Parallel β-Sheet

Research Article Cite This: ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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Toxic Amyloid Tape: A Novel Mixed Antiparallel/Parallel β‑Sheet Structure Formed by Amyloid β‑Protein on GM1 Clusters Yuki Okada,† Kaori Okubo,† Keisuke Ikeda,‡ Yoshiaki Yano,† Masaru Hoshino,† Yoshio Hayashi,§,∥ Yoshiaki Kiso,§,⊥ Hikari Itoh-Watanabe,# Akira Naito,# and Katsumi Matsuzaki*,† †

Graduate School of Pharmaceutical Sciences, Kyoto University, 46-29 Yoshida-Shimoadachi-cho, Sakyo-ku, Kyoto 606-8501, Japan Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan § Center for Frontier Research in Medicinal Science, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8412, Japan ∥ Department of Medicinal Chemistry, Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan ⊥ Laboratory of Peptide Science, Nagahama Institute of Bio-Science and Technology, Nagahama, Shiga 526-0829, Japan # Graduate School of Engineering, Yokohama Naitional University, 79-5 Tokiwadai, Hodogaya-ku, Yokohama 240-8501, Japan

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‡

S Supporting Information *

ABSTRACT: The abnormal aggregation of amyloid β-protein (Aβ) is considered central in the pathogenesis of Alzheimer’s disease. We focused on membranemediated amyloidogenesis and found that amyloid fibrils formed on monosialoganglioside GM1 clusters were more toxic than those formed in aqueous solution. In this study, we investigated the structure of the toxic fibrils by Aβ-(1−40) in detail in comparison with less-toxic fibrils formed in aqueous solution. The lesstoxic fibrils contain in-resister parallel β-sheets, whereas the structure of the toxic fibrils is unknown. Atomic force microscopy revealed that the toxic fibrils had a flat, tape-like morphology composed of a single β-sheet layer. Isotope-edited infrared spectroscopy indicated that almost the entire sequence of Aβ is included in the βsheet. Chemical cross-linking experiments using Cys-substituted Aβs suggested that the fibrils mainly contained both in-resister parallel and two-residue-shifted antiparallel β-sheet structures. Solid-state NMR experiments also supported this conclusion. Thus, the toxic fibrils were found to possess a novel unique structure. KEYWORDS: amyloid-β (Aβ), GM1 ganglioside, cross-linking, isotope-edited FTIR, solid-state NMR, antiparallel β-sheet

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in Aβ-positive nerve terminals from the AD cortex,16 and lipid rafts from the frontal cortex and the temporal cortex of AD brains contained a higher concentration of GM1 compared to an age-matched control.17 We reported that cholesteroldependent GM1 clusters facilitate amyloidogenesis by Aβ18 and that, importantly, amyloid fibrils formed on GM1 clusters (membrane fibrils or M-fibrils) were different from those formed in aqueous solution (water fibrils or W-fibrils). Mfibrils were more toxic, thicker, and contained some antiparallel β-sheet structures19,20 in contrast to in-resister parallel β-sheet structures for less-toxic W-fibrils.21 This study investigated the structure of M-fibrils by transmission electron microscopy (TEM), atomic force microscopy (AFM), isotope-edited Fourier-transform infrared (FTIR) spectroscopy, chemical cross-linking, and solid-state NMR, and we propose a novel unique “amyloid tape” structure composed of mixed antiparallel/parallel β-sheets.

INTRODUCTION One of the pathological hallmarks of Alzheimer’s disease (AD), the most common form of dementia, is the deposition of senile plaques in the brain, the major component of which is fibrillar aggregates of amyloid-β protein (Aβ). According to the amyloid cascade hypothesis, the conversion of the soluble, nontoxic Aβ monomer to aggregated, toxic Aβ rich in β-sheet structures (oligomers and fibrils) is considered central to the development of AD.1−3 Recently, both types of aggregates have been implicated in the pathogenesis of AD.4 Accumulating evidence has suggested that the binding of Aβ to cell membranes plays an important role in the aggregation of Aβ,5−11 as for other amyloidogenic proteins.12 Yanagisawa et al. identified a specific form of Aβ bound to monosialoganglioside GM1 (GM1) in brains exhibiting the early pathological changes associated with AD and also suggested that the GM1bound form of Aβ may serve as a seed for the formation of Aβ aggregates.13 This hypothesis was confirmed in vivo: the antibody 4396C specific for GM1-bound Aβ stained AD brains but not control brains,14 and the administration of modified 4396C significantly reduced plaque formation in AD model mice.15 Furthermore, significant increase in GM1 was reported © XXXX American Chemical Society

Received: August 21, 2018 Accepted: September 27, 2018

A

DOI: 10.1021/acschemneuro.8b00424 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 1. AFM image of (A) W-fibrils and (B) M-fibrils. The height profiles along the lines are shown at the bottom.

Figure 2. Isotope-edited FTIR spectra. FTIR spectra of (A, B) W-fibrils and (D, E) M-fibrils by (A, D) Aβ-(1−40) and (B, E) 1-13C-F20-Aβ-(1− 40). Observed spectra are shown by solid lines, and deconvolved bands and their summed spectra are denoted by dotted lines. Difference spectra of (C) B − A and (F) E − D are also shown. Solid and open arrows indicate bands originating from antiparallel β-sheets and 1-13C-F20, respectively.

B

DOI: 10.1021/acschemneuro.8b00424 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 3. Amino acid alignments of (A) 0-, (B) 1-, (C) 2-, and (D) 3-residue-shifted antiparallel β-sheets by Aβ-(1−40). Possible salt bridges, hydrophobic, and π− π interactions are indicated in blue, green, and brown, respectively. The Cys-substituted sites are shown in red.

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RESULTS Morphology of Fibrils. Aβ-(1−40) was incubated with or without GM1-containing liposomes (GM1:sphingomyelin:cholesterol = 1:1:1) at an Aβ-to-GM1 ratio of 1 at 37 °C, and the formation of amyloid fibrils was monitored with the amyloid-specific fluorescent dye thioflavin-T (Th-T). We used this model membrane system mimicking lipid rafts because fibril formation occurs in GM1-rich, cholesterol-rich regions on neuronal cells.22,23 In the absence of GM1-liposomes, fibrils were formed after a long lag time, whereas in its presence, ThT fluorescence increased much faster, confirming the catalytic activity of the membrane in amyloidogenesis, as seen with other membrane systems.24 The formed fibrils were examined in terms of morphology, toxicity against neuronal SH-SY5Y cells, and secondary structures. The M-fibrils were significantly more toxic and thicker (11.6 ± 1.8 nm) than the W-fibrils (7.8 ± 1.7 nm), and the presence of a weak peak at 1695 cm−1 in the FTIR spectrum suggested the presence of antiparallel βsheets in the former, confirming our previous reports (Figure S1 in the Supporting Information and also Figure 2).19,20,25 It should be noted that the toxicity was not due to contaminating oligomers because toxic oligomers positive to the oligomer specific antibody A11 were not detected19 and, even if present, oligomers formed on membranes are nontoxic. 26 The observation that W-fibrils were not cytotoxic also supports the conclusion that contamination of oligomers was minimal. The heights of fibrils were measured by AFM to get an insight into their three-dimensional structure (Figure 1). Note that the width information by AFM is inaccurate because of the finite size of the probe. The height of W-fibrils was 7.1 ± 1.7 nm (n = 20 including data from different snapshots), in accordance with previous studies.21,27 In contrast, the height of M-fibrils was extremely short (0.6 ± 0.1 nm, n = 20), suggesting that M-fibrils had flat, tape-like structures composed of a single β-sheet layer, the height of which was ∼1 nm for Aβ-(1−40).21 Note that this value is likely diminished by the

interaction between the protein and the solid support for AFM measurements (mica). Isotope-Edited Infrared Spectroscopy. The structures of fibrils were analyzed by 13C-edited FTIR spectra. The carbonyl group of F4, V12, F19, F20, V24, or V36 of Aβ-(1− 40) was labeled with 13C, and W-fibrils and M-fibrils were prepared. If fibrils were composed of an in-resister parallel βsheet, a 13C−13C coupling was expected to be observed. Otherwise, 13C−12C coupling was expected. The former is characterized by a larger isotope shift, whereas the intensity of the 13C peak is higher in the latter case.28 Figure 2 shows the results for [1-13C] F20-Aβ as an example. The spectra were deconvolved into component peaks to estimate isotope shifts. Unlabeled W-fibrils and M-fibrils exhibited intense bands at 1631.1 and 1626.0 cm−1, respectively, indicating that both fibrils were mainly composed of β-sheets and the hydrogen bonds were stronger for M-fibrils. Additional minor bands at around 1695 cm−1 were observed for M-fibrils, suggesting the presence of antiparallel β-sheets.29 The 13C bands appeared at around 1605.7 and 1606.6 cm−1 for W-fibrils and M-fibrils, respectively (isotope shifts of 25.4 and 19.4 cm−1). The percent area values for W-fibrils (4.4%) were smaller than those for M-fibrils (6.4%). These results were compatible with the assumption that some 13C−12C coupling existed in Mfibrils. Isotope dilution experiments also supported this conclusion (Table S1). 1-13C-labeled Aβ was mixed with unlabeled Aβ at a molar ratio of 1:3, and W- and M-fibrils were prepared. For W-fibrils, the isotope shift was significantly reduced (5.6 cm−1) by isotope dilution, suggesting the presence of 13C−13C coupling in W-fibrils. In contrast, it was only marginally affected (1.4 cm−1) in M-fibrils. The difference spectra clearly indicated that the labeled residue (F20) was in the β-sheet structures (Figures 2C and F). Similar results were observed for the other labeled peptides except for W-fibrils prepared with F4- or V12-labeled Aβs (Figure S2 and Table S1). For these, 13C bands were not discernible, in accordance with the less ordered nature of the N-terminal region of WC

DOI: 10.1021/acschemneuro.8b00424 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

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ACS Chemical Neuroscience

Figure 4. HPLC profiles of cross-linked products for (A) W-fibrils and (B) M-fibrils formed by an equimolar mixture of G9C- and A30C-Aβ-(1− 40). Peaks: a, G9C-Aβ-(1−40) monomer; b, A30C-Aβ-(1−40) monomer; c, heterodimer by G9C-Aβ-(1−40) and A30C-Aβ-(1−40); d, A30C-Aβ(1−40) homodimer; e, G9C-Aβ-(1−40) homodimer.

fibrils.21 M-fibrils appeared to possess β-sheet structures almost along the entire sequence. This can explain the thickness of Mfibrils (∼12 nm) as estimated by TEM (Figure S1e). The translation per residue value for β-sheets is ∼0.33 nm.30 Thus, the length of a 40-residue β-sheet is ∼13 nm. Cross-Linking. Having confirmed that M-fibrils were a single layer of β-sheet composed of almost the entire sequence of Aβ, next we determined the interstrand structure by chemical cross-linking. We hypothesized that antiparallel βsheets were two-residue-shifted structures because they could be better stabilized by multiple salt bridges and hydrophobic/ π−π interactions compared with other structures (Figure 3). It should be noted that electrostatic interactions are augmented in a less polar membrane environment. We carried out chemical cross-linking experiments using Cys-substituted-Aβ(1−40) peptides, the Cys residues of which are expected to be in close proximity in the proposed structures. If antiparallel βsheets formed, heterodimers were expected to be formed. Small residues, the sizes of which were close to that of Cys, were chosen for Cys mutations (Figure 3). Such substitutions are known to minimally affect the self-assembly of Aβ-(1− 40),31,32 in contrast to mutations to amino acids with larger side chains such as A2 V.33 Namely, S8C/G33C-, A2C/ G38C-, G9C/A30C-, and S8C/A30C-Aβ-(1−40) peptides were used for the detection of 0-, 1-, 2-, and 3-residue-shifted antiparallel β-sheets, respectively. W- and M-fibrils were formed under reduced conditions by incubating an equimolar mixture of these mutants. The fibrils were washed to remove the reducing agent dithiothreitol and air-oxidized. The generated cross-linked products were dissolved and analyzed by reversed phase HPLC. Representative chromatograms are shown for the G9C/A30C pair (Figure 4), and the results are summarized in Table 1. For the G9C/A30C pair, which detects the most plausible two-residue-shifted structure, Wfibrils produced mainly G9C- and A30C-Aβ homodimers as cross-linked products, in accordance with the in-resister parallel β-sheet structure model.21 For M-fibrils, not only the homodimers, but also significant amounts of the heterodimer, were detected, suggesting the coexistence of in-resister parallel and two-residue-shifted antiparallel β-sheets. For the other Cys mutant pairs, differences in heterodimer formation efficiency between M- and W-fibrils were less marked (Table 1).

Table 1. Summary of Cross-Linking Experiments % heterodimer/total dimersa Cys mutant pair S8C/G33C (0-residue shifted) A2C/G38C (1-residue shifted) G9C/A30C (2-residue shifted) S8C/A30C (3-residue shifted)

M-fibrils

M-fibrils

difference (M − W)

fibrils not formed 12.8 ± 1.8

21.7 ± 0.2

8.9 ± 1.8

4.3 ± 0.5

18.0 ± 0.3

13.7 ± 0.6

5.7 ± 0.1b

11.2 ± 1.1

5.5 ± 1.1

Mean ± SD, n = 2−3. bThe total Aβ concentration was 100 μM because fibrils were not formed at 50 μM. a

REDOR. The cross-linking experiments suggested that the main antiparallel β-sheet structure present in M-fibrils was a two-residue-shifted one. To confirm this in a nonperturbing fashion, we carried out solid-state NMR measurements. 15N cross-polarization and magic angle spinning (CP-MAS) NMR spectra of M- and W-fibrils are shown in Figure 5. For Mfibrils, doublet signals (a- and b-peaks) were observed in the lower and higher fields with an intensity ratio of 1:1.6, indicating that two structures coexisted. The chemical shift of the a-peak was similar to that of the single peak of a W-fibril, indicating that the a-peak corresponds to an in-register parallel β-sheet. The normalized rotational echo double resonance (REDOR) factors (ΔS/S0) were evaluated from the full echo and REDOR 15N CP-MAS NMR signals as shown in Figure 6 for M-fibrils by [15N]F19, [1-13C]A21-Aβ-(1−40). The b-peak showed larger REDOR factors than the a-peak against REDOR evolution (NcTr) times, where Nc is the number of rotor cycles and Tr is the rotor cycle period. REDOR factors vs NcTr times are plotted in Figure 6C to examine the inter- and intrastrand 13C−15N-13C interatomic distance patterns, which reflect fibril packing patterns (Figure 7). For the β-sheet structure, it is necessary to consider three spin systems to determine parallel or antiparallel β-sheet structures.34,35 Analysis using more than three spin system36,37 was not considered because the longer distance show the smaller contribution for evaluating 15N−13C distances. Using the structure model for a mixture of two-residue-shifted antiparallel (V18 -A21 is the closest proximate pair and L17-A21 D

DOI: 10.1021/acschemneuro.8b00424 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

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Figure 5. 15N CP-MAS NMR spectra of (A) M-fibrils and (B) Wfibrils by [15N]F19, [1-13C]A21-Aβ(1−40). The spectra were obtained using CP time of 2 ms, 90° pulse of 5.50 μs, and repetition time of 4 s at 20 °C. Six thousand transients were accumulated and made Fourier transformation with 30 Hz Lorentzian line broadening. The doublet signal was deconvolved into the lower field a-peak and the higher field b-peak with an intensity ratio of 1:1.6. The chemical shift of the a-peak is the same as that of the single peak of W-fibrils.

intensity change (ΔS/S0), and the b-peak plot fitted well to the calculated REDOR curve for the APA arrangement. The REDOR curve of [15N]F19, [1-13C]A21-Aβ-(1−40) W-fibrils fitted to that for the PPP arrangement within the error range (Figure 8), which was similar to the REDOR curve of the apeak in M-fibrils. We also performed 15N REDOR experiments for [15N]L17, [1-13C]A21-Aβ-(1−40) M-fibrils (Figure S3). 15N NMR signals showed a singlet, and hence, two signals were overlapped in this case. A plot of REDOR factors vs NcTr indicated that the fibril structure was not the PPP structure alone but also a mixture of the PPP, PPA, APP and APA structures. Similarly, we performed 15N REDOR experiments for [15N]V18, [1-13C]A21-Aβ-(1−40) M-fibrils (Figure S4). The 15N NMR signal also showed a singlet, and hence, two signals were overlapped in this case as well. A plot of REDOR factor vs NcTr indicated that again the fibril structure was not the PPP structure alone but a mixture of the PPP, PPA, APP, and APA structures. Note that the REDOR factors of the PPA arrangement were different from those of the APP arrangement for the V18/A21 labeling positions. Therefore, as the largest REDOR factors appeared in the V18/A21 labeled M-fibrils among the three differently labeled Aβ-(1−40) β-strand structures (L17/A21, V18/A21 and F19/A21), the residues V18 and A21 were located in the closest proximity in the tworesidue shifted antiparallel β-sheet structure (Figures 7E, E′, and F). It should be noted that all the labeled positions were in β-sheets, and intersheet interaction was absent (vide supra). These results indicate that M-fibrils are composed of a mixture of in-register parallel and two-residue-shifted antiparallel βsheet structures as postulated by the cross-linking experiments.

and F19-A21 are the second closest pairs)/in-register parallel β-sheet structures, calculated REDOR factors against NcTr values were obtained for four types of β-strand arrangements, P′(NC)P(NC)P(NC)[PPP], P(NC)P(NC)A(CN)[PPA], A(CN)P(NC)P(NC)[APP], and A(CN)P(NC)A(CN)[APA], respectively, where N and C indicate the directions of N-and C-termini, respectively (Figure 7). The plots of the REDOR factors against NcTr (REDOR curve) for the two peaks in [15N]F19, [1-13C]A21-Aβ-(1−40) M-fibrils were different; the a-peak plot fitted to the calculated REDOR curve for the PPP arrangement by considering the error range in the small

Various three-dimensional structures have been proposed for less-toxic W-fibrils, which are essentially composed of inresister parallel β-sheets.21,38−40 In each case, clusters of hydrophobic amino acids are sequestered from water. In contrast, structural information is lacking for more toxic Mfibrils, except for the presence of some antiparallel β-sheets.20 Cross-linking experiments suggested that a two-residueshifted antiparallel β-sheet structure was most probable for Mfibrils (Table 1). However, significant amounts of heterodimer were also observed for the one-residue shifted structure. This

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DISCUSSION

Figure 6. (A) Full echo and (B) REDOR 15N NMR spectra of [15N]F19, [1-13C]A21-Aβ(1−40) M-fibrils at NcTr = 20 ms. (C) The plots of ΔS/ S0 against NcTr values. Three calculated REDOR curves were obtained for PPP, PPA (APP), and APA β-sheet units by considering shortest interatomic distances for the PPP unit and two shortest interatomic distances for PPA (APP) and APA units as shown in the solid arrows in Figure. 7. E

DOI: 10.1021/acschemneuro.8b00424 ACS Chem. Neurosci. XXXX, XXX, XXX−XXX

Research Article

ACS Chemical Neuroscience

Figure 7. Three β-strand packing units for the mixtures of antiparallel and parallel β-sheet structures of M-fibrils in [15N]L17, [1-13C]A21-Aβ(1− 40), [15N]V18, [1-13C]A21-Aβ(1−40), and [15N]F19, [1-13C]A21-Aβ(1−40). 15N−13C interatomic distances used for calculation of the REDOR curves are as follows. (A) 15NV17-13CA21 (P) = 16.3 Å. (B) 15NL17-13CA21 (PA) = 5.85 Å, 15NL17-13CA21 (PP) = 16.3 Å, and < 13C−15N−13C = 47.5°. (B′) 15NL17-13CA21 (AP) = 5.85 Å, 15NL17-13CA21 (PP) = 16.3 Å, and < 13C−15N−13C = 47.5°. (C) 15NL17-13CA21 (AP) = 6.0 Å, 15NL17-13CA21 (PA) = 6.0 Å, and