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Heparin-binding Properties of the Amyloidogenic Peptides Aβ and Amylin

1997; Elsevier BV; Volume: 272; Issue: 50 Linguagem: Inglês

10.1074/jbc.272.50.31617

1083-351X

Deborah Watson, Arthur D. Lander, Dennis J. Selkoe,

Amyloidosis: Diagnosis, Treatment, Outcomes

Aggregation and deposition of the 40–42-residue amyloid β-protein (Aβ) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Aβ plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Aβ and other amyloid deposits at all stages. However, the nature of the Aβ-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Aβ to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Aβ and the glycosaminoglycan heparin and found that the aggregation state of Aβ governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Aβ. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The “Dutch” mutant AβE22Q peptide formed fibrils more readily than wild type Aβ and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AβE22Q was reversible with incubation in a solvent that promotes α-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Aβ-heparin interaction on the conformation and aggregation state of Aβ rather than primary sequence alone, and suggest methods of interfering with this association. Aggregation and deposition of the 40–42-residue amyloid β-protein (Aβ) are early and necessary neuropathological events in Alzheimer's disease. An understanding of the molecular interactions that trigger these events is important for therapeutic strategies aimed at blocking Aβ plaque formation at the earliest stages. Heparan sulfate proteoglycans may play a fundamental role since they are invariably associated with Aβ and other amyloid deposits at all stages. However, the nature of the Aβ-heparan sulfate proteoglycan binding has been difficult to elucidate because of the strong tendency of Aβ to self-aggregate. Affinity co-electrophoresis can measure the binding of proteoglycans or glycosaminoglycans to proteins without altering the physical state of the protein during the assay. We used affinity co-electrophoresis to study the interaction between Aβ and the glycosaminoglycan heparin and found that the aggregation state of Aβ governs its heparin-binding properties: heparin binds to fibrillar but not nonfibrillar Aβ. The amyloid binding dye, Congo red, inhibited the interaction in a specific and dose-dependent manner. The “Dutch” mutant AβE22Q peptide formed fibrils more readily than wild type Aβ and it also attained a heparin-binding state more readily, but, once formed, mutant and wild type fibrils bound heparin with similar affinities. The heparin-binding ability of aggregated AβE22Q was reversible with incubation in a solvent that promotes α-helical conformation, further suggesting that conformation of the peptide is important. Studies with another human amyloidogenic protein, amylin, suggested that its heparin-binding properties were also dependent on aggregation state. These results demonstrate the dependence of the Aβ-heparin interaction on the conformation and aggregation state of Aβ rather than primary sequence alone, and suggest methods of interfering with this association. A hallmark of both Alzheimer's disease (AD) 1The abbreviations used are: AD, Alzheimer's disease; Aβ, amyloid β-peptide; HSPG, heparan sulfate proteoglycan; ACE, affinity co-electrophoresis; HCHWA-D, Hereditary Cerebral Hemorrhage with Amyloidosis, Dutch-type; HPLC, high performance liquid chromatography; CSF, cerebrospinal fluid; NaMOPSO, 3-[N-morpholino]-2-hydroxypropanesulfonic acid, sodium salt; LMW, low molecular weight; HFIP, (1,1,1,3,3,3)-hexafluoroisopropanol. and Down's syndrome is the presence of numerous extracellular deposits of the amyloid β-protein (Aβ), termed senile or neuritic plaques, in the brain parenchyma. In most cases, Aβ is also deposited in the walls of parenchymal and meningeal blood vessels. In these two types of deposits, Aβ exists largely in a fibrillar form consisting of 40 or 42 amino acid monomers aggregated into insoluble filamentous polymers. Aβ, which is derived by endoproteolysis from the β-amyloid precursor protein (1Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Greschik K.-H. Multhaup G. Beyreuther K. Müller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3968) Google Scholar, 2Haass C. Schlossmacher M.G. Hung A.Y. Vigo-Pelfrey C. Mellon A. Ostaszewski B.L. Lieberburg I. Koo E.H. Schenk D. Teplow D.B. Selkoe D.J. Nature. 1992; 359: 322-325Crossref PubMed Scopus (1767) Google Scholar), is by far the major constituent of plaques (3Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3699) Google Scholar, 4Selkoe D.J. Abraham C.R. Podlisny M.B. 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Acta Neuropathol. 1982; 57: 239-242Crossref PubMed Scopus (387) Google Scholar). The order in which these various proteinaceous components are added to the senile plaques is not well understood, but some clues can be obtained from the composition of another type of Aβ deposit found in AD brains. “Diffuse” plaques are composed of Aβ in a particulate but not fibrillar form and do not react with the classic amyloid-staining dyes, Congo red and thioflavin S. Because the brains of young ( 30–40 year old) Down's syndrome subjects (11Mann D.M.A. Brown A. Prinja D. Davies C.A. Landon M. Masters C.L. Beyreuther K. Neuropathol. Appl. Neurobiol. 1989; 15: 317-329Crossref PubMed Scopus (64) Google Scholar, 12Lemere C.A. Blusztajn J.K. Yamaguchi H. Wisniewski T. Saido T.C. Selkoe D.J. Neurobiol. Dis. 1996; 3: 16-32Crossref PubMed Scopus (467) Google Scholar). Snow and colleagues (8Snow A.D. Mar H. Hochlin D. Kimata K. Kato M. Suzuki S. Hassell J. Wight T.N. Am. J. Pathol. 1988; 133: 456-463PubMed Google Scholar, 13Snow A.D. Mar H. Nochlin D. Sekiguchi R.T. Kimata K. Koike Y. Wight T.N. Am. J. Pathol. 1990; 137: 1253-1270PubMed Google Scholar) have detected immunohistochemically the presence of a specific HSPG, perlecan, in the compacted plaques and cerebrovascular amyloid of Alzheimer's disease brain. In addition, diffuse plaques in the hippocampus and cerebral cortex, but not in the cerebellum, were shown to contain perlecan (13Snow A.D. Mar H. Nochlin D. Sekiguchi R.T. Kimata K. Koike Y. Wight T.N. Am. J. Pathol. 1990; 137: 1253-1270PubMed Google Scholar, 14Snow A.D. Sekiguchi R.T. Nochlin D. Kalaria R.N. Kimata K. Am. J. Pathol. 1994; 144: 337-347PubMed Google Scholar). Because compacted plaques are rarely found in the cerebellum even in end stage AD brains (15Joachim C.L. Morris J.H. Selkoe D.J. Am. J. Pathol. 1989; 135: 309-319PubMed Google Scholar), it is postulated that HSPGs such as perlecan could play a role in the transition of diffuse Aβ deposits into compacted amyloid. The finding that cortical diffuse plaques in Down's syndrome brain are also perlecan-immunoreactive (13Snow A.D. Mar H. Nochlin D. Sekiguchi R.T. Kimata K. Koike Y. Wight T.N. Am. J. Pathol. 1990; 137: 1253-1270PubMed Google Scholar) is consistent with this hypothesis. HSPGs may play a general role in the formation and stabilization of many types of amyloid, since they have also been identified in association with amyloid deposits in virtually all other human amyloid diseases (for review, see Ref. 16Snow A.D. Wight T.N. Neurobiol. Aging. 1989; 10: 481-497Crossref PubMed Scopus (188) Google Scholar). In AD cerebrovasculature, Aβ amyloid deposits have been ultrastructurally localized to the vascular basement membrane region of capillaries, arterioles, and small arteries (17Yamaguchi H. Yamazaki T. Lemere C.A. Frosch M.P. Selkoe D.J. Am. J. Pathol. 1992; 141: 249-259PubMed Google Scholar), where perlecan is a prominent constituent. In addition, one of the diseases linked genetically to β-amyloid precursor protein (Hereditary Cerebral Hemorrhage with Amyloidosis, Dutch-type (HCHWA-D)) causes a particularly severe deposition of Aβ in meningocerebral blood vessels (18Luyendijk W. Bots G.T.A.M. Vegter-van der Vlis M. Went L.N. Frangione B. J. Neurol. Sci. 1988; 85: 267-280Abstract Full Text PDF PubMed Scopus (97) Google Scholar, 19Levy E. Carman M.D. Fernandez-Madrid I.J. Power M.D. Lieberburg I. van Duinen S.G. Bots G.T.A.M. Luyendijk W. Frangione B. Science. 1990; 248: 1124-1126Crossref PubMed Scopus (1167) Google Scholar), again suggesting an important role for a vascular basement membrane factor in Aβ deposition. Previous attempts to characterize the binding between HSPGs and Aβ (20Brunden K.R. Richter-Cook N.J. Chaturvedi N. Frederickson R.C.A. J. Neurochem. 1993; 61: 2147-2154Crossref PubMed Scopus (121) Google Scholar, 21Buée L. Ding W. Delacourte A. Fillit H. Brain Res. 1993; 601: 154-163Crossref PubMed Scopus (76) Google Scholar, 22Buée L. Ding W. Anderson J.P. Narindrasorasak S. Kisilevsky R. Boyle N.J. Robakis N.K. Delacourte A. Greenberg B. Fillit H.M. Brain Res. 1993; 627: 199-204Crossref PubMed Scopus (67) Google Scholar, 23Snow A.D. Kinsella M.G. Parks E. Sekiguchi R.T. Miller J.D. Kimata K. Wight T.N. Arch. Biochem. Biophys. 1995; 320: 84-95Crossref PubMed Scopus (126) Google Scholar) have been hindered by the unique difficulties of working with synthetic Aβ, a highly hydrophobic 40–42-amino acid peptide that readily precipitates into insoluble aggregates in vitro. Affinity co-electrophoresis (ACE) is an advantageous method to characterize the binding of proteins to proteoglycans or their glycosaminoglycan side chains (24Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar, 25Herndon M.E. Lander A.D. Jackson P. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997Google Scholar). Each component is freely mobile within a highly porous native agarose gel, and no coupling of either component to any matrix, resin, or solid support is required, so all of the potential binding surfaces remain available. Importantly, Aβ has less opportunity to aggregate during the experiment than in many other types of binding assays. In this paper, we use ACE to characterize the binding of Aβ peptides to heparin (a glycosaminoglycan that is frequently used as a model for tissue heparan sulfates) at physiological pH and ionic strength. Both wild type Aβ and the HCHWA-D disease-causing mutant form were studied. We show that the heparin-Aβ interaction is critically dependent on the secondary structure and aggregation state of Aβ and is potently inhibited by the amyloid-binding dye Congo red. Aβ containing the HCHWA-D mutation binds heparin more readily than wild type peptide due to its increased tendency to form fibrils, not because of a greater affinity for heparin. Finally, we demonstrate that another amyloid-forming subunit, human amylin, also binds to heparin, whereas the nontoxic and non-amyloidogenic rat isoform of amylin does not. Wild type Aβ1–40 peptide was synthesized and HPLC-purified by Dr. D. Teplow (Biopolymer Laboratory, Brigham and Women's Hospital). Aβ1–40 containing the E22Q “Dutch” mutation (AβE22Q) was made by Dr. D. Chin (University of Missouri, Columbia) and aliquots were HPLC-purified by Dr. D. Walsh (Biopolymer Facility, Brigham and Women's Hospital). Human and rat amylin peptides were purchased from Peninsula Laboratories (Belmont, CA) or Bachem (Torrance, CA). The amino acid sequence of wild type human Aβ1–40 is DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV (see,e.g. Ref. 1Kang J. Lemaire H.-G. Unterbeck A. Salbaum J.M. Masters C.L. Greschik K.-H. Multhaup G. Beyreuther K. Müller-Hill B. Nature. 1987; 325: 733-736Crossref PubMed Scopus (3968) Google Scholar); in mutant AβE22Q, Glu22 (underlined) is changed to Gln (19Levy E. Carman M.D. Fernandez-Madrid I.J. Power M.D. Lieberburg I. van Duinen S.G. Bots G.T.A.M. Luyendijk W. Frangione B. Science. 1990; 248: 1124-1126Crossref PubMed Scopus (1167) Google Scholar). For amylin and nonfibrillar Aβ preparations, lyophilized peptides were freshly resuspended in water (initially at 1 mm) and used immediately. Alternatively, fibrillar Aβ was formed by resuspending the lyophilized peptide in water to 1 mm, then adding artificial cerebrospinal fluid (CSF; 150 mm NaCl, 3 mm KCl, 1.7 mm CaCl2, 0.9 mm MgCl2 in 1.5 mm phosphate buffer, pH 6.5) to achieve a final concentration of 100 μm Aβ peptide and rocking it for 2 days at room temperature, followed by centrifugation at 16,000 × gfor 15 min. The pellet was resuspended in ACE electrophoresis running buffer (50 mm NaMOPSO in 125 mm acetate buffer, pH 7.0; Ref. 24Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar) and sonicated before use. A similar protocol using an ultrafiltrate of CSF showed Aβ1–40 fibril formation after 48 h at room temperature (26Wisniewski T. Castaño E. Ghiso J. Frangione B. Ann. Neurol. 1993; 34: 631-633Crossref PubMed Scopus (52) Google Scholar). Duplicate aliquots of each peptide preparation were collected and the peptide concentrations were determined by amino acid analysis, performed by M. Condron (Brigham and Women's Hospital Biopolymer Laboratory). Simultaneously, aliquots of each peptide preparation were applied to a 200-mesh copper-Formvar/carbon grid (EM Sciences, Gibbstown, NJ), negatively stained with 2% uranyl acetate, and observed with a JEOL JEM 100CX-II electron microscope at 60 kV. Congo red birefringence was determined by the protocol of Castañoet al. (27Castaño E.M. Prelli F. Wisniewski T. Golabek A. Kumar R.A. Soto C. Frangione B. Biochem. J. 1995; 306: 599-604Crossref PubMed Scopus (215) Google Scholar). Tyramine was added to the reducing ends of unbleached heparin molecules (Grade I, porcine intestinal mucosa, Sigma) via a reductive amidation reaction (28San Antonio J.D. Slover J. Lawler J. Karnovsky M.J. Lander A.D. Biochemistry. 1993; 32: 4746-4755Crossref PubMed Scopus (81) Google Scholar). This method adds ≤1 tyramine per heparin molecule and does not disturb the chemistry of the heparin along its chain length. The tyramine moiety was iodinated by the IODOGEN method (Pierce), and the heparin was then size-fractionated by gel filtration. The low molecular weight (LMW) fraction (≤ 6 kDa) was used to minimize the number of potential binding sites per heparin molecule. Unlabeled LMW-heparin was from Sigma. Horizontal 1% (w/v) agarose gels were cast with two combs essentially as described (24Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar, 25Herndon M.E. Lander A.D. Jackson P. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997Google Scholar), producing a set of nine rectangular wells and a perpendicularly oriented slot (Fig. 1). Serial dilutions of each peptide preparation were prepared in ACE electrophoresis running buffer (see above) and mixed with an equal volume of molten 2% agarose, cast into the nine wells, and allowed to set. The slot was loaded with125I-LMW-heparin, which was subjected to electrophoresis through the protein-containing zones. After drying the gels, the positions of bands were measured using a PhosphorImager 400A and ImageQuant software (Molecular Dynamics). Staining the gels with Coomassie Brilliant Blue showed that Aβ itself did not noticeably migrate from its original position during the electrophoresis times used (not shown). From positions of labeled bands, a retardation coefficient,R, was calculated (R = (M o − M)/M o, where M o is the mobility of free heparin andM is heparin's observed mobility through a protein (i.e. Aβ)-containing zone; Ref. 24Lee M.K. Lander A.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 2768-2772Crossref PubMed Scopus (155) Google Scholar). Values were fit to the equation R = R ∞/(1 + (K d /[protein]) n ) (25Herndon M.E. Lander A.D. Jackson P. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997Google Scholar). In general, better fits were obtained with n = 2, suggesting either positive cooperativity in binding, or perhaps the fact that some Aβ fibrils are lost on the walls of tubes and pipette tips in the process of making serial dilutions, causing Aβ concentrations to be somewhat overestimated at the highest dilutions. Protein concentrations are given in units of molarity of monomer, and were determined by amino acid analysis. Values for K d were calculated using these units of molarity; a calculation based on the molarity of polymerized fibrils would yield a much lower K d (see “Discussion”). Preparations of fibrillar or nonfibrillar Aβ were incubated for 1 h at room temperature with125I-LMW-heparin in ACE electrophoresis buffer. In some cases, serial dilutions of Congo red or of unlabeled LMW-heparin were included; these solutions were made fresh for each experiment. The mixtures were then transferred to nitrocellulose by vacuum filtration through a dot-blot apparatus, followed by one rinse with phosphate-buffered saline. The filter was dried and the bound radioactivity retained on the filter was counted directly in a γ counter (Cobra II AutoGamma, Packard Instruments). Background radioactivity in control wells containing 125I-LMW-heparin tracer alone were subtracted from the values for Aβ-containing wells before analysis. Values from six control wells containing only Aβ and heparin tracer were averaged to provide a value for maximum Aβ-heparin binding. ACE experiments to measure the binding of Aβ to heparin were initially attempted using aliquots of lyophilized wild type Aβ1–40that were freshly resuspended in water, diluted, and assayed immediately. Samples were incorporated into the nine lanes of a 1% agarose gel, and 125I-LMW-heparin was subjected to electrophoresis through those lanes (Fig.1 A). At neutral pH, the mobility of Aβ is much less than that of heparin (data not shown), so we expected a complex of heparin and Aβ to have a mobility significantly less than that of free heparin. Assuming this is the case, any binding of Aβ to heparin should have been revealed as a series of electrophoretic bands that were progressively retarded with increasing Aβ concentration (Fig. 1 B). In contrast, no effect of Aβ on heparin mobility was seen at peptide concentrations up to 243 μm (Fig.2 A). These data indicate that either Aβ and heparin do not bind under the conditions of the assay, or that binding could not be detected because the mobility of the Aβ-heparin complex is not sufficiently different from that of heparin alone. To test the latter possibility, we reversed the roles of heparin and peptide in the experiment (25Herndon M.E. Lander A.D. Jackson P. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997Google Scholar). In this case, heparin was cast into the nine parallel lanes, and125I-Aβ introduced into a single slot perpendicular to the lanes. Because heparin is more mobile than Aβ, the peptide-containing slot was placed between the heparin lanes and the anode (Fig. 1 C). In this experiment, any binding of heparin to Aβ should have been revealed as a series of electrophoretic bands whose mobility was progressively increased by increasing heparin concentrations. However, as shown in Fig. 2 B, no change in the mobility of 125I-Aβ was seen at heparin concentrations up to 10 mg/ml (>1.5 mm). As before, the only way we could have failed to detect actual binding of heparin and Aβ in this experiment is if the mobility of the heparin-peptide complex was not significantly different from that of the labeled species, in this case Aβ. However, it is not possible for the mobility of the heparin-Aβ complex to be indistinguishable from both that of free Aβ and that of free heparin, as Aβ and heparin have mobilities that are very different from each other. Thus, the negative results in Fig. 2, A and B, together indicate that freshly resuspended Aβ and heparin do not bind each other (K d ≥ 0.25 mm). Because the close association of heparan sulfate with Aβ deposits in AD brain still suggested an important interaction between the two molecules, we next considered the possibility that the secondary structure of polymerized fibrillar Aβ creates a heparin-binding epitope. To aggregate Aβ into fibrils, aliquots of lyophilized Aβ1–40 were resuspended initially in water and then diluted into artificial CSF and rocked for 48 h at room temperature. Electron microscopy of the pelleted precipitate confirmed the formation of straight, unbranched filaments approximately 10 nm in diameter (Fig. 2 D), similar to the structure and dimensions of Aβ fibrils purified from Alzheimer's plaques (29Narang H.K. J. Neuropathol. Exp. Neurol. 1980; 39: 621-631Crossref PubMed Scopus (39) Google Scholar, 30Merz P.A. Wisniewski H.M. Somerville R.A. Bobin S.A. Masters C.L. Iqbal K. Acta Neuropathol. 1983; 60: 113-124Crossref PubMed Scopus (130) Google Scholar). Furthermore, after staining with Congo red, Aβ fibrils showed birefringence under polarized light (not shown). In contrast, electron microscopy of the freshly resuspended nonfibrillar Aβ preparation demonstrated large amorphous clumps (Fig. 2 C), indicating the presence of aggregated but not fibrillar Aβ. Upon reaction with Congo red, the nonfibrillar Aβ was not birefringent (not shown). Increasing concentrations of the Aβ fibrils were then loaded into the lanes of an ACE gel and tested for heparin binding. In sharp contrast to what was observed with the freshly resuspended peptide, low micromolar concentrations of fibrillar Aβ completely retarded the mobility of the heparin tracer (Fig. 2 E). To confirm that the retardation was due to a specific binding interaction and not to nonspecific physical blockage of the heparin by a dense network of fibrils, we demonstrated that the binding of125I-LMW-heparin to Aβ fibrils could be completely competed away by excess unlabeled LMW-heparin (Fig. 2 F). Several independently aggregated fibril preparations showed electrophoretic retardation profiles very similar to that shown in Fig.2 E. To measure binding affinity, we calculated retardation coefficients, R, from each of several ACE gels. The results from five experiments were fit to the equation R =R ∞/(1 + (K d /[protein])2) (25Herndon M.E. Lander A.D. Jackson P. Gallagher J.T. A Laboratory Guide to Glycoconjugate Analysis. Birkhauser Verlag AG, Basel, Switzerland1997Google Scholar, 31Lim W.A. Sauer R.T. Lander A.D. Methods Enzymol. 1991; 208: 196-210Crossref PubMed Scopus (58) Google Scholar), which yielded an average K d of 1.31 ± 0.10 μm (Fig. 3; this value is expressed in units of molarity of Aβ monomer (see “Experimental Procedures”)). As an independent test of the results obtained by ACE, a solution-phase binding assay was also used. Increasing concentrations of fibrillar or nonfibrillar Aβ were incubated with 125I-LMW-heparin in solution, after which the mixtures were applied to a nitrocellulose membrane by vacuum filtration and counted to measure the retained (protein-bound) heparin. Again, fibrillar but not non-fibrillar Aβ bound heparin with a low micromolar affinity (not shown). Congo red is a histochemical dye used for the detection of amyloids of all types in tissue sections. The binding of Congo red to Aβ has been postulated to depend in part on fibrillar Aβ structure (32Klunk W.E. Pettegrew J.W. Abraham D.J. J. Histochem. Cytochem. 1989; 37: 1273-1281Crossref PubMed Scopus (571) Google Scholar). We tested whether Congo red could inhibit the binding of heparin to fibrillar Aβ. Equal aliquots of fibrillar Aβ1–40 were incubated with increasing concentrations of Congo red, and then loaded into the lanes of an ACE gel. Compared with the control lanes of Congo red alone (Fig. 4 A, lane 7) and fibrils alone (lane 8), a dose-dependent inhibition of heparin binding to the fibrils was observed (lanes 1–6). We also used the solution-phase binding assay (above) to confirm the interaction of Congo red with fibrillar Aβ. In the ACE gels, an inhibition of heparin-protein binding is most readily visualized when the protein concentration in the lanes containing no inhibitor is high enough to completely retard the migration of heparin (see,e.g., Fig. 4 A, lane 8). However, using the solution-phase binding assay, we were able to test a range of Aβ concentrations to show that the IC50 for Congo red inhibition of heparin binding increases with increasing protein concentration (Fig. 4 B). When similar concentrations of Aβ were compared in the two assays, ACE gels showed a higher IC50 for Congo red than the solution-phase assay (Fig. 4,A versus B, middle trace). This may be due to the migration of the negatively charged Congo red out of the well during ACE electrophoresis, decreasing the total amount of drug in the lane. HPLC-purified Aβ1–40containing the E22Q (HCHWA-D) substitution was either freshly resuspended from a lyophilized aliquot or aggregated into fibrils as described above. Serial dilutions of each preparation were loaded into the lanes of ACE gels and tested for binding to125I-LMW-heparin. The freshly resuspended peptide did not bind to heparin up to peptide concentrations of 15.2 μm(Fig. 5 A, lane 1), whereas 5.7 μm fibrillar AβE22Q almost completely retarded the mobility of heparin (Fig. 5 B,lane 4). From autoradiograms of ACE gels such as that in Fig. 5 Bretardation coefficients were calculated and plotted against peptide concentration (Fig. 5 C). Values of K d were calculated as described above, and results from several independent preparations of fibrillar AβE22Q yielded an average K d of 3.45 ± 0.08 μm. This is in the low micromolar range, similar to the value measured for heparin binding to wild type Aβ fibrils prepared by the same method. In contrast, when Aβ was subjected to a milder aggregation protocol, we observed a distinct difference in the heparin-binding behavior of wild type and mutant peptides. In this case, each peptide was neutralized, incubated for 48 h at 4 °C at a concentration of 1 mm in water and then lyophilized. Immediately after resuspension in water, the wild type Aβ remained in a non-heparin binding state (data not shown). However, after identical treatment, the mutant peptide was able to bind heparin (Fig.6 A) and showed an affinity similar to that of preformed AβE22Q fibrils. We interpreted this result as indicating that water-aggregated AβE22Q adopted a structure similar to that of wild type Aβ that had been aggregated in artificial CSF (i.e.fibrils with substantial β-pleated sheet conformation). Further evidence for the presence of this conformation came from the fact that the interaction of heparin with the treated AβE22Q was blocked by Congo red in a dose-dependent manner (Fig.6 B). In addition, the ability of these AβE22Qsamples to bind heparin was reversed by overnight incubation in (1,1,1,3,3,3)-hexafluoroisopropanol (HFIP) for 72 h and lyophilization (Fig. 6 C). HFIP is a solvent that is thought to promote or stabilize α-helices (33Barrow C.J. Yasuda A. Kenny P.T.M. Zagorski M. Mol. Biol. 1992; 225: 1075-1093Crossref Scopus (612) Google Scholar), and that can also reverse the neurotoxicity of fibrillar Aβ in cell cultures (34Pike C.J. Burdick D. Walencewicz A.J. Glabe C.G. Cotman C.W. J. Neurosci. 1993; 13: 1676-1687Crossref PubMed Google Scholar). The presence of heparan sulfate has been demonstrated immunohistochemically in amyloid deposits from most human amyloidotic diseases (for review, see Ref.16Snow A.D. Wight T.N. Neurobiol. Aging. 1989; 10: 481-497Crossref PubMed Scopus (188) Google Scholar). One well known example is the islet amyloid of type II diabetes (35Young I.D. Ailles L. Narindrasorasak S. Tan R. Kisilevsky R. Arch. Pathol. Lab. Med. 1992; 116: 951-954PubMed Google Scholar). In this disease, the 37-residue amyloid subunit protein, called amylin, is the major constituent of the extracellular amyloid deposits that surround pancreatic islet cells. In vitro, the human isoform of amylin readily forms fibrils that are toxic to cultured islet cells (3