Amyloid-beta Interactions with Chondroitin Sulfate-derived Monosaccharides and Disaccharides

IMPLICATIONS FOR DRUG DEVELOPMENT*

Paul E. FraserDagger §, Audrey A. DarabieDagger , and JoAnne McLaurinDagger ||**

From the Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

In Alzheimer's disease, the major pathological features are diffuse and senile plaques that are primarily composed of the amyloid-beta (Abeta ) 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 Abeta -GAG interactions, understanding the Abeta 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 Abeta that are associated with GAG interactions. Our results demonstrate that the disaccharides GalNAc-4-sulfate(4S), Delta UA-GalNAc-6-sulfate(6S), and Delta 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 Delta UA-GalNAc, induce the fibrillar features of Abeta -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 Abeta binding, as illustrated by competitive inhibition ELISAs. Therefore, the development of therapeutics based on the model of Abeta -chondroitin sulfate binding may lead to effective inhibitors of the GAG-induced amyloid formation that is observed in vitro.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

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-beta (Abeta ).1 Abeta 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 Abeta . Two mechanisms have been proposed for the nucleation of Abeta fibrils. The first involves the self-assembly of Abeta 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-Abeta seeds (1).

Many proteins are associated with amyloid plaques; their presence may result in heterogeneous nucleation of Abeta (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). Abeta -proteoglycan interactions are mediated predominantly through Abeta -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 beta -sheet conformation thereby increasing the number of nucleation seeds, as demonstrated by a virtually instantaneous structural transition in Abeta 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 Abeta -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 Abeta 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 Abeta interactions. Therefore, these GAGs were used as the prototype to determine Abeta binding and potentially to develop compounds that could compete with all identified proteoglycans associated with plaques.

The GAG binding site on the Abeta peptide has been investigated using amino acid substitution of the Abeta 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 Abeta folding and aggregation (22). Furthermore, GAG-Abeta interactions are more sensitive to the conformation and aggregation state of Abeta rather than the primary sequence (22, 23). Together these results suggested that inhibition of Abeta -GAG interactions through targeting of the GAG binding site on Abeta may not provide a viable therapeutic. Alternatively, the Abeta -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 Abeta (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 Abeta precipitation into nontoxic plaques and thereby decreasing the presence of toxic Abeta species. To develop more specific therapeutics directed toward Abeta fibrillogenesis, we determined the minimum GAG unit necessary for Abeta binding, fibrillogenesis, and lateral aggregation. Here, we examine the interaction of chondroitin sulfate-derived monosaccharides and disaccharides with Abeta 40 and Abeta 42. Fluorescence spectroscopy and electron microscopy demonstrate a potent effect of both monosaccharides and disaccharides on the formation and structure of Abeta fibrils. In addition, competitive inhibition ELISAs demonstrate that the binding of both monosaccharides and disaccharides to Abeta inhibits interaction with the polymeric chondroitin sulfate and heparan sulfate GAGs.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Abeta Peptides-- Abeta 40 and Abeta 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.

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 Delta UA-GlcNAc, Delta UA-GalNAc, Delta UA-GalNAc-4S, Delta UA-GalNAc-6S, and Delta UA-GalNAc-4S,6S were purchased from Dextra Laboratories (Reading, United Kingdom), and Delta UA-2S-GalNAc, Delta UA-2S-GalNAc-4S, Delta UA-2S-GalNAc-6S, and Delta 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.

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 Abeta 40 or Abeta 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.

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 Abeta in the presence and absence of GAGs (29, 30) and GAG-derived disaccharides and monosaccharides was used to evaluate the similarity between Abeta -GAG fibrils and classical amyloid fibers. Samples were incubated at a 1:1 ratio by weight with a final Abeta 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.

Competitive Inhibition ELISAs-- Nunc Immunosorp plates were coated with 100 µl of GAGs (5 µg/ml) and incubated overnight, 4 °C. Simultaneously, Abeta 40 and Abeta 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. Abeta 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 Abeta , 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
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES

Induction and Morphology of Abeta 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 beta -structural transition in Abeta 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 Abeta 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.

To investigate the effect of chondroitin sulfate-derived saccharides on Abeta nucleation, the intrinsic tyrosine fluorescence of Abeta 40 and Abeta 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 Abeta 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 Abeta 42 (Fig. 2) over Abeta 40 (data not shown). At 24 h, the disaccharides Delta UA-GalNAc-4S, Delta UA-GalNAc-6S, and Delta UA-GalNAc-4S,6S significantly increased the amount of aggregated Abeta 40/42 with Delta UA-GalNAc-4S,6S being the most effective (Fig. 2C). It is interesting to note that all disaccharides and in particular Delta 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 Abeta .



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Fig. 2.   Tyrosine fluorescence was used to determine the extent of Abeta 40 and Abeta 42 aggregation in the presence of chondroitin sulfate-derived monosaccharides, disaccharides, and glycosaminoglycans. Abeta 42 was incubated with the various GAGs at a 1:1 ratio (weight/weight) for 24 h. Abeta aggregation was determined using the ratio of tyrosine fluorescence before and after centrifugation. The extent of Abeta aggregation when incubated alone (A) and in the presence of D-GalNAC-4S,6S (B) was minimal, whereas in the presence of Delta UA-GalNAc-4S,6S (C) and dermatan sulfate (D) virtually all Abeta tyrosine fluorescence was lost due to peptide precipitation.

The characteristics of Abeta 40 and Abeta 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 Abeta 40 and Abeta 42 (18). Unseeded samples of both Abeta 40 and Abeta 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 Abeta 42 fibrils were 50-70 Å in diameter with an average length of 750 Å (Fig. 3A). These were indistinguishable from those of Abeta 42 in the presence of the desulfated Delta UA-GalNAc (Fig. 3B) or Delta UA-GlcNAc (data not shown). The monosulfated disaccharides, Delta UA-GalNAc-6S and Delta 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 Delta UA-GalNAc-4S,6S, Abeta 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 Abeta 42 in the presence of chondroitin sulfate-derived disaccharides. Abeta 42 incubated in buffer alone (A) demonstrates many long fibers. When incubated in the presence of Delta 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 Delta UA-GalNAc-4S (C) and Delta UA-GalNAc-4S,6S (D). In the presence of sulfated iduronic acid disaccharides Delta UA-2S-GalNAc-4S (E) and Delta UA-2S-GalNAc-4S,6S (F) demonstrate fibers that are similar to the unsulfated iduronic acid derivatives. Scale bar is 50 nm.

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 Abeta binding (28, 31). Therefore, the introduction of a sulfate group onto the iduronic acid may present a more favorable or deleterious surface for Abeta 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 Delta UA-2S-GalNAc had no effect on the Abeta 42 fibers formed in comparison to Abeta 42 alone or in the presence of Delta UA-GalNAc (data not shown). In the presence of Delta UA-2S-GalNAc-4S and Delta UA-2S-GalNAc-6S, many short fibers could be detected displaying limited aggregation. (Fig. 3E). Incubation of Abeta 42 with Delta 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 Abeta 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 Abeta binding and enhanced fibrillogenesis.

GAGs also laterally aggregate preformed Abeta 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 Abeta 40 fibrils and examined the morphology by negative stain electron microscopy. Preincubated Abeta 40 forms long fibers that are nonaggregated (Fig. 4A). In contrast, dermatan sulfate induced a consistent lateral aggregation of Abeta 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, Abeta 40 lateral aggregation was similar to that of an extended GAG polymer, with the disulfated Delta 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 Abeta 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 Abeta . Furthermore, these small GAG-derived saccharides demonstrate the potential use of these molecules to decrease the soluble pool of Abeta in situ by enhancing the precipitation of nontoxic fibers.



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Fig. 4.   Negative stain electron microscopy of seeded Abeta 40 was examined in the presence and absence of chondroitin sulfate GAGs. Abeta 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 Delta UA-GalNAc-4S,6S, Abeta 40 fibrils were organized into thick bundles (C, arrows delineate bundles) characteristic of the intact dermatan sulfate. Scale bar is 50 nm.

Effect of Chondroitin Sulfate-derived Monosaccharides on Abeta Fibrillogenesis-- The effect of chondroitin sulfate-derived monosaccharides on the induction of Abeta 42 fibrillogenesis was investigated. To investigate the effect of chondroitin sulfate-derived saccharides on Abeta nucleation, the intrinsic tyrosine fluorescence of Abeta 40 and Abeta 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 Abeta 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 Abeta 42 pelleted in the presence of D-GalNAc-4S,6S could be detected (Fig. 2B).

The morphology of Abeta fibrils in the presence of chondroitin sulfate-derived monosaccharides was investigated using negative stain electron microscopy. Similar to Abeta 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, Abeta 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 Abeta 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 Abeta 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 Abeta 40 and Abeta 42 preformed fibrils by all monosaccharides (data not shown).



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Fig. 5.   Negative stain electron microscopy of Abeta 42 in the presence of chondroitin sulfate-derived monosaccharides. Abeta 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 Abeta 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 Abeta 42 were detected (D). Scale bar is 50 nm.

Thioflavin T specifically stains amyloid deposits in vivo and has been shown to bind both Abeta fibers and aggregates in vitro (30). We investigated the binding of Thioflavin T to chondroitin sulfate-derived saccharide-Abeta complexes to further characterize the nature of these fibers. Thioflavin T fluorescence intensity increased for both Abeta 40 and Abeta 42 in the presence of intact chondroitin sulfate GAGs (Fig. 6). In the presence of D-GalNAc, the Thioflavin T fluorescence was indistinguishable from Abeta 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, Abeta 42 demonstrated a morphology similar to protofibrils and exhibited a Thioflavin T fluorescence greater than Abeta 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 Abeta and induce fibrillogenesis without lateral aggregation.



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Fig. 6.   Thioflavin T assay of chondroitin sulfate-Abeta fibrils. Thioflavin T binding to chondroitin sulfate-Abeta complexes resulted in both an increase in fluorescence and a shift in the emission spectra. Thioflavin T fluorescence as a result of Abeta 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 Abeta 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 Abeta 42 resulted in the most intense fluorescence indicating that it is qualitatively more effective at enhancing Abeta fibrillogenesis. Values for Thioflavin T fluorescence of Abeta 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.

Characterization of the Abeta -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 Abeta 40 and Abeta 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 Abeta 40 and Abeta 42 before incubation with chondroitin-4-sulfate, chondroitin-6-sulfate, or dermatan sulfate. The amount of Abeta bound to the intact GAG chain on the microtiter plate was determined using the anti-Abeta 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 Delta UA-GalNAc were unable to compete with the chondroitin sulfate GAGs for Abeta binding, which is in agreement with our electron microscopy data in which Abeta 40/42 fibrils were indistinguishable in the presence and absence of D-GalNAc and Delta 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 Abeta 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 Abeta 40 and Abeta 42. These observations further suggest that the monosaccharides D-galacturonic acid, D-GalNAc-6S, and D-GalNAc-4S,6S are sufficient to induce Abeta binding and structural transitions associated with Abeta -GAG interactions. It was not surprising to find that D-GalNAc-4S competed poorly with all GAGs for Abeta 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).


                              
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Table I
Competitive inhibition ELISAs for Abeta 42 binding
Abeta 42 was pre-incubated with the chondroitin sulfate-derived monosaccharides or disaccharides before co-incubation with intact chondroitin sulfate GAGs. The amount of Abeta 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 Abeta 42. Values are reported as mean ± S.D. for at least three separate ELISA assays. Paired t test indicates p < 0.05.

The chondroitin sulfate-derived disaccharides had variable abilities to compete for Abeta binding with Delta 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 Abeta , 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 Abeta that allowed the intact GAG chain to elicit binding between Abeta 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 Abeta 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).

Chondroitin Sulfate-derived Saccharides Inhibit Heparan Sulfate GAG Binding to Abeta -- 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 Abeta 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 Abeta (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 Abeta -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 Abeta could represent an agent to block Abeta -proteoglycan interactions.


                              
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Table II
Competitive inhibition ELISAs between D-GalNAc-4S,6S and heparin for Abeta binding
Abeta was incubated in the presence of D-GalNAc-4S,6S before competition for heparin binding. The amount of Abeta 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.

Further investigation into the ability of chondroitin sulfate-derived saccharides to inhibit heparan sulfate binding to Abeta demonstrated similar results to those of both chondroitin and dermatan sulfate (Table III). None of the nonsulfated monosaccharides or disaccharides could inhibit Abeta 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 Delta UA-2S-GalNAc-4S and Delta 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 Delta UA-GalNAc-4S, Delta UA-GalNAc-6S, and Delta UA-GalNAc-4S,6S all competed with heparan sulfate for Abeta 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 Abeta binding to all GAGs present in the central nervous system.


                              
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Table III
Competitive inhibition ELISAs for Abeta 42 binding
Abeta 42 was pre-incubated with the chondroitin sulfate-derived monosaccharides or disaccharides before co-incubation with intact GAGs. The amount of Abeta 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 Abeta 42 binding alone.

Cumulatively, our results demonstrate that chondroitin sulfate-derived monosaccharides represent the minimal GAG subunit required for Abeta binding and that lateral aggregation between Abeta 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 Abeta . Development of drugs based on these monosaccharide compounds will have to take into account the potential for stabilization of toxic Abeta intermediates. We have previously shown that Abeta 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 Abeta fibers. These studies further emphasize the importance of investigations into the design of GAG memetics as potential amyloid therapeutics.


    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.


    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


    ABBREVIATIONS

The abbreviations used are: Abeta , amyloid-beta peptide; GAG, glycosaminoglycan; ELISA, enzyme-linked immunosorbent assay.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
REFERENCES


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