From the Centre for Research in Neurodegenerative
Diseases, the § Department of Medical Biophysics, and the
Department of Laboratory Medicine and Pathobiology, University
of Toronto, Toronto, Ontario M5S 3H2, Canada
Received for publication, September 6, 2000, and in revised form, November 29, 2000
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ABSTRACT |
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In Alzheimer's disease, the major
pathological features are diffuse and senile plaques that are primarily
composed of the amyloid- Alzheimer's disease is characterized neuropathologically by
amyloid deposits, neurofibrillary tangles, and selective neuronal loss.
The major component of the amyloid deposits is a 39-43-residue peptide, amyloid- Many proteins are associated with amyloid plaques; their presence may
result in heterogeneous nucleation of A The GAG binding site on the A A Glycosaminoglycans and Subunits--
Chondroitin-4-sulfate
(bovine trachea, 8000 Da), dermatan sulfate (bovine mucosa, 16,000 Da),
chondroitin-6-sulfate (shark cartilage), heparin (porcine intestinal
mucosa), heparan sulfate (bovine kidney), and keratan sulfate (bovine
cornea) were purchased from Sigma. Chondroitin sulfate-derived
disaccharides Tyrosine Fluorescence Assay--
Tyrosine emission spectra from
290 to 340 nm were collected (excitation wavelength 281 nm, 0.5 s/nm,
band pass = 4 nm). A cuvette with a 1-cm path length was used. For
the centrifugation studies, 1 µM A Electron Microscopy--
For negative staining, carbon-coated
pioloform grids were floated on aqueous solutions of peptides (100 µg/ml). After grids were blotted and air-dried, the samples were
stained with 1% (w/v) phosphotungstic acid, pH 7.0. The peptide
assemblies were observed in a Hitachi H-7000 operated with an
accelerating voltage of 75 kV (28).
Amyloid Staining--
Thioflavin T fluorescence assay of A Competitive Inhibition ELISAs--
Nunc Immunosorp plates were
coated with 100 µl of GAGs (5 µg/ml) and incubated overnight,
4 °C. Simultaneously, A Induction and Morphology of A
To investigate the effect of chondroitin sulfate-derived saccharides on
A
The characteristics of A
Another major component of polymeric GAGs are the 2-sulfated iduronic
acid disaccharides, which are present predominantly in heparan sulfate
GAGs. Certain repeating disaccharide motifs containing sulfated
iduronic acid are also present as part of the chondroitin and dermatan
sulfate backbones. Many studies have demonstrated that charge
distribution across small molecules represents the limiting factor for
A
GAGs also laterally aggregate preformed A Effect of Chondroitin Sulfate-derived Monosaccharides on A
The morphology of A
Thioflavin T specifically stains amyloid deposits in vivo
and has been shown to bind both A Characterization of the A
The chondroitin sulfate-derived disaccharides had variable abilities to
compete for A Chondroitin Sulfate-derived Saccharides Inhibit Heparan Sulfate GAG
Binding to A
Further investigation into the ability of chondroitin sulfate-derived
saccharides to inhibit heparan sulfate binding to A
Cumulatively, our results demonstrate that chondroitin sulfate-derived
monosaccharides represent the minimal GAG subunit required for A (A
) peptide. It has been proposed that
proteoglycans and glycosaminoglycans (GAG) facilitate amyloid fibril
formation and/or stabilize the plaque aggregates. To develop effective
therapeutics based on A
-GAG interactions, understanding the A
binding motif on the GAG chain is imperative. Using electron
microscopy, fluorescence spectroscopy, and competitive inhibition
ELISAs, we have evaluated the ability of chondroitin sulfate-derived
monosaccharides and disaccharides to induce the structural changes in
A
that are associated with GAG interactions. Our results demonstrate
that the disaccharides GalNAc-4-sulfate(4S),
UA-GalNAc-6-sulfate(6S), and
UA-GalNAc-4,6-sulfate(4S,6S), the
iduronic acid-2-sulfate analogues, and the monosaccharides
D-GalNAc-4S, D-GalNAc-6S, and D-GalNAc-4S,6S, but not D-GalNAc,
D-GlcNAc, or
UA-GalNAc, induce the fibrillar features of
A
-GAG interactions. The binding affinities of all chondroitin
sulfate-derived saccharides mimic those of the intact GAG chains. The
sulfated monosaccharides and disaccharides compete with the intact
chondroitin sulfate and heparin GAGs for A
binding, as illustrated
by competitive inhibition ELISAs. Therefore, the development of
therapeutics based on the model of A
-chondroitin sulfate binding may
lead to effective inhibitors of the GAG-induced amyloid formation that
is observed in vitro.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
(A
).1
A
fibrillogenesis in vitro is a
nucleation-dependent process consisting of a slow lag phase
for nucleation followed by faster propagation of fibrils (1-3).
However, in vivo fibrillogenesis is likely a complex pathway
involving many factors that modulate the aggregation of A
.
Two mechanisms have been proposed for the nucleation of A
fibrils. The first involves the self-assembly of A
monomers, which
undergo a conformational change to become the fibril nucleus. The
second involves an alternative pathway of heterogenous
nucleation, which results from outgrowth of fibrils from non-A
seeds
(1).
(4-11). Although heparan
sulfate proteoglycans have been extensively correlated with plaque
formation, in Alzheimer's disease at least four types of proteoglycans
are associated with amyloid plaques (12-17). A
-proteoglycan interactions are mediated predominantly through A
-glycosaminoglycan (GAG) binding with GAGs acting as a scaffold for the assembly of the
fibrils. The scaffold may function by enhancing the structural features
that favor a
-sheet conformation thereby increasing the number of
nucleation seeds, as demonstrated by a virtually instantaneous
structural transition in A
upon addition of GAGs (18). In the
later stages of the amyloid pathway, GAGs also act by enhancing
lateral aggregation of small fibrils to confer insolubility and
protection from proteolysis (18-21). A structure-activity relationship
for A
-GAG interactions is slowly emerging based on the affinities of
various GAGs. In vitro studies have shown that the
chondroitin sulfates are more effective at both nucleation and lateral
aggregation of A
fibrils than the heparin GAGs (18). Chondroitin
sulfates are sulfated on a single face of the polymer and may represent
an ideal distribution of charge for A
interactions. Therefore, these
GAGs were used as the prototype to determine A
binding and
potentially to develop compounds that could compete with all identified
proteoglycans associated with plaques.
peptide has been investigated using
amino acid substitution of the A
1-28 peptide and as a function of aggregation state (22, 23). These studies have demonstrated
that although electrostatic interactions through basic amino acids
contribute to GAG binding, nonionic interactions, such as hydrogen
bonding and van der Waals packing, play a role in GAG-induced A
folding and aggregation (22). Furthermore, GAG-A
interactions are
more sensitive to the conformation and aggregation state of A
rather
than the primary sequence (22, 23). Together these results suggested
that inhibition of A
-GAG interactions through targeting of the GAG
binding site on A
may not provide a viable therapeutic.
Alternatively, the A
-GAG interaction may be mediated by a unique
binding site on the GAG backbone that could serve as a target for
inhibition of amyloid formation. This therapeutic strategy is supported
by in vitro studies, which demonstrated that polysulfated
compounds could inhibit binding of heparan sulfate to A
(24). Also
using an in vivo model of splenic amyloidosis, small
sulfonated or sulfated molecules have been shown to be active inhibitors of amyloid deposition (25). Alternatively, small GAG-derived
saccharides may have the alternate effect of enhancing A
precipitation into nontoxic plaques and thereby decreasing the presence
of toxic A
species. To develop more specific therapeutics directed
toward A
fibrillogenesis, we determined the minimum GAG unit
necessary for A
binding, fibrillogenesis, and lateral aggregation.
Here, we examine the interaction of chondroitin sulfate-derived monosaccharides and disaccharides with A
40 and A
42. Fluorescence spectroscopy and electron microscopy demonstrate a potent effect of
both monosaccharides and disaccharides on the formation and structure
of A
fibrils. In addition, competitive inhibition ELISAs demonstrate
that the binding of both monosaccharides and disaccharides to A
inhibits interaction with the polymeric chondroitin sulfate and heparan
sulfate GAGs.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Peptides--
A
40 and A
42 were synthesized by solid
phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry
by the Hospital for Sick Children's Biotechnology Center (Toronto,
ON). Peptides were isolated by reverse phase high pressure liquid
chromatography on a C18 µbondapak column, and purity was determined
using mass spectrometry and amino acid analyses. Peptides were
initially dissolved in 0.5 ml of 100% trifluoroacetic acid
(Aldrich), diluted in distilled H2O, and immediately
lyophilized (26). Peptides were then dissolved in 40% trifluoroethanol
(Aldrich) in H2O and stored at
20 °C until use.
Alternatively, the lyophilized peptides were dissolved in distilled
H2O at 2.5 mM concentration and used immediately.
UA-GlcNAc,
UA-GalNAc,
UA-GalNAc-4S,
UA-GalNAc-6S, and
UA-GalNAc-4S,6S were purchased from Dextra
Laboratories (Reading, United Kingdom), and
UA-2S-GalNAc,
UA-2S-GalNAc-4S,
UA-2S-GalNAc-6S, and
UA-2S-GalNAc-4S,6S were
purchased from Calbiochem. Monosaccharides D-GalNAc,
D-GlcNAc D-GalNAc-4S, D-GalNAc-6S,
and D-GalNAc-4S,6S were purchased from Sigma. All GAGs and
saccharides were dissolved in distilled H2O at 10 mg/ml and
stored at
20 °C until use.
40 or A
42 was
incubated in the presence or absence of chondroitin sulfate subunits at
a 1:1 ratio for 24 h. Samples were centrifuged for 30 min at
15,600 × g to sediment aggregates and fibrils as
described previously (27, 28). The relative amount of tyrosine in the
supernatant was then determined. The fluorescence of the noncentrifuged
sample was used as a measure of the total tyrosine fluorescence.
in
the presence and absence of GAGs (29, 30) and GAG-derived disaccharides
and monosaccharides was used to evaluate the similarity between
A
-GAG fibrils and classical amyloid fibers. Samples were incubated
at a 1:1 ratio by weight with a final A
concentration of 200 µM for 3 days. Samples were vortexed and 40-µl aliquots
were added to 960 µl of 10 µM Thioflavin T in
phosphate-buffered saline, pH 6.0. Steady state fluorescence was
measured at 20 °C using a Photon Technology International QM-1
fluorescence spectrophotometer equipped with excitation intensity
correction and magnetic stirrer. Thioflavin T emission spectra from 475 to 495 nm were collected (excitation wavelength 437 nm, 0.5 s/nm, band
pass = 4 nm). A cuvette with a 1-cm path length was used.
40 and A
42 were incubated with
chondroitin sulfate-derived monosaccharides and disaccharides at a 1:1
or 1:10 ratio by weight. The plates were rinsed twice with water and
blocked with 100 µl of 1% bovine serum albumin in phosphate buffered
saline. After incubation for 1 h at room temperature, the plates
were washed 3 times with 0.05% Tween 20/phosphate-buffered saline and
twice with phosphate-buffered saline. A
was then added to the plates
and incubated for 2 h at room temperature with shaking. Plates
were washed as above before the addition of monoclonal antibody against
A
, 6F/3D (Dako), 6E10, or 4G8 (Senetek, Carpinteria, CA). The
reaction with 50 µl of horseradish peroxidase-conjugated goat
anti-mouse IgG 1:2000 was performed at room temperature for 1 h.
Color development was achieved with 100 µl of
2,2'azino-di(3-ehtyl-benzthiazoline-6-sulfonic acid) in 0.1 M acetate buffer, pH 4.2. The absorbance was monitored at
415 nm on a Bio-Rad Benchmark microtiter plate reader.
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES
Fibril in the Presence of
Chondroitin Sulfate-derived Disaccharides--
We have previously
shown that the chondroitin sulfate GAGs are the most efficient at
inducing a
-structural transition in A
and subsequent
fibrillogenesis (18). Therefore, we have used the chondroitin
sulfate-derived monosaccharides and disaccharides (Fig.
1) to elucidate the minimum sugar moiety
necessary for A
binding and fibrillogenesis. Chondroitin sulfate
subunits are derived by enzymatic cleavage of the GAG chain by
chondroitinases ABC, AC-I, -B, or -C. The saccharides used in this
study represent repeat disaccharides present in chondroitin-4-sulfate,
chondroitin-6-sulfate, and dermatan sulfate, which retain the charge
distribution present in the intact GAG (Fig. 1). The monosaccharides
are generated by removal of uronic acid from and desulfation of the
disaccharides.
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Fig. 1.
The structure of chondroitin sulfate-derived
saccharides used in this investigation. The monosaccharides are
derived by removal of the uronic acid from each disaccharide and is
replaced by a hydroxyl group.
nucleation, the intrinsic tyrosine fluorescence of A
40 and
A
42 was used to monitor the amount of soluble peptide after
incubation in the presence and absence of the disaccharides. After
24 h of incubation, soluble A
was separated from aggregated and
fibrillar peptide by centrifugation (23, 24). Different chondroitin
sulfate-derived saccharides had variable effects on the amount of
pelletable aggregates detected with greater effects seen for A
42
(Fig. 2) over A
40 (data not shown). At
24 h, the disaccharides
UA-GalNAc-4S,
UA-GalNAc-6S, and
UA-GalNAc-4S,6S significantly increased the amount of aggregated
A
40/42 with
UA-GalNAc-4S,6S being the most effective (Fig.
2C). It is interesting to note that all disaccharides and in
particular
UA-GalNAc-4S,6S induced the same extent of fluorescence
loss as the intact GAG, dermatan sulfate (Fig. 2D). These
results demonstrate that the presence of chondroitin sulfate-derived
disaccharides can be correlated with an increased amount of aggregated
A
.
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Fig. 2.
Tyrosine fluorescence was used to determine
the extent of A 40 and
A
42 aggregation in the presence of chondroitin
sulfate-derived monosaccharides, disaccharides, and
glycosaminoglycans. A
42 was incubated with the various
GAGs at a 1:1 ratio (weight/weight) for 24 h. A
aggregation was
determined using the ratio of tyrosine fluorescence before and after
centrifugation. The extent of A
aggregation when incubated alone
(A) and in the presence of D-GalNAC-4S,6S
(B) was minimal, whereas in the presence of
UA-GalNAc-4S,6S (C) and dermatan sulfate (D)
virtually all A
tyrosine fluorescence was lost due to peptide
precipitation.
40 and A
42 fibrils in the presence and
absence of chondroitin sulfate-derived saccharides were examined by
electron microscopy. Previous investigations indicated that GAG
promoted morphological changes in the fibrous structures formed by
A
40 and A
42 (18). Unseeded samples of both A
40 and A
42 were
incubated in the presence of chondroitin sulfate-derived disaccharides,
intact chondroitin sulfate GAGs, and alone for up to 96 h.
Negative stain electron microscopy demonstrated that A
42 fibrils
were 50-70 Å in diameter with an average length of 750 Å (Fig.
3A). These were
indistinguishable from those of A
42 in the presence of the
desulfated
UA-GalNAc (Fig. 3B) or
UA-GlcNAc (data not
shown). The monosulfated disaccharides,
UA-GalNAc-6S and
UA-GalNAc-4S, induced fibrils of similar size but with increased lateral aggregation as compared with control (Fig. 3C). The
bundles of fibers were similar to those seen in the presence of
polymeric chondroitin sulfate GAGs (18). In the presence of
UA-GalNAc-4S,6S, A
42 formed many fibers displaying extensive
lateral aggregation as illustrated by the heavily stained clusters of
fibrils (Fig. 3D). These results demonstrate that the
sulfated disaccharides derived from chondroitin sulfate are
representative of the intact GAG chains in terms of activity. The
extent of lateral aggregation induced by the disaccharides reflects
that of the intact GAGs, with 4-sulfate < 6-sulfate < 4/6-sulfate.
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Fig. 3.
Negative stain electron microscopy of
A 42 in the presence of chondroitin
sulfate-derived disaccharides. A
42 incubated in buffer alone
(A) demonstrates many long fibers. When incubated in the
presence of
UA-GalNAc (B) no difference could be detected
in the structure of the fibrils formed. Alternatively, lateral
aggregation was apparent in the fibrils formed in the presence
of
UA-GalNAc-4S (C) and
UA-GalNAc-4S,6S
(D). In the presence of sulfated iduronic acid disaccharides
UA-2S-GalNAc-4S (E) and
UA-2S-GalNAc-4S,6S
(F) demonstrate fibers that are similar to the unsulfated
iduronic acid derivatives. Scale bar is 50 nm.
binding (28, 31). Therefore, the introduction of a sulfate group
onto the iduronic acid may present a more favorable or deleterious
surface for A
binding. To investigate these possibilities, we
examined the effect of 2-sulfated iduronic acid-containing
disaccharides on fibril assembly and morphology. Negative stain
electron microscopy demonstrated that the extra sulfate on the
UA-2S-GalNAc had no effect on the A
42 fibers formed in comparison
to A
42 alone or in the presence of
UA-GalNAc (data not shown). In
the presence of
UA-2S-GalNAc-4S and
UA-2S-GalNAc-6S, many short
fibers could be detected displaying limited aggregation. (Fig.
3E). Incubation of A
42 with
UA-2S-GalNAc-4S,6S results in the development of thick aggregated fibers displaying a helical twist (Fig. 3F). Close examination of the fibers reveal a
length of 300-500 Å with a diameter of 100 Å, which correspond to
mature amyloid fibers. These results demonstrate that the sulfate group on the second position of the iduronic acid does not effect the morphology of A
fibrils formed in the presence of sulfated GalNAc disaccharides. Furthermore, these data suggest that using a
disaccharide with a sulfated iduronic acid does not contribute to the
minimal unit necessary for A
binding and enhanced fibrillogenesis.
fibrils into large masses
characteristic of insoluble plaques. To evaluate this phenomenon as
induced by chondroitin sulfate-derived disaccharides, we incubated the
saccharides with preformed A
40 fibrils and examined the morphology
by negative stain electron microscopy. Preincubated A
40 forms long
fibers that are nonaggregated (Fig.
4A). In contrast, dermatan
sulfate induced a consistent lateral aggregation of A
40 fibrils with
apparent helical twisting (Fig. 4B). The fiber bundles had a
cumulative diameter of up to 200 Å, but the average length of the
fibers remained unaffected. In the presence of chondroitin sulfate-derived disaccharides, A
40 lateral aggregation was similar to that of an extended GAG polymer, with the disulfated
UA-GalNAC-4S,6S being the most effective (Fig. 4C). The
fibers were aligned in large bundles rather than a haphazard
distribution across the grid. These data indicate that the binding of
chondroitin sulfate-derived disaccharides to A
fibrils is sufficient
to stabilize the macromolecular structure of pre-existing fibers by
lateral aggregation and represent the smallest unit responsible for
interfiber stabilization. Similar to polymeric GAGs, all data taken
together demonstrate that the extent of sulfation on the disaccharide
backbone defines the extent to which the disaccharide interacts with
and stabilizes A
. Furthermore, these small GAG-derived saccharides
demonstrate the potential use of these molecules to decrease the
soluble pool of A
in situ by enhancing the precipitation
of nontoxic fibers.
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Fig. 4.
Negative stain electron microscopy of seeded
A 40 was examined in the presence and absence
of chondroitin sulfate GAGs. A
40 formed long fibers
(A) and upon incubation with dermatan sulfate appeared to be
thicker as a result of lateral aggregation with a 150 nm periodic twist
(B). In the presence of
UA-GalNAc-4S,6S, A
40
fibrils were organized into thick bundles (C,
arrows delineate bundles) characteristic of the intact
dermatan sulfate. Scale bar is 50 nm.
Fibrillogenesis--
The effect of chondroitin sulfate-derived
monosaccharides on the induction of A
42 fibrillogenesis was
investigated. To investigate the effect of chondroitin sulfate-derived
saccharides on A
nucleation, the intrinsic tyrosine fluorescence of
A
40 and A
42 was used to monitor the amount of soluble peptide
after incubation in the presence and absence of the monosaccharides. At
24 h, no significant difference could be detected in the amount of
A
40/42 pelleted in the presence and absence of the
monosaccharides D-GalNAc, D-GalNAc-4S, and
D-GalNAc-6S, whereas a slight increase in the amount of
A
42 pelleted in the presence of D-GalNAc-4S,6S could be
detected (Fig. 2B).
fibrils in the presence of chondroitin
sulfate-derived monosaccharides was investigated using negative stain
electron microscopy. Similar to A
42 alone (Fig.
5A), the presence of
nonsulfated D-GalNAc and D-GlcNAc induced
fibers similar to mature amyloid fibers (Fig. 5B). In the
presence of D-GalNAc-4S or D-GalNAc-6S (Fig.
5C), the fibers were less abundant and of varying lengths
but exhibited some aggregation as demonstrated by the uneven, heavily
stained distribution of fibers across the grid. Alternatively, in the
presence of D-GalNAc-4S,6S and D-galacturonic acid, A
42 formed many protofibrils characterized by short flexible fibrils (Fig. 5D). This suggests that binding of the
sulfated monosaccharide D-GalNAc-4S,6S or
D-galacturonic acid to A
enhances the nucleation stage of
fibrillogenesis resulting in the formation of many protofibrils. The
ability of D-galacturonic acid but not D-GalNAc
to nucleate A
may not be surprising because of the differences in
charge distribution across the sugar backbone.
D-Galacturonic acid is derived by removal of the
N-acetylamine group from position 4 of the sugar backbone,
and the resultant charge distribution may represent a preferential
binding motif. These data further suggest that monosaccharides can
inhibit the formation of mature amyloid fibers by blocking the
self-association of protofibrils. Further evidence to support this
hypothesis is derived from the lack of lateral aggregation of both
A
40 and A
42 preformed fibrils by all monosaccharides (data not
shown).
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Fig. 5.
Negative stain electron microscopy of
A 42 in the presence of chondroitin
sulfate-derived monosaccharides. A
42 incubated in buffer alone
(A) demonstrates many thin fibers. When incubated in the
presence of D-GalNAc (B), a similar structure of
the fibrils to A
42 alone could be detected. Alternatively, lateral
aggregation was apparent in the fibrils formed in the presence of
D-GalNAc-6S (C). In the presence of
D-GalNAc-4S,6S, only protofibrils of A
42 were detected
(D). Scale bar is 50 nm.
fibers and aggregates in
vitro (30). We investigated the binding of Thioflavin T to
chondroitin sulfate-derived saccharide-A
complexes to further
characterize the nature of these fibers. Thioflavin T fluorescence
intensity increased for both A
40 and A
42 in the presence of
intact chondroitin sulfate GAGs (Fig. 6).
In the presence of D-GalNAc, the Thioflavin T fluorescence
was indistinguishable from A
42 alone, which is consistent with our
electron microscopy data. Thioflavin T fluorescence increased in the
presence of D-GalNAc-6S but to a lesser extent than
chondroitin-6-sulfate. When incubated with D-galacturonic acid or D-GalNAc-4S,6S, A
42 demonstrated a morphology
similar to protofibrils and exhibited a Thioflavin T fluorescence
greater than A
42 alone. These results demonstrate that the
protofibrils induced by chondroitin sulfate-derived monosaccharides
have characteristics similar to typical amyloid fibers. In summary, our
data demonstrate that chondroitin sulfate-derived monosaccharides bind
A
and induce fibrillogenesis without lateral aggregation.
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Fig. 6.
Thioflavin T assay of chondroitin
sulfate-A fibrils. Thioflavin T binding
to chondroitin sulfate-A
complexes resulted in both an increase in
fluorescence and a shift in the emission spectra. Thioflavin T
fluorescence as a result of A
binding was similar in the presence or
absence of D-GalNAc. D-GalNAc-6S induced a
modest increase in the Thioflavin T fluorescence in comparison to A
alone, indicating a slight increase in aggregation.
Chondroitin-6-sulfate and dermatan sulfate increased the extent of
fluorescence indicating enhanced fibrillogenesis. Similarly,
D-galacturonic acid enhanced the fluorescence to
approximately the same extent. D-GalNAc-4S,6S binding to
A
42 resulted in the most intense fluorescence indicating that it is
qualitatively more effective at enhancing A
fibrillogenesis.
Values for Thioflavin T fluorescence of A
alone were set at
100%, and the Thioflavin T fluorescence of all samples treated with
GAGs is reported relative to this value. Values are reported as the
mean ± S.D. of three experiments.
-Saccharide Binding Site--
To
compare the specificity of chondroitin sulfate-derived saccharide
binding, we used competitive inhibition ELISAs to determine whether the
chondroitin sulfate-derived saccharides could compete with intact GAG
chains for A
40 and A
42 binding (Table
I). Concentration-dependent studies were used to determine both the specificity of competition and
the relative binding strengths of each component. The chondroitin sulfate-derived monosaccharides and disaccharides were preincubated with A
40 and A
42 before incubation with chondroitin-4-sulfate, chondroitin-6-sulfate, or dermatan sulfate. The amount of A
bound to
the intact GAG chain on the microtiter plate was determined using the
anti-A
antibodies 6E10, 4G8, or 6F/3D. All antibodies demonstrated
similar concentration-dependent inhibition profiles indicating that the detection antibody was not a determining
factor. D-GalNAc and
UA-GalNAc were unable to compete
with the chondroitin sulfate GAGs for A
binding, which is in
agreement with our electron microscopy data in which A
40/42 fibrils
were indistinguishable in the presence and absence of
D-GalNAc and
UA-GalNAc. The monosaccharides D-galacturonic acid, D-GalNAc-6S, and
D-GalNAc-4S,6S were all effective at competing with all
chondroitin sulfate GAGs for A
binding at a 1:10 ratio (by weight).
These results suggest that the alterations in fibrous structure
detected by electron microscopy and fluorescence studies can be
attributed to the binding of these monosaccharides to the GAG binding
site on A
40 and A
42. These observations further suggest that the
monosaccharides D-galacturonic acid,
D-GalNAc-6S, and D-GalNAc-4S,6S are sufficient to
induce A
binding and structural transitions associated with A
-GAG
interactions. It was not surprising to find that
D-GalNAc-4S competed poorly with all GAGs for A
binding
because the intact chondroitin-4-sulfate is the least effective of all
the chondroitin sulfate GAGs at inducing the structural transitions
necessary for fibril formation and aggregation (18).
Competitive inhibition ELISAs for A42 binding
42 was pre-incubated with the chondroitin sulfate-derived
monosaccharides or disaccharides before co-incubation with intact
chondroitin sulfate GAGs. The amount of A
42 that bound each intact
GAG was set at 100% binding, and all other values were calculated with
respect to this value. The values are quoted using 6E10 for the
detection of A
42. Values are reported as mean ± S.D. for at
least three separate ELISA assays. Paired t test indicates
p < 0.05.
binding with
UA-Gal-4S,6S being the most effective
(Table I). The differences in binding of the disaccharides reflect the
varying abilities of the chondroitin sulfate GAGs to bind, induce a
structural change in A
, and enhance lateral aggregation. One
corollary to our results is that we cannot rule out the possibility
that the disaccharides induced a conformational change in A
that
allowed the intact GAG chain to elicit binding between A
fibrils as
has been previously suggested for GAG binding to preformed fibers (18).
The chondroitin sulfate-derived monosaccharides and disaccharides
binding strengths, as determined by the extent of competition, may
reflect the fluctuation of A
binding to surfaces with slight
variations in charge distribution. These characteristics have been
reported previously for myo-inositol and its phosphorylated analogues as well as alterations in the distribution and surface charge
of the antibiotic rifampicin (28, 31).
--
The similarities in GAG structure between
chondroitin sulfate and heparan sulfate GAGs previously have stimulated
the suggestion that proteins that bind to chondroitin sulfate should
interact with heparan sulfate and vice versa (32-34). The basic
fibroblast growth factor of the heparan sulfate-binding proteins,
platelet-derived factor 4, and fibronectin react weakly with
dermatan sulfate, whereas heparin cofactor II and hepatocyte growth
factor have a comparable high affinity for both heparan sulfate and
dermatan sulfate (35-39). To determine whether the chondroitin
sulfate-derived monosaccharides could compete with other GAGs for A
binding, we repeated the competitive inhibition ELISAs using heparin
(Table II). It is speculated that
polymeric GAGs bind to the same region or structural motif in A
(40); therefore it was not unexpected to find that the
monosaccharide D-GalNAc-4S,6S could compete to the same
extent with heparin as was seen for dermatan sulfate. Our results for
heparin competition illustrate that the competition detected between
heparin and the chondroitin sulfate-derived saccharides is independent
of the detection antibody, as both the A
-specific antibodies, 6E10
and 4G8, detect a similar concentration-dependent inhibition (Table II). These results suggest that development of an
inhibitor for GAG binding to A
could represent an agent to block
A
-proteoglycan interactions.
Competitive inhibition ELISAs between D-GalNAc-4S,6S and
heparin for A binding
was incubated in the presence of D-GalNAc-4S,6S before
competition for heparin binding. The amount of A
alone that bound to
heparin was set at 100%, and all other values were calculated with
respect to this value. The values are reported as mean ± S.D. for
at least three separate ELISAs. Paired t test indicates that
p < 0.001.
demonstrated
similar results to those of both chondroitin and dermatan sulfate
(Table III). None of the nonsulfated
monosaccharides or disaccharides could inhibit A
binding to both
heparan sulfate and keratan sulfate; these results are similar to those
for dermatan sulfate competition studies. The iduronic acid-2-sulfated
disaccharides were unable to compete for heparan sulfate binding,
whereas
UA-2S-GalNAc-4S and
UA-2S-GalNac-6S were able to compete
for keratan sulfate binding. These results suggest that subtle changes
in the GAG backbone and distribution of sulfation have significant
effects on the ability of chondroitin sulfate-derived saccharides to
compete for GAG binding sites. The disaccharides
UA-GalNAc-4S,
UA-GalNAc-6S, and
UA-GalNAc-4S,6S all competed with heparan
sulfate for A
binding with the disulfated derivative being the most
effective (Table III). As was seen for competition for chondroitin
sulfate GAGs, the monosaccharide D-GalNAc-4S,6S competed
with high affinity with both dermatan sulfate and heparan sulfate.
These results suggest that a therapeutic approach based on this
structural motif may inhibit A
binding to all GAGs present in the
central nervous system.
Competitive inhibition ELISAs for A42 binding
42 was pre-incubated with the chondroitin sulfate-derived
monosaccharides or disaccharides before co-incubation with intact GAGs.
The amount of A
42 that bound each intact GAG was set at 100%
binding, and all other values were calculated with respect to this
value. Values are reported as mean ± S.D. for at least three
separate ELISA assays. Paired t test indicates
p < 0.05 when compared with A
42 binding alone.
binding and that lateral aggregation between A
fibers or the
transition of protofilaments into mature amyloid fibers requires a
sulfated GAG disaccharide. These results suggest that the size
constraints of the monosaccharide are insufficient to facilitate the
association of fibers but are sufficient to bind A
. Development of
drugs based on these monosaccharide compounds will have to take into
account the potential for stabilization of toxic A
intermediates. We
have previously shown that A
42 is stabilized in a nontoxic oligomer
in the presence of inositol stereoisomers; this illustrates the
potential for drug design based on the present methodology (28, 31,
41). Alternatively, GAG-derived disaccharides may represent a template
in which to develop drugs that will decrease available monomer in
situ by accelerating precipitation of A
fibers. These studies
further emphasize the importance of investigations into the design of GAG memetics as potential amyloid therapeutics.
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ACKNOWLEDGEMENTS |
---|
We thank Dr. N. Wang at the Hospital for Sick Children's Biotechnology Center in Toronto, Ontario, Canada for the synthesis of peptides used in this study.
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FOOTNOTES |
---|
* This work was supported by grants from the Ontario Mental Health Foundation (to J. M. and P. E. F.), the Scottish Rite Charitable Foundation (to P. E. F.), Neurochem Inc. (to P. E. F.), University of Toronto Dean's Fund (to J. M.), and The Banting Foundation (to J. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Supported by the Alzheimer Society of Ontario.
** Supported of the Alzheimer Society of Ontario and the Kevin Burke Memorial Amyloid Fund. To whom correspondence should be addressed: Centre for Research in Neurodegenerative Diseases, Tanz Neuroscience Bldg, 6 Queen's Park Crescent West, Toronto, Ontario M5S 3H2, Canada. Tel.: 416-978-1035; Fax: 416-978-1878; E-mail: j.mclaurin@utoronto.ca.
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M008128200
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ABBREVIATIONS |
---|
The abbreviations used are:
A, amyloid-
peptide;
GAG, glycosaminoglycan;
ELISA, enzyme-linked immunosorbent
assay.
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