(Received for publication, May 15, 1997)
From the Cell Biology and Anatomy Department, Medical University of South Carolina, Charleston, South Carolina 29425-2204
Apolipoprotein J (apoJ) has been
shown to be the predominant amyloid -peptide (A
)-binding protein
in cerebrospinal fluid. We have previously demonstrated that the
endocytic receptor low density lipoprotein receptor-related
protein-2/megalin (LRP-2), which is expressed by choroid plexus
epithelium and ependymal cells lining the brain ventricles and neural
tube, binds and mediates cellular uptake of apoJ (Kounnas, M. Z.,
Loukinova, E. B., Stefansson, S., Harmony, J. A., Brewer, B.,
Strickland, D. K., and Argraves, W. S. (1995) J. Biol.
Chem. 270, 13070-13075). In the present study, we evaluated the
ability of apoJ to mediate binding of A
1-40-apoJ
complex to LRP-2 in vitro. Immunoblot analysis showed that
incubation of apoJ with A
1-40 resulted in the formation
of A
1-40-apoJ complex and the inhibition of the formation of A
1-40 aggregates. Using an enzyme-linked
immunosorbent assay, an estimated dissociation constant
(Kd) of 4.8 nM was derived for the
interaction between A
1-40 and apoJ. Enzyme-linked
immunosorbent assay was also used to study the interaction of the
A
1-40-apoJ complex with LRP-2. The results showed that
A
alone did not bind directly to LRP-2; however, when
A
1-40 was combined with apoJ to form a complex, binding
to LRP-2 took place. The binding interaction could be blocked by
inclusion of the receptor-associated protein, an antagonist of apoJ
binding to LRP-2. When LRP-2-expressing cells were given
125I-A
1-40, cellular uptake of the
radiolabeled peptide was promoted by co-incubation with apoJ. When the
cells were provided purified
125I-A
1-40-apoJ complex, the complex was
internalized and degraded, and both processes were inhibited with
polyclonal LRP-2 antibodies. Furthermore, chloroquine treatment
inhibited the cellular degradation of the complex. The data indicate
that apoJ facilitates A
1-40 binding to LRP-2 and that
the receptor mediates cellular clearance of A
1-40-apoJ
complex leading to lysosomal degradation of A
1-40. The
findings support the possibility that LRP-2 can act in vivo
to mediate clearance of the complex from biological fluids such as
cerebrospinal fluid and thereby play a role in the regulation of A
accumulation.
A hallmark feature of Alzheimer's disease is the accelerated
cerebral accumulation of amyloid -protein
(A
),1 a small 39-42-residue
proteolytically derived fragment of amyloid
-precursor protein (1,
2). The accumulation takes the form of spherical extracellular deposits
of A
fibrils in the vicinity of morphologically abnormal axons and
dendrites. Associated with these so-called plaques are microglia and
astrocytes. The mechanisms that lead to accumulation of A
are still
obscure but represent an area of intense investigation. Whereas much
emphasis is currently placed on trying to determine the mechanism(s) of
A
biosynthesis from amyloid
-precursor protein processing (3, 4),
little is being done on determining possible mechanisms that mediate the catabolism of A
. Catabolic processes may prevent the
extracellular accumulation of A
that is expressed under normal
physiological conditions yet does not accumulate to the extent seen in
Alzheimer's disease or Down's syndrome.
A can be found in cerebrospinal fluid and blood in complex with
apolipoprotein J (apoJ) or apolipoprotein E (apoE) (5, 6). Whereas apoE
has been reported to promote A
fibrilogenesis (7-9), apoJ has been
shown to slow the formation of A
aggregates and may therefore act to
maintain A
in a soluble form and prevent it from forming
pathological fibrils (10). Our discoveries that LRP-22 is an endocytic receptor for apoJ
(11) and LRP-2 is expressed by cells that are in contact with
cerebrospinal fluid (choroid plexus and ependymal cells) (12) prompted
us to hypothesize that LRP-2 may mediate clearance of A
complexed
with apoJ, thereby controlling the accumulation of A
. In the present
study, we used in vitro solid phase binding assays as well
as cellular internalization and degradation assays to evaluate the
roles of apoJ and LRP-2 in mediating cellular clearance of A
.
Human apoJ was purchased from Quidel (San Diego,
CA). Synthetic A fragment 1-40 and ovalbumin were obtained from
Sigma. Bovine serum albumin was purchased from U. S. Biochemical Corp.
Human RAP was expressed as a glutathione S-transferase
fusion protein in bacteria and prepared free of glutathione
S-transferase as described by Williams et al.
(13). LRP-2 was purified from extracts of porcine kidney by affinity
chromatography using a column of RAP coupled to Sepharose as described
previously (36).
The mouse monoclonal antibody to LRP-2
designated 1H2 was provided by Dr. Robert McCluskey (Massachusetts
General Hospital, Boston, MA). Mouse monoclonal antibody to human apoJ
(mAb 1D11) was obtained from Dr. Judith Harmony (University of
Cincinnati College of Medicine, Cincinnati, OH). Mouse monoclonal
antibody to human A (mAb 4G8) was purchased from Senetek (Maryland
Heights, MO). Rabbit anti-LRP-2 IgGs (rabbit 6286) were isolated by
immunoaffinity chromatography on a column of porcine LRP-2 coupled to
CNBr-activated Sepharose (Pharmacia Biotech Inc.) with minor
modification to a previously described procedure (11). IgG was
sequentially eluted using 100 mM glycine, pH 2.3, followed
by 100 mM triethylamine, pH 11.5, and the combined eluates
were dialyzed against 50 mM Tris, pH 7.4, 150 mM NaCl. The polyclonal anti-LRP-2 IgG preparation was
absorbed on a column of RAP-Sepharose followed by selection on a column
of protein G-Sepharose. Control rabbit IgG was isolated from the
preimmune serum of rabbit 6286 by protein G-Sepharose chromatography.
ApoJ was combined with
synthetic A1-40 at a 1:15 molar ratio in PBS as
described previously (14) and incubated for 24 h at 37 °C.
Typically, a 1:15 molar ratio was maintained for preparing complexes of
unlabeled A
1-40 and apoJ, although the total protein
concentration varied according to the assay. For SDS-PAGE and
immunoblotting analyses the concentration of apoJ was 0.95 µM and A
1-40 was 15 µM,
whereas for solid phase binding assays, apoJ was 0.095 µM
and A
1-40 was 1.5 µM. As a control,
ovalbumin was substituted for apoJ.
A1-40 peptide (300 µg in Dulbecco's
PBS (dPBS)) was radioiodinated by the IODO-GEN (Pierce) method using 2 mCi of Na125I (Amersham Life Science, Inc.). Unincorporated
[125I]iodine was removed by Sepharose G-15 chromatography
using a 0.7 × 18-cm column equilibrated with dPBS. Radioiodinated
A
1-40 peptide (100 µg) was combined with apoJ (50 µg in dPBS) at a 30:1 molar ratio and incubated for 36 h at
37 °C. Following the incubation, radiolabeled A
-apoJ complex was
separated from free 125I-labeled A
by gel filtration
chromatography using a model 650E Waters protein purification system
(Waters, Milford, MA) and a Superdex-200HR column (Pharmacia)
equilibrated with dPBS. The integrity of the complex was evaluated by
electrophoresis under non-denaturating conditions on 4-12%
polyacrylamide, Tris/glycine-containing gels (Novex, San Diego, CA)
followed by autoradiography.
Microtiter wells
were coated with LRP-2, A, or BSA (each at 3 µg/ml) in 150 mM NaCl, 50 mM Tris, pH 8.0 (TBS) containing 5 mM CaCl2 for 18 h at 4 °C. Unoccupied
sites were either blocked with TBS containing 3% nonfat milk or
treated with PBS containing 0.1%
N-octyl-
-D-glucopyranoside (OG) (Calbiochem).
For those wells that were blocked with TBS containing 3% nonfat milk,
all subsequent incubations were carried out in TBS containing 3%
nonfat milk plus 0.1% OG. For wells treated with PBS containing 0.1% OG, all subsequent incubations were carried out in PBS containing 0.1%
OG. Bound proteins were detected using mouse monoclonal antibodies, sheep anti-mouse IgG-horseradish peroxidase (Amersham International, Buckinghamshire, UK), and the chromogenic substrate
o-phenylenediamine (Sigma) (1 mg/ml in 12 mM
citric acid monohydrate, 25 mM dibasic sodium phosphate, pH
5.0, 0.0014% hydrogen peroxide). Binding data from ELISA were analyzed
using a form of the binding isotherm as described by Ashcom et
al. (15).
To analyze A-apoJ complexes by SDS-PAGE,
samples were electrophoresed on 10-20% acrylamide gradient,
Tricine-containing gels (Novex) in the presence of SDS. The separated
proteins were electrophoretically transferred to ProtranTM
nitrocellulose membranes (Schleicher & Schuell) in
Tris/glycine-methanol buffer for 2 h at 70 V. After transfer, the
membranes were incubated with 5% nonfat dry milk in TBS (pH 7.4). The
membranes were then incubated with monoclonal A
or apoJ antibodies
followed by sheep anti-mouse IgG-horseradish peroxidase (Amersham)
diluted in 5% nonfat milk, TBS, 0.1% Tween 20. To detect bound
antibodies, the membranes were incubated with ECLTM Western
blotting chemiluminescent reagent (Amersham) and exposed to
BiomaxTM MR film (Eastman Kodak Co.).
To evaluate the effect of apoJ on the internalization of
125I-A, mouse teratocarcinoma F9 cells (ATCC CRL 1720)
were treated for 6 days with retinoic acid (RA) and dibutyryl cyclic
AMP (Bt2cAMP) as described previously (30), released by
trypsin-EDTA, and reseeded onto gelatin-coated 383-mm2
wells of 12-well plates (1.5 × 105 cells/well) in
Dulbecco's modified Eagle's medium (DMEM, Life Technologies, Inc.)
containing 10% bovine calf serum (Hyclone Laboratories), 20 mM HEPES, pH 7.4, 100 units/ml penicillin, 100 µg/ml
streptomycin (Life Technologies, Inc.) and no RA/Bt2cAMP. The cells were cultured for 18 h at 37 °C, 5% CO2
and then washed with serum-free DMEM containing penicillin and
streptomycin. 125I-A
(70 nM) in DMEM, 1.5%
BSA, 1% Nutridoma serum substitute (Boehringer Mannheim) (DMEM/BSA/SS)
containing various amounts of apoJ (1-40 nM) was added to
the cells and incubated for 5 h at 37 °C, 5% CO2.
The amount of radiolabeled A
that was internalized by cells was
defined as the amount of radioactivity that remained associated with
the cell pellets following trypsin/proteinase K/EDTA treatment (11,
30).
To evaluate the role of LRP-2 in the cellular internalization and
degradation of 125I-A-apoJ complex,
RA/Bt2cAMP-treated mouse teratocarcinoma F9 cells were
cultured as described above. 60 min prior to the addition of
125I-A
-apoJ complex, the medium was removed and the
cells were treated with DMEM/BSA/SS containing either anti-LRP-2 IgG
(200 µg/ml), control rabbit IgG (200 µg/ml), or chloroquine (0.1 mM). 125I-A
-apoJ complex (10 nM)
in DMEM/BSA/SS or in DMEM/BSA/SS containing anti-LRP-2 IgG (200 µg/ml), rabbit IgG (200 µg/ml), or chloroquine (0.1 mM)
was then added and incubated with the cells for 5 or 18 h at
37 °C, 5% CO2. The amount of radiolabeled complex that was internalized was measured as described above. Radioactivity released into the conditioned culture medium that was soluble in 10%
trichloroacetic acid was taken to represent degraded ligand. Total
ligand degradation values were corrected for non-cellular mediated
degradation by subtracting the amount of degradation that occurred when
the radiolabeled complex was incubated in wells lacking cells.
ELISAs
were used to determine whether synthetic A1-40 peptide
was capable of binding directly to purified LRP-2. As shown in Fig.
1A, LRP-2 did not bind to microtiter wells
coated with A
1-40. Likewise, A
1-40 (at
concentrations up to 125 nM) did not bind to LRP-2-coated
wells (Fig. 1B). In parallel assays, A
1-40
was shown to bind to apoJ in a dose-dependent manner,
either when A
1-40 was coated onto microtiter wells and
apoJ was introduced in solution phase (Fig. 1A) or when apoJ was coated and A
1-40 was introduced in solution phase
(Fig. 1B). The data for solution-phase apoJ binding to
immobilized A
1-40 (Fig. 1A) were fit using a
hyperbolic function (16), and the half-saturating level of binding
(estimated Kd) was determined to be 4.8 nM. This value is in good agreement with the value of 2.0 nM reported by Matsubara et al. (6). By
contrast, the binding of solution-phase A
1-40 to
immobilized apoJ was not saturable (Fig. 1B). Such
non-saturable binding can be expected given that A
1-40
has the ability to self-associate (17). The results indicate that
A
1-40 does not bind to LRP-2 but does bind with high
affinity to apoJ.
Amyloid
To
generate A-apoJ complex, A
1-40 was incubated with
apoJ for 24 h at 37 °C, and complex formation was evaluated by SDS-PAGE and immunoblot analysis. As shown in Fig. 2,
this incubation resulted in the formation of an approximately 70-kDa
band, immunoreactive with monoclonal A
antibody (Fig. 2B,
lane 4). The anti-A
-reactive 70-kDa band displayed a
similar electrophoretic mobility to the 70-kDa apoJ (Fig.
1C, compare lanes 4 and 6). Incubation
of A
with ovalbumin did not produce a band having a similar
molecular mass (Fig. 2B, lane 5). Coomassie Blue
staining showed that in the lane containing A
, which had been
incubated alone for 24 h at 37 °C, there was a single band
having a mobility corresponding to ~4 kDa (Fig. 2A,
lane 3), consistent with Mr of 4392. However, immunoblot analysis of this lane (Fig.
2B, lane 3) using monoclonal A
antibody
revealed several immunoreactive species having
Mr values of ~8000 and ~12,000 and a high
molecular mass band that just entered the gel. These species likely
correspond to A
dimer, trimer, and aggregate, respectively. Although
the dimer, trimer, and aggregated species were not detectable by
Coomassie Blue staining, they were immunoreactive with A
antibody,
indicative perhaps of its preference for multimerized peptide
versus the monomeric form. Each of these immunoreactive
species was also present in the profile of the A
incubated with
ovalbumin. However, the A
aggregate was missing in the
lane containing A
incubated with apoJ (Fig.
2B, lane 4). The data indicated that incubation
of apoJ with A
under the conditions that we described resulted in the formation of a complex of A
and apoJ that is stable in SDS. The
conditions of SDS-PAGE were insufficient to permit resolution of the
A
-apoJ complex as a discrete species having a
Mr ~4000 greater than apoJ. The results also
showed that apoJ inhibited the formation of aggregated A
while not
perturbing the formation of A
dimer and trimer.
To evaluate the ability of A-apoJ complex to interact with LRP-2,
microtiter wells coated with LRP-2 were incubated with mixtures of A
and apoJ or A
and ovalbumin that had been preincubated for 24 h
at 37 °C. As shown in Fig. 3A, A
binding to immobilized LRP-2, as detected by A
monoclonal antibody,
occurred when A
was preincubated with apoJ but not with ovalbumin.
Experimental controls showed that neither of the mixtures, A
and
apoJ or A
and ovalbumin, bound to wells coated with BSA (Fig.
3B). When monoclonal antibody to apoJ was used to detect
apoJ binding to LRP-2, the half-saturating level of binding of apoJ to
LRP-2 was not significantly modified by the inclusion of A
(Fig.
3C). The results indicate that apoJ mediates binding of A
to LRP-2 and that the affinity of the A
-apoJ complex for LRP-2 does
not appear to be different from that of apoJ alone.
The Antagonist of ApoJ Binding to LRP-2 Blocks Binding of A
RAP has been shown to inhibit the
binding of apoJ to LRP-2 (11). As shown in Fig. 4,
incubation of A-apoJ complex with RAP completely blocked the binding
of the complex to immobilized LRP-2. This, taken together with the
above data, indicates that apoJ can function to bridge the interaction
of A
with LRP-2.
Cellular Endocytosis of A
To
determine whether apoJ might facilitate cellular internalization of
A, 125I-A
was administered to cultured
LRP-2-expressing cells in the presence of varying concentrations of
apoJ. As shown in Fig. 5, exogenously added apoJ
promoted the internalization of 125I-A
in a
dose-dependent manner.
LRP-2 Mediates Cellular Endocytosis and Degradation of A
We next examined the cellular clearance of
A-apoJ complex and evaluated the role of LRP-2 in the process.
Radioiodinated A
was combined with unlabeled apoJ, and the resulting
complex was purified by gel filtration chromatography. Fig.
6A shows the chromatographic profiles of the
individual components and the complex-containing mixture. Native gel
electrophoretic analysis of the complex-containing fraction is shown in
the inset of Fig. 6A. The results indicated that
the chromatography procedure permitted isolation of the
125I-labeled complex from the bulk of the unincorporated
A
; however, free radiolabeled peptide did copurify with the
complex.
Equimolar amounts of 125I-A or
125I-A
-apoJ were administered to LRP-2-expressing cells,
and the level of internalization of each was measured. As shown in Fig.
6B, there was a 2-fold higher level of
125I-A
-apoJ complex internalized as compared with
125I-A
alone. In separate experiments, the amount of
125I-A
internalized was unchanged by the inclusion of a
1000-fold molar excess of unlabeled peptide (data not shown). Complex
internalization was also measured in the presence of function blocking
LRP-2 antibodies or control IgGs. As shown in Fig. 6B (and
Fig. 7A), anti-LRP-2 IgG inhibited
125I-A
-apoJ complex internalization by 59% as compared
with treatment with control rabbit IgGs. The results indicate that
A
-apoJ complex is internalized by LRP-2-expressing cells to a
greater extent than A
alone and that the internalization of the
complex can be inhibited by LRP-2 antibodies, implicating LRP-2 in the
clearance process.
We also evaluated whether the internalized complex was lysosomally
degraded as is the case for other LRP-2 ligands including apoJ (11). As
shown in Fig. 7, administration of 125I-A-apoJ complex
to LRP-2-expressing cells resulted in the internalization and
degradation of the complex. Degradation was evidenced by the appearance
of trichloroacetic acid-soluble radioactivity in the conditioned
culture medium that could be blocked by treatment with chloroquine, an
inhibitor of lysosomal proteinase activity (Fig. 7B). Both
internalization and degradation of the complex were also inhibited with
LRP-2-antibodies. Taken together, the findings indicate that LRP-2
mediates endocytosis of A
-apoJ complex leading to its degradation in
lysosomes.
In this study, we document the ability of the A-apoJ complex to
bind to the endocytic receptor LRP-2 in both cell-free and cultured
cell assays. Although the physiological relevance of this interaction
remains to be established, we have previously hypothesized that it is
part of a mechanism in which LRP-2-expressing epithelial cells, such as
those of the choroid plexus and ependyma, can clear the A
-apoJ
complex from the cerebrospinal fluid (11). In support of such a
hypothesis is the fact that both apoJ and LRP-2 are expressed at high
levels in the choroid plexus epithelium as well as ependymal cells that
line the ventricles of the brain and neural tube (12, 18, 19) and are
therefore in direct contact with cerebrospinal fluid. In addition to
LRP-2 having a possible role in clearance of A
-apoJ complex from the
cerebrospinal fluid, there is evidence that LRP-2 may have a role in
uptake of the complex from the blood by cells of the cerebral vascular endothelium (14, 20). Following our previous report on the identification of LRP-2 as the receptor for apoJ, Zlokovic et al. (14) introduced radiolabeled A
-apoJ complex into rat brain vasculature and observed that RAP or monoclonal antibody to LRP-2 decreased the brain uptake of the radiolabeled complex. Whereas these
findings indirectly implicate LRP-2 as being responsible for the
observed vascular clearance, evidence is needed to show that LRP-2 is
indeed expressed by brain vascular endothelial cells. Nevertheless, the
results presented in the present study provide direct evidence that
LRP-2 is a receptor for the A
-apoJ complex, thus establishing that
it could serve to mediate A
-apoJ endocytosis in any LRP-2-expressing
tissue.
Our results also indicate that apoJ inhibits the formation of high
molecular weight aggregates of A1-40 (Fig. 2). This activity is consistent with observations made using a sedimentation assay (10) and with the hypothesis that apoJ serves to maintain A
in
a soluble form, preventing it from forming insoluble amyloid filaments.
Such filaments are the hallmark component of the senile plaque found in
brain parenchyma and deposited in the cerebrovasculature of patients
with Alzheimer's disease (21, 22). In addition, the A
-apoJ
interaction may have a cytoprotective effect given that the insoluble
aggregated form of A
has been shown to be toxic to cultured neuronal
cells (23). By contrast to the anti-amyloidogenic and cytoprotective
actions of apoJ, apoE has been shown to promote the formation of A
fibrils (7, 9). Further studies are required to understand the factors
that affect the in vivo interaction of A
with apoJ or
apoE. It is possible that under normal physiological conditions, the
interaction between apoJ and A
is favored over that of apoE to
prevent amyloidogenesis. Moreover, clearance of A
-apoE by endocytic
apoE receptors such as LRP-1 and LRP-2 may also function to limit the
extracellular level of complex as we have proposed for the A
-apoJ
complex. In this regard, the endocytic action of LRP-1 and the recently
described low density lipoprotein receptor family member termed apoER2
(24) may be more relevant to clearance of A
-apoE complex from brain
parenchyma than LRP-2 since LRP-1 and apoER2 have widespread expression
in the parenchyma (24-27), whereas LRP-2 expression in the brain is
restricted to epithelial cells of the choroid plexus and ependyma (12,
28, 29).
Herein, evidence is presented indicating that one consequence of
LRP-2-mediated endocytosis of A-apoJ is that A
is targeted for
lysosomal degradation. This is the end result for other LRP-2 ligands
following their endocytosis (e.g. urokinase and plasminogen activator inhibitor-1 complex, low density lipoprotein, and apoJ (11,
30-32)). It may, however, seem paradoxical that A
could be degraded
in lysosomes, considering that a number of studies indicate that it can
be created in lysosomes as a result of proteolytic processing of
amyloid
-precursor protein (33, 34). It is possible that the
lysosomal presentation of A
in the form of a complex with apoJ may
make it more susceptible to proteinase degradation. It is important to
note that LRP-2 is apparently not the only means by which extracellular
A
can be internalized. For example, fibroblasts presumably lacking
LRP-2 can internalize exogenously added
125I-A
1-42 (35). This internalization
pathway was shown to lead to intracellular accumulation of A
in the
form of aggregates that are resistant to lysosomal proteinase
degradation. LRP-2-mediated internalization may avoid this outcome by
both preventing intracellular aggregate formation and promoting
lysosomal degradation of A
.