(Received for publication, November 7, 1994; and in revised form, January 25, 1995)
From the
The main component of Alzheimer's amyloid deposits,
A, has been found also as a soluble (sA
) normal constituent
of biological fluids and cell culture supernatants. Whether or not
sA
is the immediate precursor of A
, it is clear that peptides
with the same amino acid sequence can have both fibrillar and
non-fibrillar conformations. The interconversion mechanism from one
form to another is presently under intensive investigation. We have
previously described that (i) a synthetic peptide A
immobilized on affinity matrices was able to retrieve
apolipoprotein J (apoJ) from plasma and cerebrospinal fluid; and (ii)
the interaction of sA
with apoJ occurs in vivo, as
demonstrated by the ability of anti-apoJ to co-precipitate sA
from
normal cerebrospinal fluid. We have characterized the binding between
A
and apoJ and found that the interaction is
saturable, specific, and reversible. The dissociation constant of 2
10
M is indicative of high affinity
binding. The stoichiometry of the reaction is 1:1; apoJ has five times
more affinity for fresh A
than for the
aggregated peptide. Competitive inhibition studies carried out with
apolipoprotein E (isoforms E2, E3, and E4), transthyretin, vitronectin,
and
-antichymotrypsin indicate that the complex
apoJ
A
cannot be dissociated by any of
these competitors at physiologic concentrations. The data strongly
suggest that apoJ plays an important role as a carrier protein for
sA
.
Amyloid (A
) (
)peptide (39-44
residues) is the main component of the two major neuropathological
lesions present in AD, senile plaques, and cerebrovascular amyloid
deposits(1, 2) . Although A
has high tendency to
aggregate and make fibrils, a soluble form has been detected in
biological fluids (soluble A
,
sA
)(3, 4, 5) . Whether or not sA
is
the immediate precursor of A
, it is clear that the same amino acid
sequence can have both fibrillar and non-fibrillar conformations;
therefore, the knowledge of the factors that influence its behavior in
solution will be a step forward in the understanding of AD pathology.
In this regard, the existence of specific components named
``desaggrins'' was previously suggested based on the fact
that A
spontaneous fibril formation in
vitro is inhibited in the presence of CSF(6) .
Extensive immunohistochemical studies indicate that other proteins
are co-deposited with A in senile plaques. Amyloid P-component,
-antichymotrypsin (ACT), apolipoprotein E (apoE),
apolipoprotein J (apoJ), complement components, vitronectin (Vn),
glycosaminoglycans, and extracellular matrix proteins are among the
amyloid-associated proteins described so
far(7, 8, 9, 10, 11, 12, 13, 14, 15) .
It is not clear whether they are innocent bystanders or their presence
is related to the mechanism of amyloidogenesis. Several lines of
investigation favor the latter notion, at least for some of them (i.e. amyloid P-component and apoE are present in several
types of fibrillar deposits but absent in non-fibrillar accumulations
representing pre-amyloid lesions)(16) . In addition, the apoE
gene on chromosome 19, particularly the apoE allele
4, has been
linked to sporadic and late-onset AD(17) . The inheritance of
the apoE4 allele is today considered a risk factor for AD(1) .
Biochemical studies performed in vitro have demonstrated a
certain degree of binding affinity between A and different
proteins, among them apoJ, apoE, transthyretin (TTR), and
ACT(12, 18, 19, 20, 21, 22, 23) .
The interactions have been considered in the range of ``high
avidity binding,'' although they were not quantitatively
evaluated. Using immobilized synthetic peptides homologous to A
(A
), we have shown previously its binding
association with plasma and CSF apolipoproteins J and E; moreover, the
presence of the complex apoJ
sA
was confirmed in CSF,
indicating that the interaction takes place in
vivo(18, 20) . We are reporting herein the
characterization of the complex formation between apoJ and
A
, in the presence and absence of other
amyloid-associated proteins.
Human plasma apoJ was purchased from Quidel (San Diego, CA); ACT and
TTR were obtained from Calbiochem (La Jolla, CA); Vn was purchased from
Chemicon (Temecula, CA). Recombinant apoE isoforms 2, 3, and 4 (apoE2,
apoE3, and apoE4) were obtained from PanVera (Madison, WI). In all
cases, protein purity was corroborated by SDS-PAGE and
NH-terminal sequence.
In separate experiments, variable amounts of fresh
A or 24-h self-aggregated
A
(0-1136 nM) were combined
with 25 nM apoJ in PBS and incubated for 3 h at 37 °C. The
mixture was then transferred to A
-coated
wells and incubated for 3 h at 37 °C. Bound apoJ was detected with
monoclonal IF12 and alkaline phosphatase-labeled F(ab`)
goat anti-mouse IgG, as described above.
Competitive
inhibition assays were performed with apoE2, apoE3, apoE4, ACT, Vn, and
TTR. 0-2500 nM of the various competitors in PBS were
coincubated with 25 nM of apoJ in PBS in
A-coated wells (400 ng/well) at 37 °C for
3 h. Bound apoJ was determined as described above.
The interaction between apoJ and A was characterized by means of solid-phase ELISA experiments. A
dose-response relationship that reached saturation was obtained when
increasing concentrations of apoJ at pH 7.4 were allowed to interact
with a constant amount of immobilized A
(Fig. 1). Non-linear regression analysis of the specific
binding data fitted to a rectangular hyperbola and allowed the
calculation of the corresponding dissociation constant K
of 2 nM. The specificity and reversibility of the
interaction were assessed at an apoJ concentration of 25 nM (at which 100% saturation of the
A
-coated plates was achieved) by means of
inhibition experiments with either A
or apoJ.
When a constant concentration of apoJ in PBS was preincubated with
increasing concentrations of freshly prepared A
before the addition to A
-coated wells,
apoJ-binding to immobilized A
followed a
one-site competition curve (Fig. 1, inset). The
calculated value for half-maximal inhibition (IC
) was 63
nM. In a separate set of experiments, the binding of
biotin-labeled apoJ to immobilized A
was
competitively inhibited by increasing concentrations of native apoJ.
The remaining bound biotinylated apoJ was quantitated with alkaline
phosphataselabeled streptavidin. The data fitted into a one-site
competition curve with IC
= 77 nM (Fig. 1, inset).
Figure 1:
Saturation
curve for apoJ-A interaction. Variable
concentrations (0-25 nM) of apoJ were incubated with
A
-coated wells for 3 h at 37 °C. Bound
apoJ was detected with monoclonal IF12 and alkaline phosphatase-labeled
anti-mouse, as described under ``Materials and Methods.''
Each point represents the mean (±2 S.D.) of five independent
duplicate experiments. Inset, inhibition of apoJ binding to
immobilized A
. Increasing concentrations
(0-1136 nM) of A
were
preincubated with apoJ (25 nM) for 3 h at 37 °C. The
mixture was added to A
-coated wells and
incubated for another 3 h at the same temperature. Bound apoJ was
determined as described under ``Materials and Methods.'' On
separate experiments, native apoJ (0-1136 nM) was
coincubated with 25 nM of biotin-labeled apoJ in
A
-coated wells for 3 h at 37 °C. Bound
biotinylated apoJ was detected with alkaline phosphatase-labeled
streptavidin. Results are expressed as percentage of binding compared
with controls incubated with apoJ alone. Data represent the mean of
three independent duplicate experiments. S.D. never exceeded
±6%.
The formation of the
apoJA
complex was visualized by
Coomassie Blue staining after electrophoresis on non-denaturing
polyacrylamide gels. As shown in Fig. 2, native apoJ exhibits
two molecular forms in this non-SDS system: a monomeric component of
80 kDa and a dimeric form of
160 kDa (lane
2). When complexed to A
, both components
shifted their electrophoretic mobility toward higher molecular masses,
resulting in
85 and 170 kDa bands (an increase of
5 and 10 kDa, respectively). Amino-terminal sequence
analysis of these 85- and 170-kDa components rendered the sequences
DQTVSDNELQEMSNQ, SLMPFSPYEPLNFH, and DAEFRHDSGYEVHHQ corresponding to
the first 15 residues of the apoJ
-chain, apoJ
-chain, and
A
, respectively. Recovery calculations
performed for the first 10 steps of the sequence indicated a 1 to 1
stoichiometry (Table 1).
Figure 2:
Identification and characterization of
apoJA
complexes under non-denaturing
conditions. ApoJ (5 µg) and A
(5 µg)
were incubated at 37 °C for 18 h and the resultant complexes
separated on 8% non-SDS-PAGE, transferred to Immobilon P, and stained
with Coomassie Blue. The apparent molecular masses were calculated from
the Ferguson plots constructed with known molecular mass standards
(
-lactalbumin, 14,200 Da; carbonic anhydrase, 29,000 Da; chicken
egg albumin, 45,000 Da; bovine serum albumin, 66,000 Da monomer and
132,000 Da dimer; urease, 272,000 Da monomer and 545,000 Da dimer). The
complexes (arrowheads) were excised from the membrane and
their NH
-terminal sequence determined. Lane 1,
A
; lane 2, apoJ; lane 3,
apoJ
A
complex.
The influence of the degree of
A aggregation in its ability to form a
complex with apoJ was tested using fresh and aggregated peptide; the
resulting complex was visualized via immunoblot analysis after SDS-PAGE
using anti-A
(monoclonal 6E10), as indicated
in Fig. 3. Fresh A
exhibited a major
monomeric component and a minor dimeric form while the tetrameric
aggregates were almost negligible (lane 1, arrowheads). A similar aliquot of the synthetic peptide that
had been incubated for 24 h at 37 °C showed an increase in the
amount of dimers and tetramers in addition to the typical smear-like
electrophoretic appearance which indicates the presence of multiple
minor components of higher molecular mass (lane 3). When fresh
A
was incubated with apoJ for 18 h at 37
°C (lane 2), the presence of the 85-kDa
apoJ
A
complex was immunodetected by
anti-A
(arrow); the free peptide
exhibited the same polymerization pattern as the one shown in lane
1. When 24-h-aggregated A
was incubated
with apoJ under identical conditions, the presence of a less intense
85-kDa complex was detected by anti-A
(lane 4, arrow) while the peptide aggregation
pattern resembled the one in lane 3. Densitometric evaluation
of both complexes (lanes 2 and 4, arrow)
indicated that the amount of apoJ-A
formed
was 4.6 times lower when aggregated peptide was used. To confirm this
apparent different affinity of apoJ for aggregated and non-aggregated
A
, inhibition assays were carried out on
ELISA plates. Both fresh and aggregated peptides were allowed to
interact with apoJ in fluid-phase for 3 h at 37 °C; the remaining
free apoJ was tested for its ability to bind to
A
-coated wells. As depicted in Fig. 4,
aggregated A
exhibited five times less
efficiency to form complexes with apoJ (IC
= 315
nM) than the fresh peptide (IC
= 63
nM).
Figure 3:
Immunodetection of the
apoJA
complex formed with fresh or
aggregated peptide. ApoJ
A
and
apoJ
A
(agg) complexes were prepared in
PBS using 1.1 µmol of apoJ and 1.1 µmol of either fresh or 24-h
heat-aggregated peptide. After 18 h incubation at 37 °C, complexes
were separated on Tris-Tricine 10% SDS-PAGE and immunoblotted with
anti-A
(6E10). Visualization was carried out
with peroxidase-labeled anti-mouse followed by ECL. Lane 1,
fresh A
(100 ng); lane 2, apoJ-fresh
A
complex (1:1 molar ratio); lane 3,
24-h heat-aggregated A
(100 ng); lane
4, apoJ
A
(agg) complex (1:1 molar
ratio). Solid arrowhead, monomeric
A
; open arrowheads, dimeric and
tetrameric forms of A
; arrow,
apoJ
A
complex.
Figure 4:
Inhibition of
apoJA
interaction by either fresh or
aggregated A
. ApoJ (25 nM) was
preincubated with various concentrations of either fresh or
24-h-preaggregated A
prior to the addition to
A
-coated plates. Bound apoJ was detected as
described under ``Materials and Methods.'' ApoJ binding is
expressed as percentage of binding compared to control wells incubated
in the absence of inhibitor. Data represent the mean of three
independent experiments duplicate experiments. S.D. never exceeded
±6%.
Competitive inhibition experiments using other
plasma/CSF proteins with demonstrated binding affinity for A
(apoE2, apoE3, apoE4, ACT, Vn, and TTR) were performed by solid-phase
assays. Increasing concentrations (0-2.5 µM) of each
competitor were mixed with a constant amount of apoJ and immediately
added to A
-coated wells. Bound apoJ was
detected with monoclonal IF12 after 3 h of incubation. As indicated in Fig. 5, none of the proteins tested exhibited higher affinity
for A
than ApoJ. The competition curves
distributed themselves into two very well defined groups: the first
composed of the three apoE isoforms and the second contained the other
proteins. ApoE2 (IC
= 316 nM) was the
strongest competitor of all the apoE isoforms according to the
calculated IC
values, followed by apoE3 (IC
= 502 nM) and apoE4 (IC
= 794
nM), indicating that they have 4-10 times lower relative
affinity than apoJ for A
. None of the other
proteins tested reached 50% inhibition of apoJ-A
binding under the conditions tested (TTR, IC
= 9550 nM; Vn, IC
= 9820
nM; ACT, IC
= 11340 nM).
Figure 5:
Competitive inhibition of the
apoJA
complex formation by amyloid
associated proteins. ApoJ (25 nM) was combined with variable
concentrations (0-2500 nM) of apoE2 (
), apoE3
(
), apoE4 (
), TTR (
), ACT (
), and Vn
(
) and coincubated with A
-coated
wells. Bound apoJ was determined as described under ``Materials
and Methods.'' The inhibition of biotin-labeled
apoJ
A
complex formation by native apoJ
(
) presented on Fig. 1(inset) is shown here for
comparison. Results are expressed as percent of the maximum specific
binding obtained in the absence of competitors. Data represent the mean
of five independent duplicate experiments. S.D. never exceeded
±7%.
ApoJ (also known as clusterin or SP-40, 40) is a
multifunctional disulfide-linked heterodimeric glycoprotein composed of
two 40-kDa subunits (named
and
chains)(28, 29, 30) . The gene for apoJ maps
to chromosome 8(31, 32, 33) . A single mRNA
molecule (34) codifies a 449 amino acid chain, and the final
apoJ structure is generated by post-translational cleavage at peptide
bond Arg
-Ser
. ApoJ message is expressed in
almost all mammalian tissues(35, 36) , and the protein
has been found in nearly all body fluids(36, 37) . The
normal concentration of apoJ in plasma ranges between 35 and 105
µg/ml (0.44-1.3 µM) (28) , and it is
primarily distributed in the high density lipoproteins (29) ;
it is several times concentrated in seminal fluid while in CSF the
values vary between 1.2 and 3.6 µg/ml (15-45 nM) (10, 38) . ApoJ is involved in a variety of
physiological processes, including lipid transport(36) ,
secretion(39) , membrane recycling (40) ,
spermatogenesis(34, 35) , and modulation of the
complement activity(41) . Within the central nervous system,
apoJ is synthesized in neurons (42) and astrocytes (43, 44) and is able to cross the blood-brain barrier
via a specific receptor/transport mechanism(45) . Its
production is up-regulated in the degenerative (46, 47) and/or regenerative
processes(48, 49) . The CSF concentration of apoJ is
slightly elevated in AD patients (1.8-4.4 µg/ml), although
the differences with the normal population are not statistically
significant(10) .
Biochemical data obtained in vitro indicate that apoJ is a major ligand for sA in plasma and CSF
and that the complex apoJ-sA
exists in vivo(18) ,
suggesting that apoJ may act as a carrier protein for sA
in plasma
and CSF. Our results demonstrate that apoJ binds A
with high affinity; the calculated K
obtained from the saturation curve is 2
10
M. Monomeric and dimeric forms of apoJ (28) formed complexes with A
; both
complexes were constituted by equimolar amounts of apoJ and
A
, compatible with a 1:1 stoichiometry. The
binding was specific and reversible; both, native apoJ and freshly
prepared A
, inhibited the interaction of apoJ
to immobilized A
with a similar
IC
. The fact that 100% inhibition can be achieved by
incubation of apoJ with A
in solution
indicates that the interaction indeed occurs in fluid-phase.
The
self-aggregation rate of A is pH-, ionic
strength-, temperature-, and
concentration-dependent(50, 51, 52) . The
time course of aggregation of A
in PBS at 37
°C, evaluated by SDS-PAGE, indicated that our freshly prepared
peptide was mainly monomeric while the number of dimers, tetramers, and
higher association forms increased as a function of incubation time,
reaching a plateau at 24 h that remained without major changes for at
least 72 h. ApoJ exhibited five times lower affinity for aggregated
A
than for freshly prepared peptide when
tested by solid-phase ELISA and by gel electrophoresis followed by
scanning evaluation. The presence of dimers, tetramers, and high
molecular mass components indicated that apoJ was unable to reverse the
aggregation of A
. All these data suggest that
apoJ has the capability to bind with high affinity to non-aggregated
forms of A
but not to A
polymers.
In order to balance the biological importance of the
apoJ-sA interaction, competition experiments using other
plasma/CSF proteins with demonstrated affinity to A
were carried
out at physiologic pH. ApoE isoforms were 4-10 times less
efficient than apoJ itself in inhibiting the formation of the complex
apoJ
A
, being apoE4 the least avid
competitor under the conditions tested. The rest of the proteins
assayed (TTR, ACT, and Vn) exhibited an almost negligible competitive
effect; none of them induced more than 10% inhibition at concentrations
where apoJ in fluid-phase inhibited 100% the complex formation. It
should be noted that all the proteins used in these competition
experiments were either purified from plasma or expressed in Sf9 cells.
It would be interesting to determine whether other factors such as the
association with other proteins, lipoproteins, and/or lipids may affect
their interaction with A
. These data in the context of the mean
physiologic concentrations of apoJ and apoE in plasma as well as in CSF
(apoJ
= 0.87 ± 0.43 µM;
apoJ
= 30 ± 15 nM; apoE
= 0.77 ± 0.26 µM; apoE
= 41 ± 16
nM)(10, 28, 53) , suggest that
normal physiologic conditions favor the formation of apoJ
A
complex. In fact, the presence of sA
in apoJ-containing HDL
particles was recently shown(54) .
The data indicate the
existence of a high affinity binding between apoJ and a peptide with
identical primary structure to sA. It is conceivable that the
interaction is not only related to the transport of the soluble peptide
in plasma by apoJ-containing HDL but to the delivery of sA
through
the blood-brain barrier. In this regard, recent in vivo studies performed in guinea pigs have demonstrated the existence
of cerebrovascular permeability for A
, human
apoJ as well as for the apoJ
A
complex(45, 55) . Since apoJ has lower affinity
for aggregated A
, the biological implications for the alteration
of the interaction apoJ-sA
under pathologic conditions should be
further investigated as one of the possible mechanisms of peptide
aggregation and deposition in AD tissue.