(Received for publication, January 16, 1996; and in revised form, February 19, 1996)
From the
Polymerization of amyloid -peptide (A
) into amyloid
fibrils is a critical step in the pathogenesis of Alzheimer's
disease. Here, we show that peptides incorporating a short A
fragment (KLVFF; A
) can bind full-length
A
and prevent its assembly into amyloid fibrils. Through alanine
substitution, it was demonstrated that amino acids Lys
,
Leu
, and Phe
are critical for binding to
A
and inhibition of A
fibril formation. A mutant A
molecule, in which these residues had been substituted, had a markedly
reduced capability of forming amyloid fibrils. The present data suggest
that residues A
serve as a binding sequence
during A
polymerization and fibril formation. Moreover, the
present KLVFF peptide may serve as a lead compound for the development
of peptide and non-peptide agents aimed at inhibiting A
amyloidogenesis in vivo.
The preeminent neuropathological feature of Alzheimer's
disease is the deposition of amyloid in the brain parenchyma and
cerebrovasculature(1, 2) . The basic components of the
amyloid are thin fibrils of a peptide termed
A(3, 4) . This peptide is a 40- to 42-amino
acid-long proteolytic fragment of the Alzheimer amyloid precursor
protein (APP), (
)a protein expressed in most
tissues(5) . Genetic and neuropathological studies provide
strong evidence for a central role of A
amyloid in the
pathogenesis of Alzheimer's disease(6) , but the
pathophysiological consequences of the amyloid deposition are unclear.
However, it has been suggested that A
polymers and amyloid are
toxic to neurons, either directly or via induction of radicals, and
hence cause neurodegeneration(7, 8, 9) .
Previous studies indicate that A polymerization in vivo and in vitro is a specific process that probably involves
interactions between binding sequences in the A
peptide(10, 11, 12) . A rational
pharmacological approach for prevention of amyloid formation would
therefore be to use drugs that specifically interfere with
A
-A
interaction and polymerization. We hypothesized that
ligands capable of binding to and blocking such sequences might inhibit
amyloid fibril formation as outlined schematically in Fig. 1.
Our strategy in searching for an A
ligand was to identify binding
sequences in A
and then, based on their primary structures,
synthesize a peptide ligand. Binding sequences were identified by
systematically synthesizing short peptides corresponding to sequences
of the A
molecule. The minimum length of an identified binding
sequence was determined by truncating the peptide. Residues critical
for binding were identified by alanine scanning. These critical
residues were then substituted in an A
fragment
(A
) that normally is capable of forming
amyloid fibrils (13, 14) in order to determine if they
indeed are important for A
amyloid fibril formation. Finally, it
was determined if the identified ligand, in addition to binding to the
A
molecule, was capable of inhibiting fibril formation of
A
.
Figure 1:
In vitro and in vivo studies of A amyloid have shown that the A
molecules
(shown in gray) interact during polymer growth and fibril
formation. This probably involves interaction between one or several
binding sequences in the A
molecules (A). It is therefore
reasonable to assume that ligands (shown in black) that can
bind to these sequences are capable of arresting further A
polymerization (B) and, assuming that A
polymerization is
a dynamic and reversible process, possibly also dissolve A
polymers in situ.
Figure 2:
Identification of binding sequences in the
A molecule. Ten-mers corresponding to consecutive sequences of
A
were synthesized on a cellulose membrane
matrix using the SPOT technique. Following blocking with 0.05% Tween 20
in TBS, the cellulose membrane was incubated in the presence of 20
µM
I-A
at 20
°C for 12 h in TBS, pH 7.4, supplemented with 1% bovine serum
albumin. The cellulose membrane was then washed repeatedly in the same
buffer containing 0.5 M NaCl and dried. Radioactivity bound to
the cellulose membrane was visualized by autoradiography and
quantitated using densitometry.
Being located in the center of the
binding region, A was selected for further
studies of the structural requirements for binding. This peptide, as
well as N- and C-terminally truncated fragments, were synthesized using
the same technique as described previously (Fig. 3A).
The shortest peptide still displaying consistent high A
binding
capacity had the sequence KLVFF (corresponding to
A
). In order to confirm binding between
A
and the KLVFF peptide, it was decided to
study this interaction in an additional test system. Surface plasmon
resonance spectroscopy is a technique suitable for real-time studies of
molecular interactions(19) . By adding a cysteine residue via a
linker of two
-alanine residues to the C terminus of AcKLVFF, the peptide could be attached through a
disulfide bond to the sensor chip of the surface plasmon resonance
spectroscope. As control for nonspecific binding, cysteine alone was
coupled to another channel of the sensor chip. A solution of
A
was injected onto the sensor chip.
A
was found to bind to the AcKLVFF peptide and not nonspecifically to the sensor
chip (Fig. 3B).
Figure 3:
Identification of the shortest possible
peptide capable of binding A. The
A
molecule (EVHHQKLVFF) and indicated N- and
C-terminal truncated fragments were synthesized using the same
technique as described above and analyzed for affinity to
I-A
(A). Sensorgram
from surface plasmon resonance spectroscopy (BIAcore 2000). Solubilized
A
, 100 µM, was injected during
10 min over a sensorchip derivatized with the peptide AcKLVFFAAC (upper trace; AA and C served as spacer
and linker to the chip, respectively) or cysteine alone (lower
trace). Arrows indicate start and stop of injection (B).
Figure 4:
Identification of amino acid residues
mediating binding. Each amino acid residue in KLVFF was systematically
replaced with alanine and analyzed for affinity to I-A
as described in the legend
to Fig. 2. 100% represents
I-A
binding to nonsubstituted KLVFF.
Figure 5:
Aggregation and fibril formation of wild type and substituted A. Wild
type A
(A and C) and
A
(B and D) were
incubated at 200 µM in TBS for 24 h at 37 °C in a
shaking water bath. After incubation, the tubes were centrifuged at
20,000
g for 20 min. The content of nonaggregated
peptide in the supernatants (A and B) was analyzed
using an established C-4 RPLC system, whereas the aggregated peptides
in the pellets were analyzed by electron microscopy after adsorption to
formvar-coated grids and negative staining with 2% uranyl acetate in
water (C and D; scale bars, 100
nm).
Figure 6:
Arrest
of fibril formation by AcQKLVFFNH.
A
was incubated at 100 µM in TBS
for 48 h at 37 °C in a shaking water bath, either alone (A) or together with 100 µMAcQKLVFFNH
(B). The
polymerized material was adsorbed to formvar-coated grids and
negatively stained with 2% uranyl acetate in water. Scale
bars, 100 nm.
The aim of the present study was to identify regions in the
A molecule being important for binding during polymerization and,
based on the structure of such a binding sequence, synthesize a small
peptide ligand capable of binding to full-length A
and inhibiting
its polymerization into amyloid fibrils.
The binding sequence
identified in the present study is located in a region of the A
molecule that previously has been shown to be important during
proteolytic processing of APP. During nonamyloidogenic processing of
APP (i.e.
-secretase cleavage), the molecule is cleaved
between amino acid residues Lys
and
Leu
(20) . This leads, after further processing, to
the formation of an A
fragment termed p3, corresponding to
A
or
A
(21) . Through this metabolic
pathway, the present binding sequence is disrupted. This may explain
why p3 is not capable of forming amyloid in vitro or in
vivo(11, 12) . However, experimental studies show
that p3 is capable of forming fibril-like structures in
vitro(12) . It is therefore highly probable that binding
sequences other than the KLVFF sequence are involved in A
and p3
polymerization. The C terminus of the A
peptide may be of great
importance in that respect (10, 18) .
Previous
studies of putative inhibitors of amyloid fibril formation showed that
cyclodextrins (22) and Congo red (23) may have such
properties. The usefulness of these molecules as lead substances in
development of anti-Alzheimer amyloid drugs is, however, compromised by
their lack of specificity. Cyclodextrins have primarily been used to
increase the solubility of a wide range of drugs, and it is unlikely
that they will display any specificity for A in vivo.
Congo red, which is used to detect amyloid histochemically, binds to a
wide array of non-A
amyloids as well as to other proteins with a
high content of
-pleated sheet structures(24) .
Due to
the extreme insolubility of A amyloid (strong chaotropic agents or
potent organic solvents are required for its dissolution (4) ),
the concept of breaking up amyloid deposits in situ under
physiological conditions may seem futile. However, the bulk of the
individual molecules in amyloid are probably not joined by covalent
bonds, and the deposition of A
into amyloid is, at least at some
stages, a dynamic and reversible process(25) . Hence, a
molecule capable of binding to a site in the A
molecule being
critical for fibril formation, and with an affinity higher than native
A
, may inhibit amyloid growth and possibly also specifically
dissolve amyloid fibrils in situ.
Previous studies suggest
that amino acid residues within or close to A are important for the adoption of the correct
-pleated sheet
structure of A
(26, 27) and the proteolytic
processing of its precursor(20) . Here, it was shown that this
region harbors a binding sequence required for the polymerization of
A
into amyloid fibrils. It was also demonstrated that short
peptides incorporating A
can function as
ligands that bind to A
and inhibit the formation of amyloid
fibrils. Since these peptide ligands are relatively small, they are
amenable to investigation using organic chemistry. Non-peptide
homologues of KLVFF may thus turn out to be useful as pharmacological
tools for the treatment of Alzheimer's disease in the future.