DOI: 10.1002/chem.200700643

Design of Peptides That Form Amyloid-Like Fibrils Capturing Amyloidb1–42 Peptides

Junichi Sato, Tsuyoshi Takahashi, Hideo Oshima, Sachiko Matsumura, andHisakazu Mihara*[a]

Introduction

The amyloid fibril formation of protein is related to fataldiseases.[1] Alzheimer�s disease (AD) is a progressive neuro-degenerative disease that is characterized by the abnormalaccumulation of amyloid b-peptide (Ab) in senile plaques.[2]

Moreover, the pathogenesis of AD is closely related to theaging process.[3] Amyloid fibrils are believed to play a cen-tral role in the pathogenesis of AD since amyloid fibrils pre-dominantly exist in senile plaques in the brains of patientswith AD.[4] Ab is produced by proteolysis of amyloid b pre-cursor protein (APP) and is mostly composed of either 40or 42 amino acids.[5]

Ab spontaneously self-assembles into amyloid fibrils. Al-though the concentration of the 42 amino acid form (Ab1–42) is approximately 10% that of the 40 amino acid form(Ab1–40), Ab1–42 is the predominant component of senileplaques.[6] Kinetic studies of Ab have revealed that Ab1–42forms amyloid fibrils significantly faster than Ab1–40.[6] Onthe other hand, recent studies suggest that soluble ligandssuch as Ab oligomers might be the toxic species in amy-loids.[7–10] Amyloid-derived diffusible ligands (ADDLs),composed of Ab1–42, are potent neurotoxins that kill neu-rons in cultured hippocampal brain slices from mice atnanomolar concentrations.[7,8] Protofibrils and low-molecu-lar-weight species of Ab are also neurologically active.[9,10]

Moreover, it has been reported that Ab1–42 can form oligo-meric aggregates more rapidly and stably than Ab1–40.[11]

Therefore, the construction of molecules capable of captur-ing Ab1–42-generated amyloid fibrils and toxic oligomers isa potentially important approach for the characterization ofthe various aggregated species of Ab and for developing adiagnostic methodology for AD.

Abstract: Amyloid b-peptide (Ab)plays a critical role in Alzheimer�s dis-ease (AD). The monomeric state of Ab

can self-assemble into oligomers, proto-fibrils, and amyloid fibrils. Since the fi-brils and soluble oligomers are be-lieved to be responsible for AD, theconstruction of molecules capable ofcapturing these species could provevaluable as a means of detecting thesepotentially toxic species and of provid-ing information pertinent for designingdrugs effective against AD. To thisaim, we have designed short peptideswith various hydrophobicities based onthe sequence of Ab14–23, which is acritical region for amyloid fibril forma-

tion. The binding of the designed pep-tides to Ab and the amplification ofthe formation of peptide amyloid-likefibrils coassembled with Ab are eluci-dated. A fluorescence assay utilizingthioflavin T, known to bind specificallyto amyloid fibrils, revealed that two de-signed peptides (LF and VF, with theleucine and valine residues, respective-ly, in the hydrophobic core region)could form amyloid-like fibrils effec-tively by using mature Ab1–42 fibrils

as nuclei. Peptide LF also coassembledwith soluble Ab oligomers into peptidefibrils. Various analyses, including im-munostaining with gold nanoparticles,enzyme-linked immunosorbent assays,and size-exclusion chromatography,confirmed that the LF and VF peptidesformed amyloid-like fibrils by captur-ing and incorporating Ab1–42 aggre-gates into their peptide fibrils. In thissystem, small amounts of mature Ab1–42 fibrils or soluble oligomers could betransformed into peptide fibrils and de-tected by amplifying the amyloid-likefibrils with the designed peptides.

Keywords: Alzheimer�s disease ·amyloid b-peptide · amyloid fibrils ·oligomers · peptide design

[a] J. Sato, Dr. T. Takahashi, H. Oshima, Dr. S. Matsumura,Prof. H. MiharaGraduate School of Bioscience and BiotechnologyTokyo Institute of Technology4259-B40, Nagatsuta, Yokohama (Japan)Fax: (+81)45-924-5833E-mail : hmihara@bio.titech.ac.jp

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Although structures of the soluble oligomers are notavailable, structural analyses of Ab amyloid fibrils havebeen reported.[12,13] Tycko and co-workers reported that resi-dues 1–10 are approximately disordered and that residues12–24 and 30–40 form parallel b-sheet structures in the fi-brils.[12] This model contains a bent structure and showsAsp23 and Lys28 making electrostatic interactions. Residues11–25 are located in the central region of Ab and are knownto be important for fibril formation; the peptide Ab11–25has been found to form amyloid fibrils with morphologysimilar to that of full-length Ab1–40 and Ab1–42 fibrils.[14]

The structure of this kind of fragment peptide has been ana-lyzed by X-ray diffraction and solid-state NMR spectrosco-py.[15–17] These data suggest that the peptide fibrils are com-posed of antiparallel b sheets in a cross-b arrangement andthat the hydrophobic core (LVFFA) participates in an anti-parallel b-sheet structure. The electrostatic interaction madeby His14 and Asp23 might promote an antiparallel b-sheetstructure in Ab11–25. It was reported that the sequence in-cluding this hydrophobic core region could be utilized to designmolecules that inhibit the fibrillization of Ab.[18] These in-sights into the structural basis driving Ab fibrillogenesis pro-vide a foundation for the design of molecules that can coas-semble with and capture Ab fibrils and toxic oligomers.

In the present study, short peptides capable of formingamyloid-like fibrils containing an antiparallel b sheet weredesigned and synthesized. The peptides were designed byvarying the hydrophobicity, with the expectation that differ-ent hydrophobicities would alter fibrillogenesis and bindingto Ab. If the designed peptide can form amyloid-like fibrilsby using mature Ab1–42 fibrils and Ab1–42 soluble oligo-mers as nuclei, then these designed peptides can captureand amplify small amounts of Ab fibrils and other toxic spe-cies of Ab into stable and larger peptide fibrils. This uniqueproperty of the peptide ligands may be useful for the designof detection and diagnostic agents for AD.

Results and Discussion

Peptide design : Four peptides with the sequence Ac-KQKLLXFLEE-NH2 (X=L, V, A, or T; peptides designat-ed LF, VF, AF, and TF, respectively) were designed basedon the structure of the core region of Ab (amino acid resi-dues 14–23; Figure 1). The cationic histidine and anionic as-partic acid residues of the Ab14–23 were replaced by lysineand glutamic acid, respectively, to form favorable electro-static interactions upon formation of the expected antiparal-lel structure.[16,17,19] It was predicted that variation in the hy-drophobicity of the hydrophobic core region (LLXFL)could change the fibril-forming abilities of the designed pep-tides. The glutamine and phenylalanine residues were con-served due to their importance in fibril formation.[20] All ofthe designed peptides were synthesized by the 9-fluorenyl-methoxycarbonyl (Fmoc) solid-phase method and identifiedby matrix-assisted laser-desorption/ionization time-of-flightmass spectrometry (MALDI-TOF MS).

Structural and fibril-forming properties of the designed pep-tides : Circular dichroism (CD) spectra were measured toobtain information about the secondary structure of the de-signed peptides (100 mm) in 20 mm tris(hydroxymethyl)ami-nomethane–HCl (Tris-HCl) buffer (pH 7.4) containing 10%(v/v) trifluoroethanol (TFE); the CD spectra are shown inFigure 2. Small amounts of TFE (10–20% v/v) enhance thefibril formation of Ab.[21] The LF and VF peptides showedtypical b-sheet signals, with a negative minimum at 215 nmand a positive maximum at 200 nm. The intensity of the b-

Figure 1. a) Sequences of Ab1–42 and the designed peptides. The core se-quence of Ab for amyloid fibrils is underlined. b) Model for the antipar-allel b-sheet structure of the designed peptides. The circled amino acidsare the residues on the upper side of the strand, and those surrounded bydotted circles are the ones on the opposite side of the strand.

Figure 2. Circular dichroism spectra of the designed peptides. [peptide]=100 mm in 20 mm Tris-HCl buffer (pH 7.4) containing 10% TFE at 25 8C.The CD spectra were measured just after preparation (c) and after24 h of incubation at room temperature (a)

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sheet signal of the LF peptide was twofold stronger thanthat of the VF peptide. After 24 h at room temperature, theCD spectrum of the LF peptide did not change but the spec-trum of the VF peptide showed a slightly enhanced b-sheetstructure. By contrast, the AF and TF peptides showed CDsignals typical of a random-coil structure, even after 24 h ofincubation. The YF peptide, which has tyrosine at the X po-sition, was also synthesized and provided results similar tothose of AF and TF,[20] a result indicating that A, T, and Yinhibit the formation of stable b-sheet structures. These re-sults suggest that the amino acid residue at position 6 in thehydrophobic core significantly affects the conformationalproperties of the designed peptides, and hydrophobic inter-actions between segments of the hydrophobic core are criti-cal for the intermolecular assembly of b sheets in the pep-tides.

To evaluate the fibril-forming abilities of the designedpeptides, each peptide (100 mm) was incubated in 20 mm

Tris-HCl buffer (pH 7.4) containing 10% (v/v) TFE at roomtemperature. Fibril formation was monitored by using thethioflavin T (ThT) fluorescence assay (see the ExperimentalSection). ThT is known to bind to amyloid fibrils and emit astrong fluorescence near 485 nm after binding.[22] After incu-bation of LF in 10% TFE for 6 h, the fluorescence intensityof ThT increased significantly, a result suggesting amyloid-like fibril formation had occurred (Figure 3). ThT fluores-cence in the presence of VF was slightly higher than that inthe absence of the peptide. These results suggest that the LFand VF peptides have the potential to form amyloid-like fi-brils. By contrast, the AF and TF peptides showed nochanges in fluorescence, even after overnight incubation,thereby indicating that the AF and TF peptides cannot self-assemble into amyloid-like fibrils, possibly due to their lowhydrophobicity. When the designed peptides were incubatedin buffer containing 5% dimethylsulfoxide (DMSO) without

TFE, the fluorescence intensity of ThT in the presence ofLF after 24 h incubation was slightly higher that in the ab-sence of peptide, but it did not change in the presence ofthe VF peptide. Stock solutions of the designed peptideswere prepared in neat DMSO, so the peptides were essen-tially monomeric before the incubation in aqueous buffer,thus making it difficult for the peptides to form amyloid-likelong fibrils in the absence of TFE.

TEM images of the designed peptides : To evaluate the fea-tures and morphologies of the LF and VF fibrils, transmis-sion electron microscopy (TEM) images were obtained(Figure 4). The LF peptide (100 mm) incubated with 10%TFE generated long (several mm in length) tangled fibrils(Figure 4a), similar to mature Ab fibrils. By contrast, theVF peptide (100 mm) incubated with 10% TFE formed shortfibrils, pieces of which assembled into thick fibers (Fig-

Figure 3. Fibrillogenesis of the designed peptides monitored by ThT fluo-rescence. The designed peptides (100 mm) were incubated for 6 h at roomtemperature in 20 mm Tris-HCl buffer (pH 7.4) containing 10% TFE(gray bars) and for 24 h in buffer containing 5% DMSO (white bars).The fluorescence of 6.3 mm ThT was measured with 5.0 mm peptides at25 8C. lex=440 nm and lem=485 nm.

Figure 4. TEM images of the designed peptides LF (a, c, e, and g) and VF (b, d, f, and h) incubated with 10% TFE (a, b) and without TFE (c–h). The fi-brils were prepared in the absence (a–d) and presence (e–h) of mature Ab1–42 fibrils. Fibrils were stained with 2% uranyl acetate or 2% phosphotungs-tic acid. Scale bar=1 mm.

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ure 4b). Furthermore, the LF and VF fibrils formed in theabsence of TFE, from DMSO stock, had short, flat-tape-likemorphologies (0.2–1.5 mm in length; Figure 4c and d). Theseresults suggest that the peptide sequence and incubationconditions alter the fibril morphology dramatically. Thesedifferent morphologies might also change the affinity andfluorescence properties of ThT toward the fibrils : for exam-ple, the LF peptide in 10% TFE formed longer fibrils whichbind to ThT, thereby enhancing the ThT fluorescence signifi-cantly, while the same peptide in 5% DMSO formed shorterfibrils which cannot enhance ThT fluorescence.

Fibril formation of designed peptides with mature Ab1–42fibrils : To evaluate the ability of the designed peptides toform fibrils with mature Ab1–42 fibrils, the LF and VF pep-tides (100 mm each) were incubated either with a smallamount of Ab1–42 fibrils (1, 5, and 10 mm) or in the absenceof Ab ; all incubations took place in 20 mm Tris-HCl buffer(pH 7.4) containing 5% (v/v) DMSO. The ThT fluorescencewas measured before and after 8 h of incubation of the pep-tides with or without the Ab fibrils. The fluorescence inten-sities of ThT in the presence of the LF and VF peptideswere significantly increased by incubation with Ab fibrils(Figure 5), and the fluorescence intensity of LF with Ab fi-

brils was higher than that of VF with Ab fibrils. Further-more, the fluorescence increase was dependent on the in-crease in the amount of Ab1–42 fibrils. No fluorescencechange was observed with mature Ab1–42 fibrils (10 mm)alone (data not shown). These results suggest that the LFand VF peptides can form amyloid-like fibrils by usingAb1–42 fibrils as nuclei, although the peptides alone did notform ThT-sensitive fibrils in the absence of TFE (DMSOstock). LF and VF presumably interact with the Ab1–42 fi-brils due to the sequence similarity of their hydrophobiccores, and these interactions effectively promote fibrillogen-esis of the LF and VF peptides. LF alone has a higher pro-

pensity to self-assemble than VF and this leads to easierfibril formation with Ab1–42 fibrils. Thus, formation of theAb-like fibrils was accelerated and fibrils were accumulatedby the designed peptides LF and VF. A small amount ofAb1–42 fibrils could be detected by amplification of thepeptide fibrils.

TEM images of the designed peptides with Ab1–42 fibrils :TEM images of the LF and VF peptides with mature Ab1–42 fibrils were obtained (Figure 4e–h). When either LF orVF was incubated with Ab fibrils, long tangled fibrils (amy-loid-like fibrils) were clearly observed. Furthermore, theamount of fibrils on the TEM grid increased with increasingAb concentrations, and fewer fibrils were formed in the ab-sence of LF and VF. These results, confirmed by ThT fluo-rescence analyses (Figure 5), suggest that the Ab1–42 fibrilsassist LF and VF in forming amyloid-like fibrils by acting astemplates.

To confirm whether the Ab1–42 fibrils were absorbedinto amyloid-like fibrils composed of the designed peptides,immunostaining by using the 6E10 anti-Ab antibody, whichrecognizes the N terminal of Ab (residues 1–16), conjugatedwith biotin (biotin–6E10) and gold nanoparticles (10 nm di-ameter) conjugated with the antibiotin antibody was carriedout on a TEM grid (Figure 6). Although the gold nanoparti-cles could not be observed in the LF or VF peptide fibrilsalone, the gold particles were observed in significantamounts on fibrils of LF or VF mixed with a small amountof Ab. Furthermore, the gold particles on the peptide fibrilsincreased with increasing Ab concentrations, a result indi-cating that the Ab1–42 fibrils can be captured and coassem-bled with the peptide fibrils.

Fibril formation of designed peptides with Ab1–42 oligo-mers : It has been reported that Ab1–42 readily aggregatesinto metastable oligomers at near physiological concentra-tions (1–20 nm)[23] and that these soluble oligomers might bethe toxic species of amyloids.[7–10] We therefore evaluatedwhether the designed peptides could form amyloid-like fi-brils by capturing the Ab1–42 soluble oligomers. To gener-ate the Ab1–42 oligomers, Ab1–42 treated with 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP; see the Experimental Sec-tion) was incubated in Dulbecco�s phosphate-buffered saline(PBS) for 1 day at room temperature. The oligomers weredetected by size-exclusion chromatography (SEC). The peaknear the void volume consisted of Ab1–42 oligomers (seeFigure 8b); TEM images of the oligomers are shown in Fig-ure 7a.

The LF and VF peptides (100 mm) were individually incu-bated with the Ab1–42 oligomers in the Dulbecco�s PBS at37 8C, and fibril formation by the peptides was examined byusing the ThT binding assay. The fluorescence intensity ofThT in the presence of the LF peptide was significantly in-creased by incubation with the Ab oligomers in a concentra-tion-dependent manner (Figure 7b), similar to the result ob-tained by incubation with mature Ab1–42 fibrils (Figure 5).By contrast, ThT fluorescence in the presence of VF was

Figure 5. Fluorescence increase of ThT before and after 8 h of incubationof the LF (gray bars) and VF (white bars) peptides (100 mm) with andwithout mature Ab1–42 fibrils (0, 1, 5, 10 mm per monomer) at 37 8C. Thefluorescence of 6.3 mm ThT was measured with 5.0 mm peptides at 25 8C.lex=435 nm and lem=485 nm.

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not obviously increased by incubation with the Ab oligo-mers. The VF peptide alone showed a lower ability to formamyloid-like fibrils than the LF peptide alone, as shown inFigure 3. Therefore, the ability of the peptide to form fibrilsis related to the fibril formation with Ab oligomers asnuclei.

When the LF peptide forms amyloid-like fibrils with Ab

oligomers, the Ab oligomers can be captured in the LF fi-brils. To evaluate whether the soluble Ab oligomers coas-semble, an enzyme-linked immunosorbent assay (ELISA)with a fluorogenic peroxidase substrate for detecting Ab

oligomers was carried out by using the supernatant after the

fibrils were removed by centri-fugation (Figure 8a). It hasbeen reported that Ab oligo-mers can be detected by usingcapture and detection antibod-ies (6E10 and biotin–6E10, re-spectively) capable of recogniz-ing the same primary sequenceepitope.[23] Nanomolar-level oli-gomerization can be detectedeasily, but monomeric Ab, witha single epitope, cannot be de-tected. By using this sandwichELISA with 6E10 and biotin–6E10, Ab oligomers in the su-pernatant were detected at ahigher level in the absence ofLF. In a sample immediatelyafter mixing with LF, however,

the fluorescence signal in the ELISA was much lower thanthat in the absence of LF. Furthermore, Ab oligomers in thesupernatant of a sample after 2 h of incubation were hardlydetectable above background level. SEC analysis also con-firmed that samples after incubation with LF contained asmaller amount of Ab oligomers than samples before incu-bation with LF (Figure 8b). These results strongly suggestthat the LF peptide captures soluble Ab oligomers effective-ly in its amyloid-like fibrils.

TEM images of the LF peptide incubated with Ab oligo-mers : To confirm whether Ab1–42 oligomers were captured

Figure 6. Immunostained TEM images of LF (a, c, and e) and VF (b, d, and f) incubated with mature Ab1–42 fibrils. The concentrations of Ab1–42 were1 (a, c), 5 (c, d), and 10 mm (e, f). The Ab1–42 species on the fibrils were immunostained with antibody-conjugated gold nanoparticles. Fibrils werestained with 2% uranyl acetate. Scale bar=500 nm (inset=100 nm).

Figure 7. a) TEM image of Ab1–42 large oligomers. Scale bar=100 nm. b) Fluorescence increase of ThTbefore and after 8 h of incubation of the designed peptides (100 mm) with and without Ab1–42 soluble oligo-mers (0, 1, 5, and 10 mm) at 37 8C. The fluorescence of 6.3 mm ThT was measured with 5.0 mm peptides at 25 8C.lex=435 nm and lem=485 nm.

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and coassembled into the amyloid-like fibrils composed ofthe LF peptide, immunostaining analysis with gold nanopar-ticles was carried out on a TEM grid (Figure 9). Gold nano-particles were observed on LF fibrils incubated with Ab1–42oligomers. The number of gold particles on the fibrils in-creased with increasing Ab1–42 oligomer concentration,similar to the result obtained with mature Ab1–42 fibrils.The nanoparticles on the oligomer–peptide fibrils weremore uniformly distributed than those on LF fibrils withmature Ab1–42 fibrils. Since the size of the gold particles(10 nm diameter) is similar to that of the large Ab1–42 olig-omers, one gold particle could bind to one Ab oligomer;therefore, the particles might be observed separately andnot assembled on the LF fibrils. Furthermore, gold particlescannot be observed on LF fibrils alone. These results con-firm that the Ab1–42 oligomers can be captured by the LFfibrils.

Discussion

Short peptides capable of forming amyloid-like fibrils bycapturing Ab1–42 fibrils and soluble oligomers have beendesigned and synthesized. The LF and VF peptides incubat-ed without TFE or Ab formed short flat fibrils. ThT, a spe-

cific binder to amyloid fibrils, cannot react with such LF andVF fibrils (DMSO stock). By contrast, the LF and VF pep-tides incubated with mature Ab1–42 fibrils formed amyloid-like fibrils to which ThT bound. The different morphologiesand ThT-binding properties of the peptide fibrils with andwithout the Ab1–42 fibrils indicate that the LF and VF pep-tides interact with the Ab1–42 fibrils and form amyloid-likefibrils. Furthermore, a TEM study with gold nanoparticlesalso revealed that LF and VF coassembled with the Ab1–42fibrils. In this system, small amounts of mature Ab1–42 fi-brils were detected by amplification of the Ab fibrils asamyloid-like fibrils composed of LF or VF.

In the case of the Ab1–42 soluble oligomers (which arebelieved to be the more toxic species), the LF, but not theVF, peptide can bind to the soluble oligomers immediatelyand form amyloid-like fibrils. The Ab1–42 soluble oligomerscaptured by the LF fibrils can be removed easily by centrifu-gation. VF cannot capture the Ab1–42 oligomers effectively.The VF peptide forms amyloid-like fibrils in Tris-HCl bufferwithout NaCl but hardly form fibrils in phosphate-bufferedsaline. The LF peptide rapidly self-assembles and can cap-ture Ab1–42 soluble oligomers into LF fibrils. Thus, thesoluble oligomers can be detected by amplification intostable peptide fibrils. The construction of capturing agentsfor amyloid fibrils and toxic oligomers of Ab1–42 is usefulfor characterization of the various species of Ab aggregatesand for developing detection and diagnostic agents for AD.In particular, since the soluble oligomers can be trappedinto the peptide fibrils, the LF peptide might transform thetoxic Ab1–42 oligomers into less toxic species. Evaluation of

Figure 8. a) ELISA assay for Ab1–42 soluble oligomers by using anti-Ab

6E10 (capture) and biotinylated 6E10 (detection) antibodies. The super-natants were measured after centrifugation of the Ab1–42 oligomers in-cubated with (gray bars) or without (white bars) LF. b) SEC analysis ofthe supernatants after centrifugation before (c) and after incubationwith LF (0 h, g ; 2 h, a) on a Superdex 75 column.

Figure 9. Immunostained TEM images of LF fibrils incubated with Ab1–42 soluble oligomers. The concentrations of Ab1–42 were a) 1 andb) 10 mm. The Ab1–42 species on the fibrils were immunostained with an-tibody-conjugated gold nanoparticles. Fibrils were stained with 2%uranyl acetate. Scale bar=100 nm. Two images are shown for each set ofconditions.

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the biological activity of the designed fibrils is currently un-derway.

Experimental Section

Chemicals and reagents : All chemicals and solvents were of reagent orHPLC grade. Amino acid derivatives and reagents for peptide synthesiswere purchased from Watanabe Chemical (Hiroshima, Japan). MALDI-TOF MS was performed on a Shimadzu MALDI III mass spectrometerby using 3,5-dimethoxy-4-hydroxycinnamic acid as a matrix. HPLC wascarried out on a YMC ODS A-302 5C18 column (4.6M150 mm; YMC,Tokyo, Japan), a YMC ODS A-323 5C18 column (10M250 mm), or De-velosil packed ODS-UG-5 columns (4.6M150 mm, 10M250 mm) by em-ploying a Hitachi L-7000 HPLC system. Amino acid analyses were per-formed by using the phenyl isothiocyanate (PTC) method on a WakopakWS-PTC column (Wako chemical, Osaka Japan). 6E10 antibody and bio-tinylated 6E10 were purchased from Signet Laboratories (Dedham, MA,USA). Electron microscopy was performed with a H-7500 electron mi-croscope at 80 or 100 kV (Hitachi High-technologies, Tokyo, Japan).Streptavidin-conjugated horseradish peroxidase was purchased fromAmersham Bioscience (Tokyo, Japan). QuantaBlu, a fluorescent sub-strate for peroxidase, was purchased from Pierce Biotechnology (Rock-ford, IL, USA). High-binding 96-well plates for the ELISA were pur-chased from Corning (Tokyo, Japan).

Peptide synthesis : The peptides, both the designed ones and Ab1–42,were synthesized by the solid-phase method by using the Fmoc strategywith O-(7-azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluoro-phosphate (HATU) or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluro-nium hexafluorophosphate (HBTU) and 1-hydroxybenzotriazole hydrate(HOBt·H2O) as the coupling reagents. To remove the resin and the pro-tecting groups, the peptide resin was treated with trifluoroacetic acid(TFA) containing m-cresol, ethanedithiol, and thioanisole as scavengersfor 1 h at room temperature. The product was solidified with diethylether in an ice bath. The designed peptides were purified with reversed-phase (RP) HPLC on the YMC ODS A-323 column by using a lineargradient of acetonitrile/0.1% TFA to give the purified peptides. Ab1–42was purified with RP HPLC on the Develosil packed ODS-UG-5 columnby using a linear gradient of acetonitrile/0.1% NH4OH. Peptides wereidentified by their molecular ion peak [M+H]+ in the MALDI-TOFmass spectrum: m/z found (calcd): LF: 1302.9 (1302.6); VF: 1287.1(1288.6); AF: 1259.4 (1260.5); TF: 1352.0 (1352.6); Ab1–42: 4518.0(4515.1).

Sample preparation : The peptide stock solution was prepared in neatTFE or DMSO at a concentration of 1 mm or 2 mm, respectively. Thepeptide concentration was determined by quantitative amino acid analy-sis. Peptides were diluted in 20 mm Tris-HCl buffer (pH 7.4) or Dulbec-co�s PBS (0.7 mm KCl, 0.4 mm KH2PO4, 137 mm NaCl, 8.1 mm Na2HPO4,pH 7.5) with or without Ab1–42 to start the incubation. Ab1–42 was dis-solved in HFIP to monomerize, and then the solution was evaporated.The monomerized Ab1–42 was dissolved in dimethylsulfoxide, and thena buffer solution was added to start the incubation (final DMSO concen-tration: 4%). Mature Ab1–42 fibrils were prepared by incubation of100 mmAb1–42 in 20 mm Tris-HCl buffer for 1 week at room temperature.Before the mature Ab1–42 fibrils were used, they were sonicated tomake a homogeneous solution. Ab1–42 soluble oligomers were preparedby incubation of 40 mmAb1–42 in Dulbecco�s PBS for 24 h and centrifu-gation (18800 g, 60 min) to remove the larger aggregates such as fibrils.

Circular dichroism measurement : CD measurements were performed ona Jasco J-720WI spectropolarimeter equipped with a thermoregulatorand with a quartz cell with a 1.0-mm path length. Spectra were recordedin terms of mean residue ellipticity (q, in degcm2dmol�1). A stock solu-tion of each peptide in TFE was diluted in 20 mm Tris-HCl buffer(pH 7.4). The final concentration of the peptides was 100 mm in the buffercontaining 10% (v/v) TFE. The concentration of the peptide solutionswas determined by quantitative amino acid analysis.

Thioflavin T fluorescence analysis : ThT (Aldrich Chemical Co.) was dis-solved at a cocentration of 250 mm in water. The ThT stock solution(10 mL) was added to the peptide solution (390 mL; the final concentra-tions of ThT and the peptide were 6.3 and 5 mm, respectively), and thenfluorescence emission spectra were recorded immediately at an excitationwavelength of 440 nm at 25 8C. Fluorescence spectra were measured on aHitachi F2500 fluorescence spectrophotometer by using a 5M5 mmquartz cell.

Transmission electron microscopy : The peptide solution (10 mL) was ab-sorbed on to a carbon-coated copper grid (200 mesh) by floating the gridon a drop of the peptide solution for 30–60 s. The excess solution was re-moved by filter-paper blotting, the grid was washed by floating on a dropof water for 10 s, and then the water was removed. The sample on thegrid was then negatively stained with a 2% (w/v) aqueous phosphotungs-tic acid or uranyl acetate solution for 30 s and the excess staining solutionwas removed. After drying, the samples were visualized with a HitachiH-7500 electron microscope operating at 80 or 100 kV.

To detect the Ab1–42 on the peptide fibrils, biotinylated anti-Ab antibod-ies (biotin–6E10) and antibiotin antibody-conjugated gold nanoparticleswere used. The peptide solution (10 mL) was absorbed on to a carbon-coated copper grid (200 mesh) by floating the grid on a drop of the pep-tide solution for 30–60 s. The excess solution was removed with filterpaper, and the grid was blocked by 20 mm Tris-HCl buffer (10 mL) con-taining 150 mm NaCl and 0.05% (v/v) Tween 20 (TBST) with 1% (w/v)bovine serum albumin (BSA) for 15 min. The grid was washed withTBST (10 mLM2), and then the peptide fibrils on the grid were treatedwith biotin–6E10 (10 mgmL�1) in TBST with 1% BSA (10 mL) for30 min. The grid was washed with TBST (10 mLM3), and then fibrils onthe grid were treated with 1% (v/v) antibiotin antibody-conjugated goldnanoparticle solution in TBST with 1% BSA (10 mL) for 30 min. Thegrid was washed with TBST (10 mLM3) and water (10 mLM1), and thenthe grid was stained by 2% (w/v) uranyl acetate aqueous solution for 1–2 min. After drying, the samples were visualized with a Hitachi H-7500electron microscope operating at 80 kV.

ELISA : To detect the Ab1–42 oligomers, an ELISA was carried out. The6E10 antibody (2 mg per well) was coated on a 96-well high-binding plateat 4 8C overnight. The wells were blocked by TBST with 1% BSA(200 mL) at room temperature for 90 min. The wells were washed withTBST (200 mLM3). The samples were centrifuged at 18800 g for 60 minat 4 8C to remove fibrils. The resulting supernatants were diluted 100-foldinto TBST, and then the solution (100 mL) was added into the wells for30 min at 4 8C. The wells were washed with TBST (200 mLM3), biotin–6E10 (1 mgmL�1) in TBST with 1% BSA (100 mL) was added, and themixture was incubated for 90 min at room temperature. The wells werewashed with TBST (200 mLM3), and then streptavidin-conjugated horse-radish peroxidase (1 mgmL�1) in TBST with 1% BSA (100 mL) wasadded and incubated for 30 min at room temperature. The wells werewashed with TBST (200 mLM3), and then QuantaBlu fluorogenic perox-idase substrate (Pierce, 100 mL) was added and incubated for 60 min. Thestop solution (Pierce, 100 mL) was added and the fluorescence was mea-sured on a Twinkle LB970 fluorometer. An excitation filter of 355 nm(bandwidth�20 nm) and an emission filter of 460 nm (bandwidth�12.5 nm) were used.

Size-exclusion chromatography : For analytical size-exclusion chromatog-raphy, a Superdex 75 10/300 GL column (10M300 mm) (Amersham Bio-science) was used with Dulbecco�s PBS as the eluent at a flow rate of0.4 mLmin�1. To detect the Ab1–42 soluble oligomers, the samples werecentrifuged at 18800 g for 60 min at 4 8C. The resulting supernatant wasinjected into the column and detected by absorbance at 220 nm on anHPLC system. The following proteins were used as molecular-weightstandards: BSA (66 kDa), carbonic anhydrase (29 kDa), cytochrome c(12.4 kDa), aprotinin (6.5 kDa), and insulin B chain (3.5 kDa).

Chem. Eur. J. 2007, 13, 7745 – 7752 D 2007 Wiley-VCH Verlag GmbH&Co. KGaA, Weinheim www.chemeurj.org 7751

FULL PAPERAmyloid Peptide Capture

Acknowledgements

This work was supported in part by a Grant-in-Aid for Scientific Re-search from the Ministry of Education, Science, Culture and Sports,Japan.

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Received: April 27, 2007Published online: July 2, 2007

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H. Mihara et al.