(Received for publication, July 10, 1995; and in revised form, August 22, 1995)
From the
A 39-43-amino acid residue-long fragment (-peptide)
from the amyloid precursor protein is the predominant component of
amyloid deposits in the brain of individuals with Alzheimer's
disease. Serum amyloid P component (SAP) is present in all types of
amyloid, including that of Alzheimer's disease. We have used an in vitro model to study the effects of purified SAP on the
fibril formation of synthetic Alzheimer
-peptide 1-42. SAP
was found to inhibit fibril formation and to increase the solubility of
the peptide in a dose-dependent manner. At a 5:1 molar ratio of
A
1-42 peptide to SAP, fibril formation was completely
inhibited, and approximately 80% of the peptide remained in solution
even after 4 days of incubation. At lower SAP concentrations, e.g. at peptide to SAP ratio of 1000:1, short fibrillar like
structures, lacking amyloid characteristics, were formed. These
structures frequently contained associated SAP molecules, suggesting
that SAP binds to the polymerizing peptide in a reaction which
prevented further fibril formation.
Serum amyloid P component (SAP) ()is a
calcium-dependent lectin, the best defined specificity of which is
4,6-cyclic pyruvate acetal of
-D-galactose. The SAP
pentamer consists of five identical 25-kDa subunits each of 204 amino
acids, the three-dimensional structure of which was determined
recently(1) . Each subunit was found to be constructed from
multiple antiparallel
-strands arranged in two sheets, and the
tertiary fold was remarkably similar to that of the legume lectins. It
binds to DNA, to chromatin, and to glycosaminoglycans such as heparin,
heparan, and dermatan sulfate, which are frequently associated with
amyloid deposits (2, 3) . It is also present in
glomerular basal membranes and associated with elastic
fibers(4) . In many SAP-ligand interactions, phosphorylated
and/or sulfated groups are
involved(5, 6, 7, 8) . After
calcium-dependent self-aggregation, or when bound to chromatin, SAP may
trigger complement activation(9, 10, 11) . In
addition, SAP also inhibits the complement regulatory function of
C4b-binding protein, a protein to which it is complexed in
blood(12, 13) . Despite the numerous properties
ascribed to SAP, its physiological function is largely unknown. SAP is
a universal constituent of amyloid deposits, including plaques from
Alzheimer's disease (AD), amorphous
deposits, and
neurofibrillar tangles(14) .
The predominant component of
amyloid deposits in the brain of individuals with AD is a
39-43-amino acid-long peptide (A-peptide) which is a
proteolytic product of the amyloid precursor
protein(15, 16) . Recently, it was shown that the
A
1-42 and A
1-43 forms are specifically found in
all kinds of AD plaques, indicating that those forms are critically
important in AD pathology(17) .
-Amyloid-related peptides
are secreted by cultured cells and are normally present in the
cerebrospinal fluid(18) . The local fibril formation is
probably a multistep process which is influenced by the rate of
A
-peptide production and also involves conformational changes of
the peptide. The presence of amyloid-associated proteins such as
-antichymotrypsin (ACT) and apolipoprotein E (apoE)
appears to enhance fibril
formation(19, 20, 21, 22) . ACT is
present only in amyloid plaques from AD, while a specific allele of
apoE (apoE4) has been associated with late onset AD. It has been
suggested that ACT and apoE may act as pathological chaperones
promoting fibril formation(22) . Although SAP is a universal
constituent of all types of amyloid, its physiological role in fibril
formation is not known.
SAP binds to preformed amyloid fibrils and
to Alzheimer A1-40 peptide in vitro in a
calcium-dependent reaction (23, 24) . It has been
suggested that SAP protects amyloid from proteolytic degradation in
vivo by binding to fibrils and masking fibrillar
conformation(1, 25) . We now wish to report that SAP
inhibits amyloid fibril formation from the Alzheimer A
1-42
peptide in an in vitro model.
To examine the effect of SAP on A1-42 fibril
formation, an in vitro model was used in which the Alzheimer
A
1-42 peptide spontaneously adopts a
-pleated sheet
conformation and forms elongated, approximately 7-8 nm thick,
fibrils. The fibril formation of the A
1-42 peptide was
monitored by light-scattering and electron microscopy (Fig. 1).
In the absence of SAP, the light scatter increased to a maximum after
72 h (Fig. 1A), and elongated fibrils, having amyloid
characteristics(29) , were observed in the electron micrographs (Fig. 1B). The addition of SAP resulted in a
dose-dependent inhibition of fibril formation. At a peptide to SAP
ratio of 5:1, there was almost no increase in light scattering (Fig. 1A), and the fibril formation was completely
inhibited (Fig. 1B). A distinct inhibitory effect was
observed also at the highest molar ratios of peptide over SAP. Even at
a peptide to SAP ratio of 1000:1, the solubility of the peptide was
increased as compared to the control, and short, flexible fibrils were
formed as revealed by electron microscopy (Fig. 1D) and
as reflected by a slight but significant increase in light scatter (Fig. 1A). These fibrils were 1-2 nm thicker than
those formed by A
1-42 alone. Moreover, they did not exhibit
green birefringence after Congo red staining, which is characteristic
of amyloid fibrils.
Figure 1:
Inhibition of fibril formation from
Alzheimer A1-42 peptide by SAP. Synthetic Alzheimer peptide
A
1-42 was incubated for 4 days at 37 °C in the presence
of increasing concentrations of SAP (the peptide to SAP molar ratios
were 5:1
, 50:1
, 100:1
, and 1000:1
.
, A
1-42 alone as described under ``Experimental
Procedures.'' In the absence of SAP, the peptide polymerized into
extended fibrils, a process which was monitored by light scattering at
400 nm (A) or by electron microscopy after a 48-h incubation (B, C, and D): B, A
1-42
alone; C, A
1-42 and SAP, 5:1 ratio; D,
A
1-42 and SAP, 1000:1 ratio. A dose-dependent inhibition of
fibril formation was observed with both techniques, and, at a peptide
to SAP ratio of 5:1, the fibril formation was essentially completely
inhibited. At a peptide to SAP ratio of 50:1 up to 1000:1, short
fibrils (120 nm long, 10-12 nm thick) were formed which yielded
intermediate light scattering. In the electron micrographs, it was
noteworthy that SAP molecules often appeared associated with the short
fibrils. In the upper panels of B, C, and D, bars represent 200 nm, whereas, in the bottom
panels, bars represent 100
nm.
Although the precise mechanism of the molecular
interaction between SAP and A1-42 has not been elucidated,
the changes in fibril morphology suggest that SAP affects the packing
mechanism of A
1-42 and disturbs the typical uniformity of
the fibrils. SAP is unique in its ability to inhibit fibril formation
at all concentrations reported in this study. The amyloid-associated
proteins apoE and ACT have been reported either to inhibit or to
stimulate the
-peptide amyloid fibril formation. At a low ratio
between the A
1-42 peptide and apoE (1000:1), a significant
delay in the onset of amyloid fibril formation was observed, whereas
under other conditions, apoE stimulated fibril
formation(20, 22, 30) . Similar results have
been reported for ACT(19, 21) .
To study the effect
of SAP on the overall solubility of A1-42 peptide, the
amount of radiolabeled peptide remaining in solution after
centrifugation and filtration was measured at various times of
incubation (Fig. 2). After a 96-h incubation, the solubility of
A
1-42 peptide reached its minimum with 20% of radioactivity
remaining in solution. Addition of increasing amounts of SAP resulted
in increased solubility demonstrating that SAP may be able to prevent
A
1-42 aggregation. To ensure that the experimental
conditions (10-min centrifugation at 13,000
g and
filtration through 0.2-µm filters) were sufficient to remove small
aggregates of A
1-42 peptide, a second experiment was
performed in which the peptide:SAP mixtures after a 72-h incubation
were centrifuged at 120,000
g for 1 h. The results
confirmed the ability of SAP, at an A
1-42:SAP ratio of 5:1,
to keep essentially all the peptide in solution (89%) for up to 72 h.
At A
1-42:SAP ratios of 100:1 and 1000:1, the amounts of
peptide remaining in solution were 81 and 60%, respectively. In this
experiment, 54% of the peptide remained soluble in the absence of SAP.
Figure 2:
Effect
of SAP on solubility of Alzheimer A1-42 peptide. The
radiolabeled Alzheimer A
1-42 peptide (final concentration 8
µM) was incubated with increasing concentrations of
purified SAP in 100 µl of TBS. The amount of peptide remaining in
solution was determined as described under ``Experimental
Procedures.'' A, the soluble peptide was determined after
centrifugation (10 min at 13,000
g) and filtration
through 0.2-µm filters of the samples. The peptide to SAP molar
ratios were 5:1
, 10:1
, 50:1
, 100:1
, 200:1
, 1000:1
, and A
1-42 alone
. Each point
represent the mean of two experiments.
Direct binding between SAP and the A1-42 peptide was
demonstrated by nondenaturing agarose gel electrophoresis, but, in
contrast to the complexes between ACT and the A
1-42
peptide(21) , complexes between SAP and the
-peptide were
not stable in SDS (results not shown). Recently, it has been shown that
SAP is able to bind to A
1-40 immobilized in microtiter
plates(24) . Together, these data suggest that SAP is able to
bind A
1-42 under nondenaturing conditions.
The inhibitory
effect of SAP on Alzheimer A1-42 peptide fibril formation
appears to reflect a general ability of SAP to inhibit amyloid fibril
formation. In support for this concept, SAP was found to inhibit the
formation of fibrils from a
-pleated sheet containing peptide
derived from
-antitrypsin(31) . Complete
inhibition of fibril formation was observed at a peptide to SAP ratio
of 5:1. Even at a peptide to SAP ratio of 1000:1, a clear attenuation
of fibril formation was observed (Fig. 3). It was noteworthy
that at this latter experimental condition, the morphology of the
aggregated peptide was different from that observed in the experiment
using A
1-42 (compare Fig. 1D and
3C). Instead of short flexible fibers, dense aggregates of
fibers were observed.
Figure 3:
Inhibition of fibril formation from
-antitrypsin C-terminal peptide by SAP. Synthetic
-antitrypsin C-terminal peptide was incubated at 37
°C (final concentration 12 µM) in the presence of
increasing concentrations of SAP as described for A
1-42
under ``Experimental Procedures.'' In the absence of SAP, the
peptide polymerized into extended fibrils as observed by electron
microscopy (A). No fibrils were observed at a peptide to SAP
ratio of 5:1 (B). At a peptide to SAP ratio of 1000:1, a few
number of dense clusters of fibrils were observed (C). It was
noteworthy that SAP molecules often appeared associated with the
fibrils, as observed in Fig. 1(arrow). In the upper and lower panels, bars represent 200
and 100 nm, respectively.
Our present data suggest SAP both to impede
the seeding process, which initiates fibril formation(32) , and
to inhibit fibril growth by binding to the peptide and thus preventing
peptide-peptide polymerization. Both processes presumably involve
binding of SAP to -pleated sheet structures formed by the
polymerizing peptide. If the physiological function of SAP is to
inhibit amyloid fibril formation, this process is probably imbalanced
in AD, either due to changes in the metabolism of the
-peptide or
to the presence of other SAP ligands in the plaques, such as
glycosaminoglycans which would inhibit SAP function. Moreover, in such
pathological situations, the attachment of SAP to amyloid fibrils may
lead to increased resistance to proteolysis (1, 25) .
SAP has been extremely well conserved through evolution (33) and no deficiency of SAP has been described, suggesting
SAP to have important functions. The ability of SAP to inhibit
pathological deposition of amyloid-forming peptides may be such a
function.