 4 M acetic acid was added, and the solution was applied to a column
(2.5
180 cm) consisting of 1:1 (wt) Sephadex G-75 and Sephadex
G-100 (Pharmacia Biotech Inc.), equilibrated with 5 M guanidine hydrochloride, 1 M acetic acid. AL amyloid
fragments isolated by size exclusion chromatography and AA fibrils were
subjected to reverse phase chromatography on a Deltapak C
column (0.78
30 cm, Waters) with a gradient of
30-80% acetonitrile in 0.1% (v/v) trifluoroacetic acid, pH 2.5.
The column eluents were monitored at 214 nm, and protein peaks were
pooled and lyophilized.
light chain (Dako Corp.),
goat anti-human apolipoprotein E (Fitzgerald), and monoclonal
antibodies ID7 and 6C5 raised against residues 142-158, and
1-15 of apoE, respectively (a generous gift of Dr Y. Marcel).
Horseradish peroxidase-conjugated sheep anti-mouse (Amersham Corp.),
goat anti-rabbit, and rabbit anti-goat IgG (Biosource International)
were used as the second antibody at a dilution of 1:5000. Immunoblots
were visualized with an ECL chemiluminescence kit (Amersham Corp.),
according to the manufacturer's specifications. Anti-apoE
antiserum was adsorbed by incubating 15 µg of purified human apoE
with anti-apoE diluted 1:100 in 0.3% bovine serum albumin (BSA) in
Tris-buffered saline, pH 7.4, for 3 h at room temperature.
Protein Sequence Analysis
Automated Edman
degradation sequence analysis was carried out on a 477A Protein/Peptide
Sequenator, and the resulting phenylthiohydantoin amino acid
derivatives were identified using the on-line 120A PTH Analyzer
(Applied Biosystems, Foster City, CA).
In Vitro Incubation of ApoE and Amyloid
Peptides
Human ApoE was purchased from Cortex Biochem, and
bovine ubiquitin and BSA were purchased from Sigma. The purity (>
95%) of these proteins was assessed by SDS-PAGE and
NH-terminal sequence analysis. Since human pooled apoE was
used, it seems likely that its major isoform is apoE3 according to the
statistical distribution in the normal population. Stock solutions of
amyloid peptides were prepared in 0.1% trifluoroacetic acid, 50%
acetonitrile and quantitated by amino acid analysis using a Pico-Tag
analyzer (Waters) or by using a microbicinchoninic acid assay kit
(Pierce). ApoE stock solution was made at 0.7 mg/ml in 0.1 M Tris, pH 7.4, and aliquots were stored at -20 °C.
Aliquots from these stock solutions were lyophilized and used for the
co-incubation experiments as described(21) . In brief, 0.5
µg of apoE were incubated with 10 µg of AA or 15 µg of AL
in 12 µl of 0.1 M Tris, pH 7.4, for the indicated time at
room temperature. After incubation, 15 µl of 4% SDS-sample buffer
were added, and the mixture was run on 12.5% SDS-Tricine gels or on
nondenaturing 7.5% polyacrylamide gels without SDS. Proteins were
transferred to Immobilon P and detected using polyclonal anti-apoE or
anti-AA and anti-AL antibodies as described above. As a control for the
co-incubation experiments, ubiquitin (molecular mass, 8 kDa) and BSA
were used instead of amyloid peptides and apoE, respectively.
Immunohistochemistry
Sections of spleen tissue
from cases of amyloid A (COH) and AL (RAM) were deparaffinized in
xylene and rehydrated in ethanol and Tris-buffered saline. After
rehydration, endogenous peroxidase was quenched by incubation with 0.3%
hydrogen peroxide in methanol for 30 min. After blocking with 3% BSA in
Tris-buffered saline, sections were incubated with goat anti-human apoE
(Fitzgerald) at a 1:100 dilution overnight at 4 °C and rabbit
anti-goat labeled with horseradish peroxidase (Biosource International)
at 1:1000 for 1 h. The reaction was detected using 0.03%
3,3`-diaminobenzidine tetrachloride and 0.01% hydrogen peroxide, 50
mM Tris, pH 7.4. Anti-apoE antiserum was adsorbed with
purified human apoE as described above.
Amyloid L from patient RAM was purified
by the procedure of saline-water extraction and gel chromatography on
Sephadex G-100 equilibrated with 5 M guanidine. SDS-PAGE of
the fractions revealed that the main amyloid subunit had a molecular
mass of 12-13 kDa, and amino-terminal sequence analysis showed
its homology to a III or IV Ig light chain (Fig. 3). HPLC
separation of RAM AL yielded a broad peak between 45 and 73% solvent B.
This peak was divided into four fractions, A, B, C, and D, which were
rechromatographed (Fig. 2a).
) and amyloid A (
) proteins and of
the apoE peptides that co-purified with each of
them.
column
(0.78
30 cm) using a 30-80% linear gradient of
acetonitrile in 0.1% (v/v) trifluoroacetic acid. RAM amyloid L eluted
as a broad peak between 45 and 73% of solvent B. b, Coomassie
Blue stain of 12.5% Tricine-SDS-PAGE of RAM AL fibrils (F) and
of HPLC fractions A, B, C, and D. RAM amyloid L subunit has a mass of
12-13 kDa (K) and is present in all the fractions
together with a minor dimeric component of 26 kDa. The 8-9-kDa
band present in fraction C is a carboxyl-terminal truncated fragment of
AL. c, Western blot analysis of HPLC fractions A, B, C, and D
using an antibody to human apoE and developed with chemoluminescence
showed a major 8-9-kDa band and a minor 18-kDa band in fraction
D. The approximate yield of the apoE fragment obtained from RAM amyloid
was estimated as follows. The ratio of amyloid to apoE in fraction D
was determined by densitometry of the Coomassie Blue-stained
12-13-kDa and 26-kDa AL bands and the 8-9 kDa apoE band in
a PDI optical densitometer. This ratio was applied to the area of
fraction D in the HPLC profile. Then the proportional area of fraction
D to the total area of the HPLC profile was
determined.
SDS-PAGE analysis of
these fractions revealed that in addition to the major AL subunit of
12-13 kDa, a 26-kDa component was seen in all the fractions. The
latter probably corresponded to a dimer of AL subunit since it was
recognized by anti- chain antibody (not shown). In addition, minor
bands of approximately 8-9 kDa were present in fractions C and D (Fig. 2b). Western blot analysis of the same fractions
using anti-apoE antibody and visualized by chemiluminescence detection
revealed a major band at 8-9 kDa and a minor 24-kDa component in
fraction D (Fig. 2c). The failure to detect apoE by
direct amino-terminal sequence analysis of RAM fraction D, despite the
positive apoE immunoreactivity of this fraction, indicated that it was
present in a very low concentration. Therefore, the HPLC fractions A,
B, C, and D were run on SDS-Tricine gels and transferred to Immobilon
membrane, and the bands were excised and subjected to amino-terminal
sequence analysis. Amino-terminal sequence of the 8-9-kDa
component of fraction C revealed that it was a fragment of AL starting
at position 1 (not shown). The 8-9-kDa band of HPLC fraction D
yielded two major fragments of apoE starting at positions 225 and 227
and a minor one starting at position 216 in addition to the amyloid
protein (Fig. 3). The estimated relative yield of apoE
carboxyl-fragment extracted from these amyloid deposits was
approximately a 1:50 molar ratio, apoE:amyloid.
column (0.78
30 cm) using a 30-80% linear gradient
of acetonitrile in 0.1% (v/v) trifluoroacetic acid. COH amyloid A
eluted as a broad peak between 60 and 78% of solvent B. b,
12.5% Tricine-SDS-PAGE of HPLC fractions A, B, C, and D. A major COH
amyloid A subunit of 6-8 kDa (K) was present in all the
fractions. A minor 16-kDa component, possibly a polymer of the former,
was detected in fractions B and C. c, Western blot analysis of
HPLC fractions A, B, C, and D using an antibody to human apoE and
developed by chemiluminescence revealed a broad 8-9-kDa component
in fraction D.
The
failure to detect apoE by direct amino-terminal sequence analysis of
COH fraction D, despite the positive apoE immunoreactivity, indicated
that it was present in a very low concentration. Therefore, a similar
approach as for RAM AL was used, and proteins were separated on
SDS-Tricine gels and transferred to Immobilon membrane. The upper
portion of the 6-8-kDa band of HPLC fraction D yielded two
sequences corresponding to the N terminus of amyloid A and to an apoE
fragment starting at position 199, respectively (Fig. 3). In
order to investigate whether intact apoE was present in the crude A and
L amyloid fibril fraction before HPLC separation, we performed Western
blots using monoclonal antibodies ID7 and 6C5, specific for the
amino-terminal domain of apoE. A band corresponding to the 34-kDa
intact apoE was observed only in association with COH fibrils.
Laemmli sample buffer, and samples were run on 12.5% Tricine SDS-PAGE.
After transferring to Immobilon P, proteins were detected with
anti-apoE antibody. Lane1, apoE alone; lane2, apoE incubated with amyloid A. Leftmargin indicates molecular mass in kilodaltons (kD). E, apoE; EA, apoE-amyloid A complex. b, apoE and amyloid A were incubated as described above and
run on nondenaturing 7.5% polyacrylamide gels. Proteins were detected
on Western blots with anti-apoE antibody. A shift in the
electrophoretic mobility of apoE reflects the formation of an
amyloid-apoE complex. Lane1, apoE alone; lane2, apoE incubated with amyloid A. c, amyloid A
was incubated alone or with apoE as described above. Samples were run
on 12.5% SDS-Tricine PAGE and transferred to Immobilon P membranes.
Proteins were detected with anti-AA antibody. A higher degree of
polymerization of amyloid A peptide was present upon incubation with
apoE. Lane1, amyloid A alone; lane2, amyloid A incubated with apoE. Leftmargin indicates molecular mass in
kDa.
(b)(20) .
We propose that the apoE fragments that co-purify with
AA and AL are bound to these amyloid proteins in vivo. The
remainder of the apoE molecule is cleaved by thrombin and/or other
serine proteases in situ either before or after the
amyloid-apoE complex is formed. Alternatively, we cannot rule out that
apoE was partially degraded during purification. Intact apoE and apoE
fragments, as determined by Western blot analysis, are present in the
crude AA and AL fibril fractions before HPLC. However, after
purification only the carboxyl-terminal apoE fragment co-purifies with
the AA and AL amyloids. The question of whether the apoE-amyloid
association precedes tissue deposition remains to be addressed.
of AD remains to be
tested.
through a
strong interaction resistant to SDS(21) . A similar interaction
between apoE and A
of the Dutch variant of cerebral amyloid
angiopathy, a rare form of familial AD, has also been documented (36) (details of apoE-A
binding will be published
elsewhere). By using a set of truncated recombinants, the purported
binding region to A
has been located within the carboxyl-end of
apoE between amino acids 244 and 272 (21) . Our findings
suggest that the binding of this region to A
is not specific for
this peptide but rather reflects a hydrophobic interaction between apoE
carboxyl end and a common conformation shared by different amyloids.
Secondary structure predictions indicate that a putative amphipathic
helix with high affinity for lipids (37) is located within the
region that co-purifies with amyloid peptides. Possibly, other
exchangeable apolipoproteins sharing similar amphipathic helices can
also have amyloid-apolipoprotein interactions. It is noteworthy that
genetic variants of apoAI can form amyloid in certain hereditary forms
of human amyloidosis (38, 39) and that SAA, the
precursor of AA, is itself an apolipoprotein associated to high density
lipoprotein(40) . We recently found that apoAI can bind to
A
and promote A
fibrillogenesis in vitro.
(
)Presumably, in the same way that there is a large
number of amyloidogenic precursor proteins that can end as insoluble
amyloid fibrils, there is also a group of amyloidogenic apolipoproteins
and acute phase reactants that can actively participate in this process
either as constituents of fibrils or as modulators of fibrillogenesis.
(22, 23, 24, 36) points to a general
role of apoE in amyloidogenesis. The molar ratios of apoE and AA used
in our in vitro experiments are opposite of those found in
plasma in normal conditions for apoE and SAA. However, SAA is an acute
phase reactant, and its concentration in plasma is capable of rising
1000-fold during tissue injury or inflammation(41) . AL, in
turn, is derived from monoclonal light chains, which are known to have
very high levels in the circulation in most of the cases. Therefore, we
believe that the concentrations that we used in vitro can
resemble more closely the pathological conditions in which amyloidosis
develops than the physiological levels of apoE and amyloid precursors.
-pleated sheet conformation of amyloid fibrils.
The absence of apoE and AP in the nonfibrillar, Congo red negative
monoclonal deposits of light chain deposition disease and light and
heavy chain deposition disease, which are considered preamyloid forms
of AL disease(26) , suggests that apoE and AP may be essential
catalysts for the modulation of the amyloid generation process. The
interaction between apoE and amyloids may also be influenced by other
local acute phase reactants such as proteoglycans to which both amyloid
precursors and apoE are known to bind(42, 43) . In
spite of this putative widespread role of apoE in amyloid formation, it
seems likely that a certain specificity exists between some of the
amyloid precursors and apoE genetic isoforms. Thus far, no preferential
associations between systemic amyloidoses and apoE isoforms have been
reported; however, the genetic association of AD with apoE4 and the in vitro higher avidity of this isoform for A
may reflect
such specificity(19, 21) . Alternatively, the apoE4
isoform may participate in the pathogenesis of AD through a different
pathway unrelated to A
formation or deposition.
aggregation in vitro at concentrations similar to those found normally in biological
fluids(44) . This apparent discrepancy with previous studies
could be due to different experimental
conditions(22, 23, 24, 36) .
However, it raises the intriguing possibility that apoE may have a dual
effect upon fibrillogenesis in vivo depending on the local
concentrations of both apoE and amyloid precursors in sites of amyloid
formation. Within physiological levels, apoE may have a protective role
on amyloid formation by sequestering soluble amyloid precursors, as has
been postulated for A
(44) . However, A
appears to be
associated to other apolipoproteins (i.e. apolipoprotein J) in
biological fluids in vivo(45) . Yet, in tissues in
which local membrane repair and lipid turnover are increased, such as
after cell injury, apoE is overexpressed and can reach higher
concentrations than those found normally in the
circulation(10, 11, 12, 46) . Under
these circumstances, apoE may have a substantially different effect
upon its association with amyloid precursors. In this pathological
setting, several other factors may affect apoE behavior, such as its
state of oxidation, lipid association, the presence of other
amyloid/apoE binding proteins, and the stage of the amyloid
process(47) .
-antichymotrypsin (22) , and proteoglycans.
These may act in conjunction with yet undefined tissue-specific factors
to modulate the conformational transition of a soluble protein into an
insoluble fibril. A better understanding of these complex interactions
between amyloids and the factors that modulate their formation may open
novel strategies for the treatment of the systemic and cerebral
amyloid-related diseases.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.