(Received for publication, April 10, 1995)
From the
Senile plaques of Alzheimer's disease brain contain, in
addition to beta amyloid peptide (A
Senile plaques are one of the classical neuropathological
features of Alzheimer's disease (AD) ( A It has been suggested that the
association of proteoglycans with A
Additional
studies were performed in which 30 µg each of chondroitin sulfate
A, chondroitin sulfate B, chondroitin sulfate C, heparin, (all from
Sigma) or heparan sulfate (Seikagaku America, Inc.) were added as above
instead of proteoglycan.
Studies examining the
pH-dependent binding of fibrillar and non-fibrillar A
To more
accurately determine binding affinities, K can be utilized to obtain accurate K [A
Figure 1:
SDS-PAGE showing proteoglycan-mediated
inhibition of A
Figure 2:
Proteoglycan-mediated protection of
A
To confirm that
the results obtained with papain and Pronase extended to more specific
and physiologically relevant proteases, we examined whether cathepsin B
degrades A The proteoglycan-mediated
inhibition of A
Figure 3:
Binding of proteoglycans to non-fibrillar
and fibrillar A
To more completely
characterize the nature of proteoglycan binding to A
Figure 4:
Binding of fibrillar and non-fibrillar
A
Since the
saturation binding studies presented in Fig. 3could not be used
to accurately determine the affinities of proteoglycan
Figure 5:
Inhibition of fibrillar A
Although the binding of both CSPG and HSPG to fibrillar A
Figure 6:
Inhibition of fibrillar A
In addition to proteoglycan
association with A A body of evidence is accumulating implicating A Senile plaques contain proteoglycans in addition to amyloid peptide (10, 11, 12) . Systemic amyloid deposits
resemble senile plaques in that polymeric The data presented here support the
hypothesis that proteoglycans play an important role in the
accumulation and persistence of plaques. We have found that mouse HSPG
and bovine CSPG have the ability to bind fibrillar A The inability of proteoglycans to
affect the degradation of non-fibrillar A The
binding of fibrillar A The
binding of proteoglycans to lysine residues within fibrillar A Buee et al.(16) demonstrated that intact HSPG and
chemically deglycosylated HSPG could bind to an A While our data indicate that
A Although proteoglycans bind fibrillar A The ability of proteoglycans to inhibit proteolysis of
A The demonstrated ability of proteoglycans to alter
the proteolytic processing of A
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
), multiple proteoglycans.
Systemic amyloidotic deposits also routinely contain proteoglycan,
suggesting that these glycoconjugates are generally involved in amyloid
plaque formation and/or persistence. We demonstrate that heparan
sulfate proteoglycan (HSPG) and chondroitin sulfate proteoglycan (CSPG)
inhibit the proteolytic degradation of fibrillar, but not
non-fibrillar, A
at physiological pH. In accordance with the
proteolysis studies, high affinity binding of proteoglycans to
fibrillar A
(1-40) and A
(1-42) is observed from pH
4 to 9, whereas appreciable binding of HSPG or CSPG to non-fibrillar
peptide is only seen at pH < 6. This differing pH dependence of
binding suggests that a lysine residue is involved in proteoglycan
association with fibrillar A
, whereas a protonated histidine
appears to be needed for binding of the glycoconjugates to
non-fibrillar peptide. Scatchard analysis of fibrillar A
association with proteoglycans indicates a single affinity interaction,
and the binding of both HSPG and CSPG to fibrillar A
is completely
inhibited by free glycosaminoglycan chains. This implies that these
sulfated carbohydrate moieties are primarily responsible for
proteoglycan
A
interaction. The ability of proteoglycans to
bind fibrillar A
and inhibit its proteolytic degradation suggests
a possible mechanism of senile plaque accumulation and persistence in
Alzheimer's disease.
)brain(1) , and a prevailing hypothesis is that
these structures lead to the dystrophic neurites and dying neurons that
cause the dementia associated with this disease. The major component of
senile plaques is polymeric fibrils of A
(2) , a
39-42-amino-acid peptide which is formed after proteolytic
processing of the amyloid precursor protein
(APP)(3, 4, 5, 6) . Other
macromolecules(7, 8, 9) , including a variety
of proteoglycans(10, 11, 12) , co-localize
with senile plaques. Interestingly, proteoglycans are also invariably
associated with plaques of peripheral systemic
amyloidoses(13, 14, 15) , suggesting they may
play an important role in plaque accumulation and/or persistence.
(16, 47) and APP (16, 17) have been shown to bind with relatively high
affinity to certain heparan sulfate proteoglycans (HSPG), and these
interactions were suggested to be mediated in part via the core protein
of the proteoglycans. Work from this laboratory (18) has
demonstrated that A
is also capable of association with a variety
of glycosaminoglycan chains. The binding of A
with proteoglycan
core protein and/or glycosaminoglycan chains provides an explanation
for the localization of CSPG(10) , DSPG(11) , and HSPG (12) with senile plaques.
might result in an enhanced
rate of fibrillation of the amyloid peptide (13) or that
proteoglycans may inhibit the degradation of
A
(13, 19) . Either of these actions could lead to
increased numbers of senile plaques, with a concomitant increase of
neuropathology. There is precedent for the glycosaminoglycan moieties
of proteoglycans affecting protein breakdown, as heparan sulfate has
been demonstrated to slow plasmin-mediated digestion of basic
fibroblast growth factor(20) . In this study, we have examined
whether proteoglycans of the classes found associated with senile
plaques are capable of decreasing the susceptibility of A
to
proteolytic degradation. The results reveal that whereas both
non-fibrillar and fibrillar A
can be readily cleaved by proteases,
complexes of proteoglycan and fibrillar amyloid peptide are relatively
resistant to breakdown. The polymeric form of A
binds
proteoglycans with significantly greater affinity than does
non-fibrillar peptide at physiological pH, and the association of CSPG
and HSPG with fibrillar A
appears to be mediated primarily via
glycosaminoglycan chains. These data thus supply the first indication
that senile plaques in AD brain may accumulate and persist due to the
inability of naturally occurring proteases to act on
A
proteoglycan complexes.
Proteoglycan Preparations
EHS mouse HSPG was
obtained commercially (Collaborative Biomedical Products). CSPG were
purified from bovine nasal septum cartilage essentially as
described(21) .Characterization of A
Fibrillar
A(1-40) (Bachem Inc.) and A
(1-42) (synthesized at
Gliatech Inc.) were prepared by resuspending lyophilized peptide in
H
O (2 mM stock) and allowing the solutions to age
for 1-3 weeks. The extent of fibril formation was quantified
using a Thioflavin T fluorescence assay as described(26) .
Non-fibrillar A
preparations were made by resuspending lyophilized
peptide in hexafluoroisopropanol, with subsequent relyophilization. The
hexafluoroisopropanol-treated lyophilized peptides were suspended in
H
O (2 mM stock) and used within 3 days of
solubilization. The non-fibrillar A
solutions did not bind
Thioflavin T as determined by the absence of specific fluorescence.
Proteolysis of A
In a typical assay, non-fibrillar or fibrillar
AProteoglycan/Glycosaminoglycan
Complexes
(1-40) (4.3 µg) and proteoglycans (30 µg) were
incubated for 1-2 h at room temperature in 10-15 µl
final volume of Tris (0.1 M) or phosphate-buffered saline
(PBS) at pH 7.4. Papain (2 µg) was subsequently added and the
reaction mixture incubated at 37 °C for 18 h. In some experiments,
papain was replaced with cathepsin B (Sigma). In these studies,
fibrillar A
(1-40) was incubated either in the presence or
absence of CSPG as above, except in 20 mM sodium acetate, pH
5.2, containing 120 mM NaCl, 0.5 mM EDTA, and 1
µM dithiothreitol. Cathepsin B (0.1 unit) was subsequently
added and incubated as above. Digested products were subjected to 16.5%
Tris-Tricine SDS-PAGE (27) and the gels were stained with
Coomassie Blue to visualize undegraded 4-kDa A
, which was
quantitated using a densitometer (PDI; model DNA 35).
A
Polyvinyl
microtiter plates (Costar high binding) were coated with proteoglycans
(2 µg/50 µl) in PBS buffer overnight at 4 °C. After
aspirating the proteoglycan solution, wells were blocked with PBS
containing 2% bovine serum albumin for 2 h at room temperature. Control
wells were prepared as above, except proteoglycan was omitted from the
initial overnight incubation. Aliquots (100 µl) of either fibrillar
or non-fibrillar AProteoglycan Binding Assay
(1-40) and A
(1-42) at varying
concentrations in blocking solution were added to the wells. Following
a 2-h incubation at room temperature, the wells were extensively rinsed
with PBS. Proteoglycan-bound A
was detected by the addition of
mouse A
antibody (4G8 at 1:2000 dilution in blocking solution;
antibody provided by Drs. K. S. Kim and H. M. Wisniewski, Institute for
Basic Research in Developmental Diabetics, Staten Island, NY), which
was allowed to incubate for 1 h at room temperature. After washing the
plate with PBS, a peroxidase-conjugated goat anti-mouse antibody
(1:2000 dilution in blocking solution; Sigma) was added and allowed to
incubate for 1 h. The plate was again rinsed thoroughly with PBS, and
peroxidase-catalyzed color development was initiated by adding 100
µl of 3,3`,5,5`-tetramethyl benzidine substrate (Kirkegard &
Perry, Inc.). The reaction was quenched by adding 100 µl of 1 M phosphoric acid, and the plates were read at 450 nm in a
microplate reader (Dynatech). The ability of proteoglycans or
glycosaminoglycans to inhibit A
binding to immobilized
proteoglycans was evaluated by adding varying concentrations of these
agents to a constant concentration of A
(typically 1-2
µM). The amount of A
bound in the wells was
determined by subtracting control wells from the experimental wells.
Data were analyzed as described below.
(1-40)
were performed as above in buffer systems containing 120 mM NaCl, 2.7 mM KCl, and 10 mM of each of the
following buffers: sodium acetate, pH 4 and 5; sodium phosphate, pH 6,
7, and 8; sodium bicarbonate/carbonate, pH 9 and 10.
Data Analysis
The estimated K of fibrillar A
-proteoglycan interaction was based on
the concentration of added A
([A
]) at 50% maximal binding
(Microcal Origin program). Scatchard analyses of binding data were
performed by plotting bound/free (OD
/[A
]) versus bound (OD
). The concentration of A
fibrils could not be accurately
determined due to the uncertainty of fibril size (i.e. [A
] =
[A
]/c, where c = number of A
monomers comprising an average fibril).
The K
values obtained based on monomeric
A
concentration (K
) are
thus greater than the true K
values for
fibrillar peptide (K
= K
/c,).
values were obtained using proteoglycans and glycosaminoglycans
as competitive inhibitors of A
binding to immobilized
proteoglycan. The K
values were derived
from , where L = the concentration of
A
([A
]) used in the inhibition
studies.
values with fibrillar A
, even with
the uncertainty of fibril concentration. In this case, L would
represent [A
], which can be replaced
by the term ([A
]/c). The K
term in would be K
, as described
above. Substituting these terms into results
in
] and K
are readily
determined parameters.
Proteoglycans Protect A
To
determine whether proteoglycans can protect A from Proteolysis
from proteolytic
degradation, either non-fibrillar or fibrillar A
(1-40) were
incubated for 1-2 h at pH 7.4 with or without HSPG and CSPG. The
nonspecific protease papain was subsequently added to the mixtures, and
after overnight incubation the extent of A
proteolysis was
evaluated by SDS-PAGE analysis. Both fibrillar A
(1-40) ( Fig. 1and 2) and non-fibrillar A
(1-40) (Fig. 2) were degraded by the protease in the absence of
proteoglycan. Addition of CSPG or HSPG to the non-fibrillar peptide
solution did not appreciably affect A
degradation (Fig. 2).
In contrast, a significant amount of fibrillar A
(1-40)
persisted following preincubation with both of the proteoglycans prior
to papain treatment ( Fig. 1and Fig. 2). Essentially
identical results were obtained when A
(1-42) was used
instead of A
(1-40) or when papain was replaced with the
nonspecific enzyme mixture Pronase (data not shown).
(1-40) proteolysis. Fibrillar
A
(1-40) (4.3 µg) was treated with papain (2 µg)
either in the absence (lanes B) or presence of 30 µg of
bovine CSPG (lanes C) or HSPG (lanes D). Lane A shows A
(4.3 µg) that was not subjected to papain
digestion. The samples were analyzed on 16.5% Tris-Tricine SDS gels as
described under ``Materials and
Methods.''
(1-40) from proteolytic degradation. Non-fibrillar or
fibrillar A
(1-40) and HSPG (lane C), bovine CSPG (lane D), or chondroitin sulfate glycosaminoglycan (lane
E) were incubated at pH 7.4 and subsequently subjected to
proteolysis with papain as described under ``Materials and
Methods.'' Lane A shows A
(1-40) in the absence
of papain, while lane B reveals A
treated with papain in
the absence of proteoglycan. The data are normalized to the amount of
A
remaining after treatment relative to lane A conditions. The inset shows a similar study in which
papain was substituted with cathepsin B. Lane A contains
fibrillar A
(1-40) in the absence of cathepsin, lane B shows fibrillar A
(1-40) treated with cathepsin in the
absence of CSPG, and lane C contains the amyloid peptide
treated with cathepsin in the presence of
CSPG.
in the absence or presence of CSPG. This enzyme has
been shown to be released by microglia (25) and thus might
represent one of the proteases found in the milieu of the brain. As
with papain, cathepsin B cleaved fibrillar A
in the absence of
proteoglycan, while CSPG inhibited cathepsin proteolysis of the amyloid
peptides (Fig. 2, inset).
degradation is most likely explained by steric
shielding of the amyloid peptide by glycosaminoglycan chains, which
should be naturally resistant to proteases. This would suggest that
free glycosaminoglycans might also reduce the papain-catalyzed
degradation of A
. However, neither chondroitin sulfate (Fig. 2, lane E), nor a variety of other
glycosaminoglycans (data not shown) inhibited the proteolysis of either
fibrillar or non-fibrillar A
. This indicates that intact
proteoglycan is required for effective protection of amyloid peptide.
Binding of Proteoglycans to A
Inhibition of
fibrillar A proteolysis by proteoglycans implied that these
glycoconjugates were binding tightly to this form of the amyloid
peptide. The reduction of A
degradation appeared to be specific to
proteoglycans, since other proteins that are associated with senile
plaques (C1q and laminin) had no effect on papain-mediated cleavage of
A
(data not shown). To directly analyze proteoglycan association
with A
, a solid-phase binding assay was developed in which
proteoglycans were immobilized to the wells of microtiter dishes.
A
was subsequently added, and bound peptide was quantitated via an
enzyme-linked immunoabsorbant methodology. As demonstrated in Fig. 3, neither HSPG or CSPG showed appreciable binding to
non-fibrillar A
(1-40) or A
(1-42) at physiological
pH. While virtually no binding of non-fibrillar amyloid peptides could
be demonstrated with CSPG, the HSPG preparation consistently bound a
small amount of both A
peptides (compare Fig. 3, A and C to B and D). However, the amounts
of non-fibrillar peptides that associated with HSPG were very low
compared to the amount of fibrillar A
(1-40) or
A
(1-42) that bound to either CSPG or HSPG (Fig. 3).
The ability of proteoglycans to bind substantial amounts of fibrillar,
but not non-fibrillar, A
at pH 7.4 coincides with the
proteoglycan-mediated inhibition of degradation of only the fibrillar
form of amyloid peptide. Scatchard transformations of the binding data (Fig. 3, insets) reveal that the association of
fibrillar A
(1-40) and A
(1-42) with both HSPG and
CSPG is governed by a single affinity interaction. The actual
affinities of fibrillar A
proteoglycan binding were not
calculated from these data, since the A
concentrations are
expressed as monomeric peptide and thus overestimate the concentration
of A
fibrils. The average amyloid fibril is likely to be composed
of hundreds of monomers(29) .
(1-40) or A
(1-42) at pH 7.4. HSPG (panels A and C) and CSPG (panels B and D) were immobilized onto microtiter wells, and the binding of
non-fibrillar A
(
) and fibrillar A
(
) were
analyzed as described under ``Materials and Methods.'' Panels A and B show data obtained with
A
(1-40), whereas panels C and D demonstrate binding with A
(1-42). Representative
binding profiles are shown. When fibrillar A
binding data were
analyzed by Scatchard transformation (inset), a linear
relationship was obtained for all combinations of proteoglycan and
amyloid peptide, indicating binding governed by a single
affinity.
, both
fibrillar and non-fibrillar peptide preparations were examined for
their ability to bind CSPG over a broad pH range. As seen in Fig. 4, the binding of proteoglycan to A
fibrils (closed circles) is relatively constant from pH 5-7,
whereas at higher pH there is a decrease in CSPG-amyloid peptide
interaction. Half-maximal binding is seen at
pH 9, suggesting that
the protonation of 1 or more lysine residues is necessary for
proteoglycan binding to A
fibrils. In contrast, non-fibrillar
A
did not associate with CSPG until pH values were 6 or lower (Fig. 4, open circles). This implies that binding of
proteoglycan to the non-fibrillar peptide was governed, at least in
part, by protonation of histidine(s). These data show that the nature
of proteoglycan binding to the two forms of A
is significantly
different, and only the fibrillar amyloid peptide would appear to play
a role in proteoglycan binding at physiological pH.
(1-40) to CSPG as a function of pH. Fibrillar
A
(1-40) (
) and non-fibrillar A
(1-40)
(
) were examined for their ability to bind immobolized CSPG at
different pH values, as described under ``Materials and
Methods.'' The optical density values obtained in the binding
assay are shown for each pH value.
A
interaction, analyses were done in which HSPG and CSPG were added as
competitive inhibitors of fibrillar A
binding to the immobilized
proteoglycans (Fig. 5). The K
values obtained for solution-phase proteoglycan inhibition
of A
binding to substrate-attached HSPG and CSPG at pH 7.4 reveal
high-affinity interactions of these proteoglycans with both
A
(1-40) and A
(1-42) (see Table 1).
Examination of the inhibition data by log-logit transformation
suggested one class of non-interacting binding sites for both
proteoglycans with either amyloid peptide (Fig. 5, insets). Therefore, the inhibition data are in agreement with
the single affinity binding observed in the A
saturation analyses.
(1-40)
and A
(1-42) binding to substrate-bound proteoglycans by
soluble proteoglycans. HSPG (panels A and C) and
bovine CSPG (panels B and D) were immobilized onto
microtiter wells. Fixed amounts (1.5 µM) of fibrillar
A
(1-40) (panels A and B) or
A
(1-42) (panels C and D) were added with
varying concentrations of HSPG (panels A and C) or
CSPG (panels B and D). The amount of bound A
was
assayed as described under ``Materials and Methods.''
Plotting logit(percent bound) versus log(inhibitor) for each curve (insets) revealed
a linear relationship with a slope of
-2.3 (Hill coefficient
of
1), suggestive of a single affinity
interaction.
is
governed by a single affinity interaction, it was not known whether the
proteoglycan association was mediated via the core-protein,
glycosaminoglycan chains, or a combination of both. To examine this
question, free heparan sulfate and chondroitin sulfate
glycosaminoglycans were utilized as competitive inhibitors in the
A
binding assay. Both of these anionic polysaccharides were
effective inhibitors of amyloid peptide binding to HSPG and CSPG (Fig. 6), with their K
values
listed in Table 1. Again, log-logit transformation of these data (Fig. 6, insets) suggested single affinity,
non-interacting binding sites. The ability of free glycosaminoglycans
to completely inhibit amyloid peptide binding to proteoglycan would
seem to indicate that the intact glycoconjugates complex with fibrillar
A
via these sulfated sugar chains. The larger K
values obtained with the glycosaminoglycans relative to the
proteoglycans is likely to reflect the fact that there are many of
these sugar moieties per proteoglycan molecule. For example, the
proteoglycans bind A
(1-42) with affinities that are
approximately 25-75-fold greater than those determined for the
free glycosaminoglycans. However, the sugar content (by weight) per
mole of the proteoglycans is 25-100 times that of the
glycosaminoglycan chain preparations.
(1-40)
and A
(1-42) binding to substrate-bound proteoglycans by
glycosaminoglycans. HSPG (panels A and C) and bovine
CSPG (panels B and D) were immobilized onto
microtiter wells. Fixed amounts (1.5 µM) of fibrillar
A
(1-40) (panels A and B) or
A
(1-42) (panels C and D) were added with
varying concentrations of heparan sulfate (panels A and C) or chondroitin sulfate (panels B and D).
The amount of bound A
was assayed as described under
``Materials and Methods.'' Plotting logit(percent bound) versus log(inhibitor) for each curve (insets)
revealed a linear relationship with a slope of
-2.3 (Hill
coefficient of
1), suggestive of a single affinity
interaction.
being prevented by glycosaminoglycans of the
same class as those of the immobilized proteoglycan, we found that free
CS and HS polysaccharides could inhibit amyloid peptide binding to
HSPG and CSPG, respectively (data not shown). The ability of dissimilar
glycosaminoglycan chains to inhibit the binding of the same
proteoglycan indicates that the binding of these polysaccharides to
A
is relatively nonspecific with regard to sugar composition.
and APP
in the etiology of
AD(30, 31, 32, 33, 34) . In
AD, A
is assembled into polymeric crossed
-fibrils within
senile plaques, and studies (35, 36) have suggested
that fibrillar A
is directly neurotoxic. It should be noted,
however, that the in vitro models used to demonstrate
A
-induced neurotoxicity are unlikely to mimic the conditions of
the AD brain, and there are alternative explanations of the role of
A
in causing AD
neuropathology(19, 37, 38, 39) .
-fibrils are found
associated with
proteoglycans/glycosaminoglycans(13, 14, 15) .
In one study of peripheral amyloidosis(40) , the temporal
deposition of proteoglycans appeared to occur concurrently with the
formation of amyloid fibrils, suggesting that proteoglycans played an
active role in plaque formation.
(1-40)
and impart protection against proteolytic attack. An identical
protective effect is elicited by CSPG purified from rat astrocytes
(data not shown), indicating that this activity is not
species-specific. Enzymes with relatively nonspecific substrate
specificity (i.e. papain and Pronase) were employed in these
studies since the stability of proteoglycan
A
complexes to
such exhaustive proteolytic conditions would suggest similar resistance
to proteases found in the brain.
reflects a relative lack
of interaction with this form of peptide at physiological pH. Previous
work from this laboratory (18) indicated that
glycosaminoglycans only associated with the truncated
A
(1-28) peptide at pH values below 7. This pH profile
suggested that 1 or more histidine residues of A
(1-28) must
be protonated for glycosaminoglycan binding. The pH dependence of
proteoglycan binding to non-fibrillar A
shows a qualitatively
similar profile, implicating histidine in the binding of
glycosaminoglycans and proteoglycans to non-fibrillar A
.
to proteoglycans at pH 7.4 would imply,
however, an interaction that is not mediated through glycosaminoglycan
association with histidine(s). A significant reduction in proteoglycan
binding to fibrillar A
is observed as the pH increases above 9,
indicating 1 or more lysine residues are involved in the association.
The critical lysine(s) is likely to reside in the A
sequence and
not in the proteoglycan core protein since our data indicate that
binding of the glycoconjugates is via glycosaminoglycans.
would suggest that anionic groups of the former ion-pair with the
amyloid fibril. It would seem likely that the sulfated sugars of
glycosaminoglycans align with a positive-charge array formed from 1 or
more lysine residues from each monomeric subunit of the assembled
amyloid fibril. Both A
(1-40) and A
(1-42) fibrils
showed comparable affinities for proteoglycans and glycosaminoglycans,
suggesting that there were not significant differences in the
arrangement of the residues critical to binding between these two forms
of amyloid peptide. The binding of proteoglycan to the longer amyloid
peptide may be of the most physiological relevance, as recent data
suggest that A
(1-42) is found in larger amounts than
A
(1-40) within the senile plaques of AD brain(41) .
(1-28)
affinity column, leading these investigators to suggest that the core
protein was involved in binding of intact proteoglycan. These authors
also showed glycosaminoglycan-mediated binding of HSPG to A
. Since
saturation binding analyses were not performed, it is not known whether
they observed a single affinity interaction between intact HSPG and the
truncated amyloid peptide. Because we have investigated binding of
proteoglycans to full-length A
peptides, it is difficult to make
direct comparisons between this prior study and the data presented
here. It should be noted, however, that the deglycosylation of
proteoglycans could expose regions of the core protein that would
normally be sterically shielded by glycosaminoglycan groups, thereby
revealing novel A
-binding sites.
fibrils bind proteoglycans through interaction with
glycosaminoglycans, we cannot exclude the possibility that the small
amount of non-fibrillar A
binding to HSPG observed at pH 7.4 (see Fig. 3, A and C) is mediated, in part, by core
protein. No attempts were made to examine the nature of non-fibrillar
A
-HSPG association through the use of glycosaminoglycan
competition studies because of the very low signal obtained in the
binding assay.
via
glycosaminoglycan chains, these polysaccharide groups alone are not
sufficient to inhibit proteolytic accessibility to the amyloid fibrils.
The binding of one or a few glycosaminoglycan groups of a proteoglycan
may allow the other sulfated polysaccharide moieties of that molecule
to form an effective barrier around the A
fibril. Such a shield
may not be formed when free glycosaminoglycan chains bind the amyloid
peptide.
has important implications with regard to AD pathology. The
association of HSPG with A
may be one of the precipitating events
in the formation of stable plaque structures. HSPG has been shown to be
present in cortical diffuse plaques(11) , and there is belief
that these structures are predecessors of senile plaques.
Interestingly, diffuse plaques of the cerebellum lack
HSPG(42) , and cerebellar dense plaques with associated
neuropathology are rare. Thus, HSPG may bind the initial few A
fibrils that form in diffuse deposits, and in doing so begin the
transformation to senile deposits. The localization of CSPG and DSPG
with senile plaques, and not diffuse deposits, suggests that these
proteoglycans may associate with fibrillar A
later in the
maturation process. Another notable difference between diffuse and
senile neuritic plaques is the clear presence of activated glia within
and around the latter(43, 44, 45) . It is
possible that glia are responsible for the release of all or some of
the proteoglycans that associate with senile plaques. CSPG are known to
be inhibitory to axon growth(46) , and their association with
plaques may cause some of the neuritic dystrophy characteristic of
AD(37) .
suggests that drugs developed to
inhibit the binding of these glycoconjugates to amyloid peptide might
increase the turnover of A
by naturally occurring proteases,
thereby providing therapeutic benefit to those suffering the
consequences of this devastating disease.
, beta
amyloid peptide; APP, amyloid precursor protein; CSPG, chondroitin
sulfate proteoglycan; DSPG, dermatan sulfate proteoglycan; HSPG,
heparan sulfate proteoglycan; PBS, phosphate-buffered saline; PAGE,
polyacrylamide gel electrophoresis; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.
We thank Drs. Stephen Yates and Clark Tedford for
their helpful discussions.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.