eral splice products of this amyloid precursor protein (APP) have been ... necessary to understand the development of AD. ..... denatured than of native APP.3.
Vol. 268, No. 35, Issue of December 15, pp. 26571-26577, 1993 Printed in U S A .
Tm JOURNAL OF Bromrcu CHEMISITIY
0 1993 by The American Society for Bioehemistry and Molecular Biology, Inc.
Amyloid-like Propertiesof Peptides Flanking the Epitopeof Amyloid Precursor Protein-specific Monoclonal Antibody 22Cll* (Received forpublication, April 29, 1993, and in revised form, August18, 1993)
Caroline HilbichSg, UrsulaMiinning$, Christine Grundn, Colin L. Masterall, and Konrad BeyreutherS From the $Center of Molecular Biology, University of Heidelberg, Im Neuenheimer Feld 282, 0-69120 Heidelberg, Federal Republic of Germany, the Wepartment of Cell Biology, German Cancer Research Center, Zm Neuenheimer Feld 280, 0-69120 Heidelberg, Federal Republic of Germany, and the lpepartment of Pathology, University of Melbourne, Parkville, Victoria 3052, Austrdlia
curs by proteolytic cleavage within the PA4 region; the resultAlzheimer’s disease is one of the prevalent forms of fragment cannot generate amyloid any more. An human dementia.Its pathology is distinguished by pro- ing teinaceous deposits (“amyloid”) in the brain. They con- alternative proteolytic breakdown of the precursor liberates tain a peptide(pA4)that is proteolytically derivedfrom the entire @A4 peptide which will then aggregate into the a larger transmembrane protein. To follow the different typicaldepositions found in AD (for a review, see Kosik metabolic pathways of this Amyloid Precursor Protein (1992)). That an aberrant catabolism of APP is not only corre(APP)may thus lead to the elucidation of the molecular lated to Alzheimer’s disease, but might be indeed a causative basis of Alzheimer’s disease. Specific antibodies are necevent, hasbeen strongly indicated by the identification of muessary tools for this task. Using synthetic peptides, we tations within APP. They have been found in families sufferhavecharacterizedtheepitopeoftheAPP-specific ing from hereditary (familial) AD, a variant in which clinical monoclonal antibody22Cll; itis localized between resi- manifestations can occur as early as in the fourth decade of dues 66 and 81 of APP.Some of the peptides flanking this life. Several of these mutations are not situated within the site exhibited properties generally associated with amyloid, i.e. low solubility, filament formation, and birefrin-PA4 sequence, but both amino- and carboxyl-terminally in its gence after Congo Red staining. Exploiting differences close vicinity (reviewed in Hardy (1992)). Thus a detailed understanding of the function and metabolism of APP will be in the peptides’ aggregational properties, we present evidence that the two dyes Eosin and Direct Red 254, in necessary to understand thedevelopment of AD. Specific antibodies against APP are important tools to inconjunction with classical amyloid staining by Congo Red, can be used to characterize aggregating, amyloid- vestigate its metabolism. One of them is themonoclonal antibody (mAb) 22C11, which was inaugurated in our laboratory like peptides in vitro. (Weidemann et al., 1989). This antibody was elicited by immunization with a bacterial APP fusion protein (Fd-APP695). Its Alzheimer’s disease (AD)’ is the most common cause for a precise epitope remained undefined. However, it has been dementia of elderly people. Its main pathological hallmarks shown to detect mature, secreted isoforms of APP lacking the
transmembrane and cytoplasmic domains as well as the signal sequence. Thus these regions cannot bear the epitope of mAb 22Cll. Moreover, the antibody was also used to detect a variety of mature as well as immatureAPP species which differ in their degree of glycosylation. Since glycosylation often prevents antibody binding not only at a glycosylation site itself, but also in neighboring regions, one would not expect the epitope to be situated within the glycosylated domain(s) of the protein. The KPI and Ox11 domains were not included in the APP fusion protein used for immunization; hence they cannot bear the epitope either. Due to theuniformity of the acidic domain, an antibody directed against this sequence would supposedly not possess such as high a specificity as mAb 22Cll does. Thus we concluded thatthe epitope ofmAb 22Cll should most probably be located within the cysteine containing domain(s) of APP. To test this hypothesis, we synthesized a peptide compris* This work was supported by the Federal Ministry for Science and Technology of the FRG, the ‘Fonds der ChemischenIndustrie,”and the ing a sequence of 87 amino acids located approximately in the Metropolitan Life Foundation. The costs of publication of this article middle of the cysteine-containing region. This peptide was inwere defrayed inpart by the payment of page charges. Thisarticle must deed detected by mAb 22Cll. The epitope was subsequently therefore be herebymarked “advertisement”inaccordancewith 18 more closely localized by a series of shorter peptides. Their seU.S.C. Section 1734 solely to indicate this fact. Fig. 1. During the 8 To whom correspondence should be addressed: Centerof Molecular quences and designations arelistedin Biology, Im Neuenheimer Feld 282, D-69120 Heidelberg, Federal Re- course of their characterization,it became apparent thatsome public of Germany of them exhibit characteristic properties of amyloid peptides. The abbreviations usedare: AD, Alzheimer’s disease; Acm, acetamidomethyl;APP, amyloid precursor protein, BSA, bovine serum albumin;They were therefore subjected not only to immunological asELISA, enzyme-linked immunosorbent assay; mAb, monoclonal anti- says,but also to some spectroscopic and physicochemical tests. body; PBS, phosphate-buffered saline. areintracellularandextracellular depositions of proteins within the brain (for a review, see Hardy and Allsop, 1991). These depositions show three properties thatcharacterize them as amyloid: 1) they are insoluble under physiological conditions; 2) visualized under polarized light,they show green birefringence after staining with thedye Congo Red; 3) they aggregate as filaments (for reviews, see Glenner (1980) and Castafio and Frangione (1988)).The main component of the extracellular depositions (amyloid plaque cores and amyloid of congophilic angiopathy) is a 4.5-kDa peptide (“PA4”) that is synthesized as part of a transmembrane protein. Several splice products of this amyloid precursor protein (APP) have been described, which appear also as secreted isoforms in extracellular compartments. Its putative domain substructure is illustrated in Fig. 1. Secretion of extracellular APP oc-
26571
26572
Epitope of APP-specificAntibody 22C11
washed 5 times using TBST (10 m~ Tris,150 m~ NaCl, 0.05% Tween 20, pH 8.0Lincubated for 1h at room temperature with a 1:10,000 dilution (in 1% BSAPBS) of secondary antibody (goat A mouse, coupled to alkaline phosphatase; Promega), and developed in APbuffer (100 nm Tris, 100 m~ NaCl, 5 rn MgCl,, pH 9.5) containing 165 m g / d 5-bromo-4chloro-3-indolylphosphate-p-toluidinesalt and 330 m g / d p-nitro blue N87 63-149 . . tetrazolium chloride. N5 1 99.149 .: ......,. m .. . .. Solubility Determinations-Lyophilized peptides (0.5-1.0 mg) were .. .. .... . .,. .. suspended in 500 pl of solvent, mixed for 30min a t room temperature, N14 T NQVEVVEY P E L O I 74-87 and centrifuged at 10,000 x g for 10 min. Half of the supernatant and N17 ~ Y C ~ E V Y P E TL N~V IV E 71-07 the remaining supernatant plus pellet were each dissolved in 550 pl of N17dV Q Y C Q E V - P E L QT IN V V E 71-87.desTyrTi 100%HCOOH. Peptide concentrations were determined via absorption N22 K E G I L ~ Y C Q E V Y P E L Q IT N V V E 66-67 at 280 nm and were corrected forthe amounts of pure pellet and 100% N16 K E G I L O Y C Q E V Y P E L Q 66-81 supernatant. N12 I D T K E G L Q I Y A A 5372.+ A M h Infrared Spectroscopy-About 1mg of lyophilized peptide was mixed with 350 mg ofanhydrous KBr and subjected to a pressure of 8.1 GPa Al PePlidSS bwIIIeeami- a d urboiy-lsrmm. Cplelne %#de c h a a are pmlecledby Ihe Acmgroup for 10 s to form a clear pellet. Dispersive infrared spectroscopy was * APPnu~nnsaocordinglol(angeiai(1987) performed within a range of 4000 to 340 cm-l on a model 580BPerkinFIG.1. Sequences and designations of peptides used in this Elmer spectrometer (spectral resolution: 2.3cm"). The spectra were study, presented in their relation to APP and its putative do- qualitatively analyzed according to Krim and Bandekar (1986). main structure. Electron Microscopy-For filament formation, solutions of peptides in PBS, pH 7.4 (0.5 mg peptideJ250 pl), were dialyzed against several changes of 50 rnNaC1, pH 6.0,at room temperature for 3 days. Peptide MATERIALS AND METHODS grids, suspensions were then applied to deionized carbon-coated copper Peptide Synthesis and Purification-Stepwise solid-phase peptide dried, negatively stained with 2% uranyl acetate, and visualized in an synthesis was performed ona peptide synthesizer Model 430A (Applied electron microscope (Zeiss EM910). Dye Staining and Detection of Birefringence-Peptides were stained Biosystems) using standard t-butoxycarbonyl chemistry (Hilbich et al., 1991a).Peptides N87 and N51 were synthesized using double coupling with the dyes Congo Red, Eosin, and Direct Red 254 (Congo Red and cycles throughout; for the shorter peptides, double coupling cycles were Eosin were purchased from Serva and Merck, respectively; DirectRed employed for Asn,Gln, and Cys-Acm, and single coupling cycles forall 254 was a gift from BASF). About 0.5mg of lyophilized peptides were other amino acids. Peptides were deprotected and cleaved from the suspended in 50 pl of distilled water and mixed with 5 pl of a dye resin by reaction with liquid hydrogen fluoride for 1h at 0 "C (Lenard solution (1% in HzO). Congo Red staining was also done according to and Robinson, 1967). 15% anisole and, inthe case of N87 and N51,5 mg Puchtler et al. (1962): peptides were suspended in a solution of 80% of methionine were added as scavengers. The cysteine protecting Acm- ethanol, 0.01% NaOH, saturated with NaCl, and Congo Red. In each group was not removed from the peptides. Peptide N12 was extracted case, the stained peptide suspensions wereplacedontomicroscope from the resin with HAC(50%,v/v). Purification by size exclusion chro- slides and inspected immediately under bright field and polarized light Photographs were taken a t a primary matography on Bio-GelP-4 (Bio-Rad)in 1M HAC(column dimensions:2 in a microscope (Axioplan, Zeiss). x 180 c m ) yielded a product that eluted as one peak in reverse-phase magnification of 1:lOO. high performance liquid chromatography. Theother peptides appeared not to be sufficiently soluble in aqueous buffers or diluted acetic acid; they were therefore extracted from the resin with HCOOH (70%,v/v) RESULTS and, except forpeptide N14, desalted by size exclusion chromatography on a Bio-Gel P-10 column (2 x 180 cm) equilibrated in 70% HCOOH. Epitope of mAb 22C11"To test whether the synthetic pepThe purity of peptides N14, N17,N17dY, N22, and N16 wastested by tides comprise the epitope of mAb 22Cl1, direct antibody bindcapillary electrophoresis (Applied Biosystems Model 270A) in 20 nm ing was initially determined by ELISA. Fig. 2, A and B, shows citrate buffer, pH 2.0. Supernatants of suspensions in PBS,pH7.4, that the antibody boundto peptide N87, but not to N51, pointshowed one majorpeak indicating that thepeptides had purities of at least 80%.Peptides N87 and N51 showed one, albeit broad, band within ing to the NH,-terminal third of N87 as the epitope region. Smaller peptides (N12 to N22) spanning this region werethen the expected molecular mass range of 10 (N87) and 6 kDa (N51) in SDS-polyacrylamidegels. They were usedwithout further purification. assessed. Of these, only peptides N22 and N16 were detectedby Identity of the shorter peptides (N12 to N22) was confirmed byEdman mAb 22Cll. Likewise, only peptidesN87, N22, and N16 could degradation of the final products (Instrumentation and reagents from abolish antibody bindingto APP on Western blots (Fig.3). Applied Biosystems). During the synthesis of peptides N87 and N51, In direct ELISAs,mAb 22Cll showed significant bindingto small resin samples were removed every 10-15 coupling cycles and peptides N22 and N16, but it was loweredin comparison to the were used directly for Edman degradation. No major coupling deficienbinding of peptide N87. This could be due to the fact that N22 cies could be detected. Enzyme-linked Immunosorbent AssapMicrotiter assay plates (96- and N16, in spite of their relatively small size, were coated onto well) were coated with 1.25 nmolof peptide per well. Nonspecificprotein ELISA plates without coupling to a carrier protein. Direct adbinding was blockedusing 1%bovine serum albumin (BSA)in PBS (137 sorption of peptides to a surface can inhibit antibody binding nm NaCl, 3 rn KCl, 8 m~ Na2HP0, x 2H20, and 2 m~ KH2P04,pH both by steric hindrance and by deformation of a peptide epi7.4). The plates were then incubated with monoclonal antibody 22Cll (clone M16)at 4 "C overnight. Dilutions of mouse ascites containing the tope. In the indirect assay, where binding between antibody N22 and N16 were as mAb ranged from 1:5,000 to 1:640,000. Following 5 washes with tap and peptide occurredin solution, peptides water, secondary antibody was applied at a dilution of 1:5,000 for 1h at potent as inhibitors of antibody bindingto APP as was N87. 37 "C. The plates were developedusing 4 m g / d 2,4-dinitrophenylphosThe epitope of mAb 22Cll could thus be localizedat residues phatein diethanolamine buffer(10% diethanolamine, v/v,0.5 n m 66-81 of APP (numbering according to Kang et al. (1987)). MgC12, pH 9.8). Neither peptide N12 nor N17, together covering the complete Western Blotting-A Western blot was applied to test whether the synthetic peptides could prevent the detection of APP by mAb 22Cll. sequence of Nl6, showed binding to mAb 22Cll; this indicates Human cerebrospinal fluid from healthy control donors was used as a that the binding region comprises amino acids located both source of APP. Cerebrospinal fluid (170$gel slot) was dried, dissolved N H 2 - and COOH-terminally of residues 71 and 72. in Laemmli sample buffer, separated on a SDSgel containing 10% Physicochemical Characterization of Peptides N12 to polyacrylamide (Laemmli, 1970) and transferred onto a nitrocellulose N22-During their synthesis and purification, peptides N14, membrane ('Ibwbin et al., 1979). f i r blocking with 1%BSAPBS, N17, N17dY, and N22 failed to be solubilized by some of the membrane slips were incubated for 2 h a t room temperature in solutions of 1%BSAPBS containing mAb 22Cll (dilution 1:10,000) and standard solvents used in peptide preparation. Thus, peptide to produce a peptide that had the synthetic peptides (20 dd). These mixtures had been incubated over- N16 was mainly synthesized night at 4 "C before application to the blots. The membranes were same antibody binding properties, but was more soluble than ~
,
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,
Epitope of APP-specific Antibody22Cll 3
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FIG.2. Binding of mAb 22Cll to the synthetic peptides as determinedby ELISA. A, results obtained with mouse ascites containing mAb 22Cll at a dilution of 1:10,000, shown for all peptides used in this study. PeptideN12 was also coupled to BSA by glutaraldehyde. B,complete ELISA results are shown for peptides N87 (O),N51 (O), N17 (O), N22 (m), N16 (A),and N12 (A).
-
N87 N51 N14 N17 N17
I
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L
dY
N22 N16
N12
FIG.3.Western blot detection of APP by mAb 22Cll after preincubation of the antibody with synthetic peptides (as indicated). The two bands correspondto the major AFP ' isoforms obtained from cerebrospinal fluid (Weidemann et al., 1989); their apparent molecular masses are105 and 125 m a .
" N14
N17
N17dY peptides
N22
N16
FIG.4. Relative solubilityof the small N-peptides in different solvents: 1 M H A C (m), H 2 0 (R), 50 m~ NaCl (D), 1 M NaCl (lV, PBS, pH 7.4 (W).
this test are presented in Fig. 5. Peptide N14 formed large amounts of apparently stablefilaments. Theyhad a diameter of about 8 nm and were associated parallel to their longitudinal axis to form pairs of filaments with an irregulartwist. As they appear in dense clusters, their accurate length could not be N22. The smaller N-peptides (N12 to N22) were accordingly determined, but filamentsof at least 500 nm could be traced. Peptides N17 and N17dY exhibited only poor ability in filasubjected to some tests to characterize their physicochemical ment formation. Filaments from peptide N17 were of about the properties. To indicate the solubility of peptides N14 to N22, relative same diameter as the ones from peptide N14, but had only values were determined as described for amyloid peptides (Hil- lengths of 50-100 nm. The preparation of N17dY showed only bich et al., 1991a) in a set of solvents differing in pH and ionic a few filamentous aggregates, which were mostly verythin and strength (see Fig. 4). Peptide N17 and N17dY were insoluble short, interspersedwith granular aggregates. For peptide N16, under acidic conditions, irrespective of salt concentration, but filaments or other aggregatescould not de detected. When native amyloid is stainedwith the dye Congo Red and were solubilized to more than 80% in PBS at pH 7.4. Peptides N14 and N22 showed elevated solubilitiesunder strongly acidic inspected under polarized light, it exhibits a green birefrinand neutral conditions; again, they were barely soluble at in- gence. Synthetic peptides bearing amyloid sequences alsoshow termediate pH values of 5 to 6. Of the peptides tested, only N16 a green or, if the peptide concentrationis very high, darkyellow could be solubilized to a reasonable degree (>60%) in all 5 birefringence (Hilbich et al., 1992). The smallN-peptides were solvents. Like N14 and N22, it showed the highest solubilities also subjected to this test. In addition, they were stained with in 1M HACand PBS. Remarkably, solubility in the intermediate the dyes Direct Red 254, whose structure has similarity to pH range (pH 5-6) decreased with increasing ionic strength. Congo Red, and Eosin. Their chemical structures are given in Peptide N12 was readily solubilized in 1M HAC;its solubility in Fig. 8. Fig. 6 shows preparations of stained peptides as viewed unother solvents was not determined. The different patterns of solubility neither correlated with the peptides' net charge nor der polarized light. PeptidesN12, N14, N17, and N17dY clearly exhibited greenand yellow birefringence after CongoRed with their theoretically expected pKI values of 5.8-6.9. The pH-dependent solubilityof peptides N14and N22 as well staining. In contrast,peptides N22 and N16 were only scarcely as theprecipitating effect of increased salt concentration stained by the dye; birefringence could not be detected. Peptide N14 alsoshowed a green birefringence uponstaining shown for peptide N16 seemed reminiscent of amyloidogenic peptides (Hilbich et al., 1991a, 1991b; Fraser et al., 1991; Bar- with Direct Red 254 and a yellow one when stained withEosin. dyes, like row et d., 1992). Peptides N14, N17, N17dY, and N16 were Viewed under nonpolarized light, staining with these therefore checked for filament formation in vitro. The results of staining withCongo Red, is proven by a red color of the peptide
26574
Epitope of APP-specific Antibody
22Cll
within all spectra is centered around1660 cm", demonstrating that the peptides contained a-helical and/or random coil conformation. A shoulder at 1630 cm", resulting from antiparallel of the spectra, but best is expressed p-sheet, could be seen in all for peptides N22 and N16. The differently accentuated peaksat 1540 to 1530 cm" (in the amide I1 region) demonstrate that all of the peptides containvarying amountsof p-turns. Thesignals of the amideI11 region are located between 1230 and 1220 cm" for peptides N12 and N16, with an additional shoulder around 1260 cm" for the otherN-peptides. They indicatethe existence of antiparallel p-sheet structure plus an amountof non-p structure which is lowest for peptides N12 and N16. In summary, the spectra show that the peptidescontain varying amounts of secondary structure elements in thesolid state. Judged from the intensity of the signal a t 1630 cm", peptides N22 and N16 contained the highest amount of well defined p-sheet structure. This unexpected result was further substantiated by circular dichroism spectroscopy. The N-peptides, solubilized in 5 m~ sodium phosphate buffer at pH 8.2, adopted mainly random coil conformations; @sheet was only reflected in the spectra of peptides N16 and especially N22 (data not shown). In solution as well as in the solid state, peptides N12, N14, N17, and N17dY seemed not to have a preference for a particular secondary structure, while N16 and N22 contained somewhat elevated levels of p-pleated sheet.
DISCUSSION
The results presented above clearly confirm our hypothesis that theepitope of mAb 22Cll islocated in theNH2-terminal, cysteine containing region of APP. Peptide N16 will be of practical use as a substitute ofAPPwhenever the specificity of mAb 22Cll has to be checked (e.g. in immunocytochemistry). The localization of the epitope (at residues 66-81of APP) explains the ability of mAb 22Cll to detect APP and APP-related proteins. Since the APP sequences of human, monkey, rat, and mouse are completely identical in theregion of peptide N16, the antibodyshould react equally well with APP from these APP homologue from Drosophila sources. Incontrast,the FIG.5. Electron microscopy of filaments prepared in v i t r o (Rosen et al., 1989) and mouse amyloid precursor-like protein from peptides N14 (a), N17dY ( b ) ,and N17 ( c ) . Magnification was (Wasco et al., 1992) show significant differences from the hu64,000 x in a and c, and 50,400 x in b, the bur corresponding to 100 nm man APPsequence in thisregion. Detection of these proteinsby in all cases. mAb 22Cll should not be expected. Since the antibodies were initially raised against denatured precipitates (datanot shown). In samplesof peptide N17, only APP that did not bear disulfide bridges, it is conceivable that a few birefringent aggregates could be seen after Eosin stain- the synthetic peptides (N87, N22, and N16) function as subing; using Direct Red 254, there wasonly weak staining and no strates for mAb 22Cl1, although they were used in their cysbirefringence. With peptide N17dY, birefringence appeared teine-protected form. However, this should not lead to the conupon staining with Direct Red 254, but only a few aggregates clusion that mAb 22Cllis directed against a completely (like theone in the upper right comer of Fig. 6i) showed bire- sequential, continuous epitope. The spectroscopic results prefringence following Eosin staining. Although it could be stained sented in this study demonstrate that only the two peptides more readily with Eosin and Direct Red 254 than with Congo (N22 and N16) that exhibit some tendency for p-sheet formation are detected by the antibody. Although it cannot be preRed, peptide N16 again did not exhibit birefringence. Birefringence after Congo Red staining is usually regarded cluded that antibody bindingoccurs when the peptides are ina as a characteristic of densely packed aggregates whose three- more globularconformational substate, itis tempting to specudimensional structure consistsmainly of p-pleated sheets late that thepeptides need to adopt an extended conformation (Glenner et al., 1972). To detect the occurrence of secondary for antibody binding. The physicochemical properties of the smallN-peptides show structure in the solid state, the N-peptides were subjected to infrared spectroscopy after dispersion in dryD r . Spectra were remarkable similarities to amyloid peptides. Peptide N14 esrecorded from 4000 to 340 cm". The amide A region showed pecially fulfils major criteria for the classification of amyloid: only a very weak shoulder at 3450 cm", indicating a low level low solubility, filament formation, and birefringence after of amide groups nonbonded within hydrogen bonds; thus, the Congo Red staining. Yet the peptides that seem to be more similar to amyloid in these respectsshow only a low tendency peptides adopted secondary structure in the solid state. The infrared spectrafrom 2000 to 1000 cm", comprising the for P-sheet formation in the solid state and none in solution, structurally sensitive areas of amide I, 11, and 111, are shown in thus not allowing a classification of any one of the N-peptides Fig. 7. Within the amide I region, the most prominent band as "amyloidogenic." Peptides exhibiting some of the aggrega-
1
Epitope of APP-specific Antibody
22Cll
26575
FIG. 6. Microscopic appearance of peptides after dye staining as viewed under polarized light.Left column and 0 , peptides after staining with Congo Red, done according to Puchtler et al.(1962); middle column (except o ) , staining with Direct Red 254; right column, staining ~me following ~ i peptides ~ were . used: N14 (a,b, and c), N17 (d, e, and f ) , N17dY ( g , h, and i), N16 (k,1 l and m ) , N12 ( n ) ,and N22 (')' with ~
tional, tinctorial, or structural properties of amyloid, but lacking a strong correlation between them, can also be derived from the sequence of the COOH-terminal domain of APP (Caputo et al., 1992).Sequences of this kind are certainly more redundant than true amyloidogenic sequences and are not confined to APP.2 Their tendency for aggregation and low solubility in aqueous solvents might indicate that they, in their native sequential context and conformation, are buried inside a protein rather thanbeing exposed on a protein surface. If the sequence of the small N-peptides was at least partly buried within the protein interior in native APP, this would also explain our observation that mAb 22Cll is more potent in the detection of C. Hilbich, unpublished results.
denatured than of native APP.3 The occurrence of birefringence after Congo Red staining is routinely used to identify natural and synthetic amyloid. The high afiinity binding of Congo Redto amyloid has raised hopes that this molecule and itsmode of binding to amyloid peptides could provide a basis for the modelling of substances interfering with and, ultimately, preventing amyloid formation. Different models for the binding of Congo Red to amyloid have been proposed (Puchtler et al., 1962; Cooper, 1974; Hunk et al., 1989).More recently, an x-ray analysis of Congo Redbinding at the hydrophobic interface of an insulin dimer has been published (Turnell and Finch, 1992).
U. Monning, unpublished observation.
Epitope of APP-specific Antibody
26576
22Cll
cooEosin FIG.
8. The chemical structuresof Congo Red, Direct Red 264,
and Eosin.
i
0
1 18001400
I 1600
wave number
I
Icm-9
I 1200
1
FIG.7. Infrared spectra of N-peptides dispersed in dry KBr.
Here, we have used two dyes other than Congo Red which also bind to amyloid sequences (e.g. PA4 peptides of Alzheimer's disease) and cause them to show birefringence under polarized light.4 This has long been known for Eosin; due to its low specificity,it cannot be used forthe histological detectionof amyloid. The properties of Direct Red 254 with regard to amyloid binding are, to the best of our knowledge, hitherto uncharacterized. Both substances might provide further clues to the binding mode of dyes to amyloid. DirectRed 254 shows somestructural similarity to Congo Red, but lacks its symmetry (see Fig. 8). If one assumes a similar binding mode for both dyes, this would cast some doubt on a recently proposed model (Turnell and Finch, 1992) emphasizing the importance of the symmetrical nature of Congo Red that would preferentially bind between The symmetrical arrays of "palindromic" amino acid sequences. same authors also pointed to the role of aromatic residues in the binding between Congo Red and amyloid. This is certainly true for their model system, but our results indicate that it cannot be generalized: peptides N17dY and N12 contain a tyrosine residue, but it is located close to the NH2orCOOH terminus of the peptides, respectively. Thus it seems rather improbable that this amino acid wouldbe essential for the C. Hilbich, unpublished observation.
binding of Congo Red. Other authors (Klunk et al., 1989) proposed that Congo Redbinding is mediated by ionic interactions. This model cannot be ruled out by a comparison of Congo Red and Direct Red 254,but it can hardly account forthe binding of a substance like Eosin, that contains only onereadily ionizable PUP. The two dyes might also be of some use in characterizing amyloid peptides or proteins in vitro. Exploiting the different aggregational properties of peptides N14, N17,N17dY, and N16, we have found a correlation between the peptides' ability for filament formation and their tinctorial properties. Peptide N14, being the only one of the peptides that formed stable filaments, was also the only one that was stained and showed birefringence with all three dyes. Peptides N17 and N17dY had a lowered tendencyfor filament formation: staining and/or birefringence was not achievedwith at least one of the two dyes Eosin or Direct Red254. Peptide N16,whichdid not form filaments, was negative for birefringence with all three dyes. However, experiments using analogues of the PA4 sequence (Hilbich et al., 1992) have produced peptidesthat formed filaments while lacking birefringence after staining with these dyes.2 The lack of staining and/or birefringence can therefore be regarded as a necessary, albeit not sufficient, criterion that can be employed as a screening test for amyloid peptideswith reduced tendency for aggregation. Eosin and Direct Red 254 could thus prove to be useful tools bothin the in vitro characterization of amyloid and in the search for substances preventing its formation. Acknowledgments-We are indebtedto W. W. Franke for his cooperation in electron microscopy. We thank E. Krambs and W. E. Hull for recording of infrared spectra, J. Reed for giving us access to circular dichroism facilities, and S. Pinto for assistance in amino acid sequencing. T. Friedrich and B. Esters-Woike influenced this work through most helpful suggestions and comments. REFERENCES Barrow. C. J.. Yasuda.. A,.. Kennv. M.G. (1992) J. Mol. Biol. _ .P. T.M.. and Zagorski, 226,' 1075-1093 Caputo, C.B., Fraser, P. E,, Sobel, I. E., and Kirschner,D.A. (1992)Arch. Biochem. Biophys. 292, 199-205 Cashio, E.M., and Frangione, B. (1988) Lab. Invest. 58, 122-132 Cooper, J. €I.(1974) Lab. Invest. 31,232-238 Fraser. P.. Nauven. J. T., Surewicz. W.T.. and Kirschner,D.A., (1991) Bwphys. . . J. m,'119&i2b1 Glenner, G. G. (1980)New Engl. J. Med. 302,1283-1292 Glenner, G. G.,Eanes, E. D., and Page, L. D.(1972) J. Hislochern. Cytochem. 20, 821-826 Hardy, J. (1992) Nature Genet. 1,233-234 Hardy, J.,and Allsop, D.(1991) Z h d s Pharmacal. Sei. 12, 383-388 Hilbich, C.,Kiaters-Woike, B., Reed, J., Masters, C.L., and Beyreuther,K (1991a) J. Mol. B i d . 218, 149-163 '
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