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Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles

Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 925073, 8 pages doi:10.4061/2011/925073 Research Article Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles 1, 2 3 4 3 Maho Yagi-Utsumi, Koichi Matsuo, Katsuhiko Yanagisawa, Kunihiko Gekko, 1, 2 and Koichi Kato Graduate school of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan Hiroshima Synchrotron Radiation Center, Hiroshima University, 2-313 Kagamiyama, Higashi-Hiroshima 739-0046, Japan Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan Correspondence should be addressed to Koichi Kato, kkatonmr@ims.ac.jp Received 13 October 2010; Revised 30 November 2010; Accepted 3 December 2010 Academic Editor: J. Fantini Copyright © 2011 Maho Yagi-Utsumi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clusters of GM1 gangliosides act as platforms for conformational transition of monomeric, unstructured amyloid β (Aβ)toits toxic β-structured aggregates. We have previously shown that Aβ(1–40) accommodated on the hydrophobic/hydrophilic interface of lyso-GM1 or GM1 micelles assumes α-helical structures under ganglioside-excess conditions. For better understanding of the mechanisms underlying the α-to-β conformational transition of Aβ on GM1 clusters, we performed spectroscopic characterization of Aβ(1–40) titrated with GM1. It was revealed that the thioflavin T- (ThT-) reactive β-structure is more populated in Aβ(1–40) under conditions where the Aβ(1–40) density on GM1 micelles is high. Under this circumstance, the C-terminal hydrophobic 39 40 anchor Val -Val shows two distinct conformational states that are reactive with ThT, while such Aβ species were not generated by smaller lyso-GM1 micelles. These findings suggest that GM1 clusters promote specific Aβ-Aβ interactions through their C- termini coupled with formation of the ThT-reactive β-structure depending on sizes and curvatures of the clusters. 1. Introduction accelerate Aβ deposition [8]. In vitro experiments have indicated that the Aβ-GM1 interaction depends on the Conformational transitions of unstructured proteins into clustering of GM1, and its carbohydrate moiety alone cannot β-structure-based oligomeric or amyloid states are crucial induce conformational changes of Aβ [15, 30, 31]. processes in the onset and development of a variety of neu- Furthermore, it has been suggested that each of the rodegenerative disorders such as Alzheimer’s disease (AD) heredity variants of Aβ reported thus far has its own and Parkinson’s disease [1, 2]. Amyloid β(Aβ), a major player specificities for gangliosides, which have been supposed to be in AD, is a 40- or 42-amino acid peptide cleaved from its associated with their ectopic deposition [9, 10]. Promotion precursor membrane protein by sequential actions of β-and of amyloid formation in membrane-bound states has also γ-secretases and has a high propensity for toxic aggregation been reported for prion and α-synuclein [11, 12]. For to form cross-β-fibrils [3, 4]. Accumulated evidence indicates example, prion protein has been reported to be localized in the membrane microdomains and caveolae enriched that the GM1 ganglioside, a glycosphingolipid abundant in neuronal cell membranes, interacts with Aβ and promotes with ganglioside, which interacts with prion protein and its assembly, resulting in pathogenic amyloid formation thereby promotes its α-to-β structural conversion [13, 14]. [5–7]. For example, high-density GM1 clustering, which Therefore, detailed conformational characterization of Aβ is exclusively observed in synaptosomes, is suggested to interacting with the ganglioside clusters not only provides 2 International Journal of Alzheimer’s Disease structural information as cues for drug development in pre- cells were grown in M9 minimal media containing [ N] venting and treating AD but also offers general insights into NH Cl (1 g/L) and/or [U- C ] glucose (2 g/L). Protein 4 6 the mechanisms underlying the disease-associated amyloid expression was induced by adding 0.5 mM isopropyl-β- - formation facilitated in membrane environments. thiogalactopyranoside (IPTG) when the absorbance reached In previous papers, we have reported nuclear magnetic 0.8 at 600 nm. After 4 hours, cells were harvested and resonance (NMR) studies of the interactions of Aβ (1–40) then suspended into buffer A (50 mM Tris-HCl, 150 mM with ganglioside clusters using lyso-GM1 micelles (approxi- NaCl, pH 8.0) containing 4-(2-aminoethyl) benzenesul- mate molecular mass 60 kDa) as model systems [15, 16]. Our fonyl fluoride hydrochloride, subsequently disrupted by NMR data showed that Aβ(1–40) is accommodated on the sonication. After centrifugation, the pellet was dissolved hydrophobic/hydrophilic interface of the ganglioside cluster in buffer A containing 8 M urea. His -Ub-Aβ(1–40) was 2+ exhibiting an α-helical conformation under ganglioside- purified by a Ni -nitrilotriacetic acid affinity column (GE excess conditions. In this state, Aβ(1–40) shows an up- Healthcare). Recombinant glutathione S-transferase- (GST- and-down topological mode in which the two α-helices ) tagged yeast ubiquitin hydrolase-1 (YUH-1) was grown 14 24 31 36 at segments His -Val and Ile -Val and the C-terminal until the absorbance reached 0.8 at 600 nm and then induced 39 40 Val -Val dipeptide segment are in contact with the to expressbyIPTG. Cell pelletsweredissolved in buffer hydrophobic interior of the micelles, whereas the remaining B (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 8.5) regions are exposed to the aqueous environment. A similar and disrupted by sonication. GST-YUH-1 was purified by tendency of Aβ(1–40) has been observed using excess a glutathione affinity column (GE Healthcare). Aβ(1–40) amounts of GM1, which forms micelles with an approximate protein was enzymatically cleaved from His -Ub by incuba- molecular mass of 140 kDa [15, 17]. These findings indicate tion with GST-YUH-1 for 1 h at 37 Catamolarratio of that ganglioside clusters offer unique platforms at their His -Ub-Aβ(1–40): GST-YUH1 = 10 : 1. The cleaved Aβ(1– hydrophobic/hydrophilic interfaces for binding coupled with 40) was purified by reverse-phase chromatography using an α-helix formation of Aβ molecules. octadecylsilane column (TSKgel ODS-80T , TOSOH) with To gain further insights into the underlying mechanisms a linear gradient of acetonitrile. The fraction containing of the amyloid formation of Aβ, it is necessary to char- Aβ(1–40) was collected and lyophilized. 15 39 acterize the conformational transition from α-helices to β- Synthetic Aβ(1–40) labelled with N selectively at Val structures on the ganglioside clusters. On the basis of the or Val was purchased from AnyGen Co. Both of recombi- circular dichroism (CD) data, Kakio et al. demonstrated nant and synthetic Aβ(1–40) proteins were dissolved at an that Aβ/GM1 ratios influence the secondary structure of approximate concentration of 2 mM in 0.1% (v/v) ammonia Aβ(1–40) on the raft-like lipid bilayers composed of GM1, solution then collected and stored in aliquots at −80 C until cholesterol, and sphingomyelin [18, 19]. Namely, Aβ adopts use. an α-helical structure at lower Aβ/GM1 ratios (≤0.025), 2.2. Preparation of Micelles. Powdered lyso-GM1 and GM1 while it assumes a β-sheet-rich structure at higher ratios were purchased from Takara Bio Inc. and Sigma-Aldrich, (≥0.05). Although more detailed structural information on respectively. These gangliosides were dissolved in methanol. Aβ bound to the GM1 cluster is highly desirable, the small Subsequently, the solvent was removed by evaporation. The unilamellar vesicles used for the CD measurements are still residual ganglioside was suspended at a concentration of too large to investigate with solution NMR techniques. 12 mM in 10 mM potassium phosphate buffer (pH 7.2) and In the present study, we attempt to characterize con- formational states of Aβ(1–40) in the presence of vary- then mixed by vortexing. Micelle sizes were determined by dynamic light scattering using a DynaPro Titan (Wyatt ing amounts of GM1 aqueous micelles using stable- technology). isotope-assisted NMR spectroscopy in conjunction with synchrotron-radiation vacuum-ultraviolet CD (VUVCD) 2.3. Thioflavin T (ThT) Assay. Aβ(1–40) was dissolved at spectroscopy. We found that GM1 micelles also induce a concentration of 0.2 mM in 10 mM potassium phosphate distinct secondary structures of Aβ(1–40) depending on the buffer (pH 7.2) in the absence or presence of 0.4–9 mM Aβ/GM1 ratios. On the basis of the spectroscopic data, we GM1 or lyso-GM1. The samples were kept on ice before will discuss Aβ behaviours on the ganglioside clusters from a measurements. 980 μLof5 μMThT (Sigma)solutionin structural point of view. 50 mM glycine-NaOH buffer (pH 8.5) was added to an aliquot of 20 μL of each sample. Fluorescence was measured 2. Materials and Methods immediately after mixing at the excitation and emission wavelengths of 446 and 490 nm, respectively, [20] using 2.1. Preparation of Aβ(1–40). Recombinant Aβ(1–40) was spectrofluorophotometer (Hitachi F-4500) at 37 C. expressed and purified as a ubiquitin extension. The plasmid vector encoding Aβ(1–40) was constructed and cloned as a fusion protein with hexahistidine-tagged ubiquitin (His - 2.4. VUVCD Measurements. Aβ(1–40) was dissolved at a Ub) using the pET28a(+) vector (Novagene), subsequently concentration of 0.2 mM in 10 mM potassium phosphate transformed into Escherichia coli strain BL21-CodonPlus buffer (pH 7.2). The CD spectra of Aβ(1–40) in the (Stratagene) [15]. Transformed bacteria were grown at presence or absence of GM1 were measured from 265 ◦ -4 ◦ 37 C in LB media containing 15 μg/mL of kanamycin. For to 175 nm under a high vacuum (10 Pa) at 37 C using the production of isotopically labelled Aβ(1–40) protein, the VUVCD spectrophotometer constructed at beamline International Journal of Alzheimer’s Disease 3 15 (0.7 GeV) of the Hiroshima Synchrotron Radiation Center (HiSOR). Details of the spectrophotometer and optical cell were described previously [21, 22]. Thepathlengthof the CaF cell was adjusted with a Teflon spacer to 50 μm or 100 μm for measurements. The VUVCD spectra were recorded with a 1.0-mm slit, a 16-s time constant, a 4- -1 nm min scan speed, and nine accumulations. The molar ellipticities of Aβ(1–40) were calculated with the average residue weight of 107.5. The secondary structure contents of Aβ(1–40) were analysed using the modified SELCON3 program [23] and the VUVCD spectra down to 160 nm for 31 reference proteins with known X-ray structures [24, 0 5 10 15 20 25 30 35 40 45 25]. The secondary structures of these proteins in crystal GM1 or lyso-GM1/Aβ(1–40) form were assigned into four classes (α-helices, β-strandes, Figure 1: ThT fluorescence enhancement by Aβ(1–40) in the turns, and unordered structures) using the DSSP program presence of varying concentrations of GM1 (open circle) or lyso- [26] based on the hydrogen bonds between adjacent amide GM1 (closed circle). Each intensity value indicates the average of groups. In this analysis, the 3 -helix was classified as an four values ± S.D. unordered structure. The root-mean-square deviation (δ) and the Pearson correlation coefficient (r) between the X-ray and VUVCD estimates of the secondary structure contents ×10 of the reference proteins were 0.058 and 0.85, respectively, confirming the high accuracy of the VUVCD estimation [27]. 2.5. NMR Measurements. NMR spectral measurements were made on a Bruker DMX-500 spectrometer equipped with a cryogenic probe as well as a Bruker AVANCE III-400 spectrometer. The probe temperature was set to 37 C. Iso- topically labelled Aβ(1–40) was dissolved at a concentration of 0.2 mM in 10 mM potassium phosphate buffer (pH 7.2) containing 10% (v/v) H O in the presence or absence of 1 15 GM1. For H- N heteronuclear single-quantum correlation (HSQC) measurements, the spectra were recorded using −10 Aβ(1–40) labelled with Nuniformlyorselectively at the 39 40 1 amide group of Val or Val at a Hobservation frequency of 500 MHz with 128 (t ) × 1024 (t ) complex points and 1 2 −20 256 scans per t increment. The spectral width was 1720 Hz 1 180 200 220 240 15 1 for the N dimension and 6000 Hz for the H dimension. Wavelength (nm) One-dimensional carbonyl C spectra were recorded 13 15 1 Figure 2: VUVCD spectra of 0.2 mM Aβ(1–40) in the absence or using uniformly C- and N-labelled Aβ(1–40) at a H presence of GM1. Aβ/GM1 molar ratios were 1 : 0 (black), 1 : 15 observation frequency of 400 MHz with a spectral width (blue), and 1 : 30 (red). of 22,000 Hz. In these experiments, 32,768 data points for acquisition and 16,384 scans were acquired. NMR spectra were processed and analysed with the program nmrPipe/Sparky. observed fluorescence intensity remained almost constant up to 12 h. These data indicated that GM1 micelles at appro- priate Aβ/GM1 ratios promote some Aβ–Aβ interaction 3. Results with formation of their β-sheet-like conformation, which, however, does not result in irreversible fibril formation. 3.1. ThT Fluorescence Enhancement. We examined whether ThT fluorescence is enhanced by Aβ(1–40) in the presence of varying concentrations of GM1 or lyso-GM1. As shown 3.2. Secondary Structure Transition. We characterized the in Figure 1, GM1 exhibited a bell-shaped dependence on conformational transition of Aβ depending on Aβ/GM1 Aβ/GM1 ratios regarding ThT fluorescence enhancement, ratios by CD measurements. The short-wavelength limit of while lyso-GM1 showed virtually no enhancement. Max- CD spectroscopy can be successfully extended using syn- imum enhancement was observed at a 1:15 molar ratio chrotron radiation as a high-flux source of photons, which of Aβ(1–40) to GM1. The dynamic light scattering data yields much more accurate data than those obtained with a confirmed that the GM1 and lyso-GM1 micelles exhibited conventional CD spectrophotometer [28, 29]. The spectral an approximate hydrodynamic radius of 6 nm and 4 nm, data indicated that Aβ(1–40) undergoes conformational respectively, irrespective of the Aβ/ganglioside ratios. The transitions depending on GM1 to Aβ(1–40) ratios (Figure 2). ThT fluorescence intensity (a.u.) (θ)(degcm /dmol) 4 International Journal of Alzheimer’s Disease consistent with the VUVCD data as well as the results of the ThT assay. 3.3. Local Structure of the C-Terminus of Aβ(1–40). To provide more detailed information on the conformational (a) transition of Aβ(1–40) on GM1 micelles, we observed H- N HSQC spectral changes of Aβ(1–40) upon titration with GM1. Interestingly, at an Aβ/GM1 molar ratio of 1 : 15, Aβ(1–40) exhibited HSQC peaks that were not observed in the spectra of free or fully micelle-bound forms (b) (Supplementary Figure 1). By using site-specifically N- labelled Aβ, these extra peaks were assigned to Val and Val (Figure 4 and Supplementary Figure 1 available online at doi:10.4061/2011/925073). Namely, the amide groups of these C-terminal residues of the micelle-bound Aβ species show double HSQC peaks under the condition where 188 186 184 182 180 178 176 174 172 170 168 13 Aβ/GM1 ratio is relatively high. More interestingly, these C (ppm) double peaks were perturbed upon the addition of ThT, (c) while the corresponding peaks originating from the free and fully micelle-bound forms showed little or no change 13 13 Figure 3: Carbonyl C spectra of uniformly C-labelled Aβ(1–40). 1 15 (Figure 4). On the other hand, many of the H- NHSQC Spectral data were obtained using 0.2 mM Aβ(1–40) titrated with 39 40 peaks from Aβ(1–40), including Val and Val ,werenot GM1 micelles at Aβ/GM1 molar ratios of (a) 1 : 0, (b) 1 : 15, and observed at an Aβ/lyso-GM1 molar ratio of 1 : 15 due to (c) 1 : 30. In (b), the spectra measured in the presence of ThT are displayed at Aβ/ThT molar ratios of 1 : 0 (black), 1 : 1 (red), and 1 : 2 intermediate chemical exchange between free and micelle- (blue). bound states of Aβ(1–40) (data not shown). 4. Discussion Table 1: Secondary structure contents (%) of Aβ(1–40) from VUVCD spectra obtained in the presence of varying concentrations Accumulating evidence, including our previous reports, of GM1. indicates that the interaction of Aβ with GM1 involves Aβ :GM1 α-Helix β-Strand Turn Unordered structure multiple steps including the initial encounter complex formation and the accommodating process on the 1 : 0 15.9 17.8 26.3 39.0 hydrophilic/hydrophobic interface of the ganglioside 1 : 15 23.6 23.6 21.6 29.3 clusters [15–17, 30]. NMRspectraldataofAβ(1–40) titrated 1 : 30 40.0 18.3 14.5 27.9 with GM1 micelles under Aβ-excess conditions indicated that they form a weak complex presumably through an interaction between the N-terminal segment of Aβ(1–40) The secondary structure contents of Aβ(1–40) at Aβ/GM1 and the outer carbohydrate branch of GM1 [15, 30]. molar ratios of 1 : 0, 1 : 15, and 1 : 30 were estimated on the Thus, it is conceivable that the outer-branch structures basis of the spectral data (Table 1). The α-helix content of of the carbohydrate moieties of gangliosides influence the Aβ(1–40) in the presence of GM1 at an Aβ/GM1 molar ratio association phase of the interaction and thereby determine of 1:30 was calculated to be 40.0%, which is consistent with the ganglioside specificities of Aβ. Nongangliosidic micelles our previous estimation based on the backbone chemical and vesicles are barely or not capable of trapping Aβ(1– shift data of lyso-GM1 [15], thus confirming close similarity 40) effectively [15, 18, 31, 32]. On the other hand, the of the binding modes of Aβ(1–40) between GM1 and lyso- α-helical conformation of Aβ(1–40) accommodated on GM1micelles. At an Aβ/GM1 molar ratio of 1:15, where sugar-lipid interface of the GM1 and lyso-GM1 micelles the maximum ThT fluorescence enhancement was observed, have been characterized by NMR under ganglioside-excess the CD data consistently indicated a significantly increased conditions (Aβ/ganglioside molar ratio of 1 : 30) [15]. content of β-strands. Because the structure of the inner part is common among The conformation of Aβ(1–40) in the presence of varying the gangliosides, non-GM1 ganglioside, for example, GM2, amounts of GM1 micelles was further characterized by can accommodate Aβ and induce its α-helical conformation 13 13 C NMR spectroscopy. The carbonyl CNMR spectral [16]. Thus, the spectroscopic characterization of the data of uniformly C-labelled Aβ(1–40) indicated that the interactions of Aβ with gangliosidic micelles has so far peaks shifted upfield, roughly corresponding to β-structures, been performed only under the extreme conditions of the are more populated at an Aβ/GM1 molar ration of 1 : 15 Aβ/ganglioside ratios. The present study attempts to bridge in comparison with the GM1-excess conditions (Figure 3). the gap in our understanding of Aβ behavior on GM1 Intriguingly, intensities of these peaks were selectively micelles by carrying out spectroscopic analyses of Aβ in the reduced upon the addition of ThT. These NMR data are again presence of varying amounts of GM1 micelles. International Journal of Alzheimer’s Disease 5 39 39 39 Val Val Val 1:15 1:30 1:0 117 117 117 118 118 118 119 119 120 120 120 8.18 7.97.8 8.18 7.97.8 8.18 7.97.8 1 1 1 H (ppm) H (ppm) H (ppm) (a) (b) (c) 40 40 40 Val Val Val 120 120 120 1:0 1:15 1:30 122 122 122 124 124 124 126 126 126 128 128 128 130 130 130 7.87.67.4 7.87.67.4 7.87.67.4 1 1 1 H (ppm) H (ppm) H (ppm) (d) (e) (f ) 1 15 39 40 Figure 4: H- N HSQC peak originating from Val (upper) and Val (lower) of Aβ(1–40) in the presence or absence of GM1 micelles and ThT. Site specifically N-labelled Aβ(1–40) proteins (0.2 mM each) were titrated with GM1 at Aβ/GM1 molar ratios of 1 : 0 (a, d), 1 : 15 (b, e), and 1 : 30 (c, f ). The spectra measured in the absence (black) and presence (red) of 0.4 mM ThT are overlaid. The peak indicated by asterisk originated from GM1. The present data all indicated that β-structure is more follows. At an extremely low concentration of GM1, most populated in micelle-bound Aβ(1–40) under the condition of Aβ(1–40) exists as a free form, which is an unstructured where the Aβ/GM1 ratio is higher. It is intriguing that the monomer and therefore is not reactive with ThT. Fraction increased β-structure is reactive with ThT. Although the of the micelle-bound form of Aβ(1–40) increases with binding mode of ThT to amyloid fibrils has yet to be fully increase of the GM1 amounts. To some extent, the micelles elucidated, it has been suggested that ThT is more likely promote intermolecular interaction of Aβ(1–40), giving rise to bind perpendicularly to parallel β-strands in a β-sheet to the ThT-reactive Aβ(1–40) species. Under GM1-excess [33–35]. In addition, recently reported solid-state NMR data conditions, however, Aβ(1–40) molecules are presumably indicate that a ThT-reactive, neurotoxic amyloid intermedi- relatively isolated from one another and therefore are not ate of Aβ(1–40) is composed of parallel β-structures [36]. capable of forming an intermolecular β-structure. The These data suggest that formation of parallel β-strands is the Aβ/GM1 molar ratio, where the maximum enhancement was minimum prerequisite for ThT fluorescence enhancement. observed, was 1 : 15, which corresponds to average number of With this in mind, the bell-shape dependence of ThT Aβ/micelle of 11.2 with the assumption of the micellar GM1 fluorescence enhancement (Figure 1) can be interpreted as aggregation number of 168 ± 4[37]. Thus, the Aβ density on N (ppm) N (ppm) N (ppm) N (ppm) N (ppm) N (ppm) 6 International Journal of Alzheimer’s Disease GM1 micelles is a crucial factor determining the occurrence tural insights into the mechanisms underlying the α-to-β of the ThT-reactive Aβ species. conformational transition of Aβ on GM1 clusters, which is Under the circumstance where the Aβ(1-40) density on associated with the nucleation process in the Aβ aggregation. GM1 micelles is high, the C-terminal dipeptide of Aβ(1– 40) shows, at least, two distinct conformational states that Abbreviations are reactive with ThT. In a previous paper, we demonstrated 39 40 Aβ:Amyloid β that the C-terminal Val -Val dipeptide is inserted into the AD: Alzheimer’s disease hydrophobic interior of the gangliosidic micelles [15]. This CD: Circular dichroism C-terminal segment is involved in the parallel β-structure GST: Glutathione S-transferase in the amyloid fibril and intermediate [36, 38]. On the His -Ub: Hexahistidine-tagged ubiquitin basis of these data, we suggest that GM1 clusters pro- HSQC: Heteronuclear single-quantum correlation mote intermolecular Aβ-Aβ interactions coupled with the IPTG: Isopropyl-β- -thiogalactopyranoside conformational transition of their C-terminal hydrophobic NMR: Nuclear magnetic resonance anchors into the ThT-reactive parallel β-structure, in which SDS: Sodium dodecyl sulfate the local chemical environments of the C-terminal segments ThT: Thioflavin T are different in different β-strands. This may account for VUV: Vacuum-ultraviolet the multiple HSQC peaks originating from the C-terminal YUH-1: Yeast ubiquitin hydrolase-1. segments (Figure 4). It has been reported that Aβ exhibits ThT-reactive β- sheet-rich aggregates in the presence of sodium dodecyl Acknowledgments sulfate (SDS) at submicellar concentrations [39, 40]. Under The authors wish to acknowledge Dr. Yoshiki Yamaguchi these conditions, all the amide peaks of Aβ(1–40) disap- 1 15 (RIKEN) for his useful discussions on the NMR analyses. peared from the H- N HSQC spectrum because of the This work was supported in part by the Nanotechnology formation of large aggregates, except for those from the Network Project and Grants-in-Aid for Scientific Research C-terminal residues that should still be mobile in this (nos. 20023033 and 20107004) from the Ministry of Edu- assembly state. On the basis of the NMR data obtained using cation, Culture, Sports, Science, and Technology of Japan, paramagnetic probes, the C-terminal segment of Aβ(1–40) the CREST project from the Japan Science and Technology bound to SDS micelles has shown to be exposed to aqueous Agency, and the Research Funding for Longevity Sciences environment, exhibiting higher mobility [41]. Taking into (22–14) from National Center for Geriatrics and Gerontol- account these data in conjunction with our present data, ogy, Japan. M. Yagi- Utsumi is a recipient of a Japan Society we suggest that different β-like structures of Aβ(1–40) for the Promotion of Science Research Fellowship for Young are induced by GM1 aqueous micelles and submicellar Scientists. concentrations of SDS. Lyso-GM1 micelles could not induce the formation of the ThT-reactive β-structure of Aβ(1–40) although the micelle- References interacting modes of Aβ(1–40) are almost identical between [1] F. Chiti and C. M. Dobson, “Protein misfolding, functional GM1 and lyso-GM1 micelles under ganglioside-excess con- amyloid, and human disease,” Annual Review of Biochemistry, ditions [15]. By inspection of the dynamic light scattering vol. 75, pp. 333–366, 2006. data on an assumption of their globular shapes, the diameters [2] G.B.Irvine,O.M.El-Agnaf, G. M. 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Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles

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Copyright © 2011 Maho Yagi-Utsumi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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SAGE-Hindawi Access to Research International Journal of Alzheimer’s Disease Volume 2011, Article ID 925073, 8 pages doi:10.4061/2011/925073 Research Article Spectroscopic Characterization of Intermolecular Interaction of Amyloid β Promoted on GM1 Micelles 1, 2 3 4 3 Maho Yagi-Utsumi, Koichi Matsuo, Katsuhiko Yanagisawa, Kunihiko Gekko, 1, 2 and Koichi Kato Graduate school of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya 467-8603, Japan Institute for Molecular Science and Okazaki Institute for Integrative Bioscience, National Institutes of Natural Sciences, 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan Hiroshima Synchrotron Radiation Center, Hiroshima University, 2-313 Kagamiyama, Higashi-Hiroshima 739-0046, Japan Department of Alzheimer’s Disease Research, National Center for Geriatrics and Gerontology, National Institute for Longevity Sciences, 36-3 Gengo, Morioka, Obu, Aichi 474-8522, Japan Correspondence should be addressed to Koichi Kato, kkatonmr@ims.ac.jp Received 13 October 2010; Revised 30 November 2010; Accepted 3 December 2010 Academic Editor: J. Fantini Copyright © 2011 Maho Yagi-Utsumi et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Clusters of GM1 gangliosides act as platforms for conformational transition of monomeric, unstructured amyloid β (Aβ)toits toxic β-structured aggregates. We have previously shown that Aβ(1–40) accommodated on the hydrophobic/hydrophilic interface of lyso-GM1 or GM1 micelles assumes α-helical structures under ganglioside-excess conditions. For better understanding of the mechanisms underlying the α-to-β conformational transition of Aβ on GM1 clusters, we performed spectroscopic characterization of Aβ(1–40) titrated with GM1. It was revealed that the thioflavin T- (ThT-) reactive β-structure is more populated in Aβ(1–40) under conditions where the Aβ(1–40) density on GM1 micelles is high. Under this circumstance, the C-terminal hydrophobic 39 40 anchor Val -Val shows two distinct conformational states that are reactive with ThT, while such Aβ species were not generated by smaller lyso-GM1 micelles. These findings suggest that GM1 clusters promote specific Aβ-Aβ interactions through their C- termini coupled with formation of the ThT-reactive β-structure depending on sizes and curvatures of the clusters. 1. Introduction accelerate Aβ deposition [8]. In vitro experiments have indicated that the Aβ-GM1 interaction depends on the Conformational transitions of unstructured proteins into clustering of GM1, and its carbohydrate moiety alone cannot β-structure-based oligomeric or amyloid states are crucial induce conformational changes of Aβ [15, 30, 31]. processes in the onset and development of a variety of neu- Furthermore, it has been suggested that each of the rodegenerative disorders such as Alzheimer’s disease (AD) heredity variants of Aβ reported thus far has its own and Parkinson’s disease [1, 2]. Amyloid β(Aβ), a major player specificities for gangliosides, which have been supposed to be in AD, is a 40- or 42-amino acid peptide cleaved from its associated with their ectopic deposition [9, 10]. Promotion precursor membrane protein by sequential actions of β-and of amyloid formation in membrane-bound states has also γ-secretases and has a high propensity for toxic aggregation been reported for prion and α-synuclein [11, 12]. For to form cross-β-fibrils [3, 4]. Accumulated evidence indicates example, prion protein has been reported to be localized in the membrane microdomains and caveolae enriched that the GM1 ganglioside, a glycosphingolipid abundant in neuronal cell membranes, interacts with Aβ and promotes with ganglioside, which interacts with prion protein and its assembly, resulting in pathogenic amyloid formation thereby promotes its α-to-β structural conversion [13, 14]. [5–7]. For example, high-density GM1 clustering, which Therefore, detailed conformational characterization of Aβ is exclusively observed in synaptosomes, is suggested to interacting with the ganglioside clusters not only provides 2 International Journal of Alzheimer’s Disease structural information as cues for drug development in pre- cells were grown in M9 minimal media containing [ N] venting and treating AD but also offers general insights into NH Cl (1 g/L) and/or [U- C ] glucose (2 g/L). Protein 4 6 the mechanisms underlying the disease-associated amyloid expression was induced by adding 0.5 mM isopropyl-β- - formation facilitated in membrane environments. thiogalactopyranoside (IPTG) when the absorbance reached In previous papers, we have reported nuclear magnetic 0.8 at 600 nm. After 4 hours, cells were harvested and resonance (NMR) studies of the interactions of Aβ (1–40) then suspended into buffer A (50 mM Tris-HCl, 150 mM with ganglioside clusters using lyso-GM1 micelles (approxi- NaCl, pH 8.0) containing 4-(2-aminoethyl) benzenesul- mate molecular mass 60 kDa) as model systems [15, 16]. Our fonyl fluoride hydrochloride, subsequently disrupted by NMR data showed that Aβ(1–40) is accommodated on the sonication. After centrifugation, the pellet was dissolved hydrophobic/hydrophilic interface of the ganglioside cluster in buffer A containing 8 M urea. His -Ub-Aβ(1–40) was 2+ exhibiting an α-helical conformation under ganglioside- purified by a Ni -nitrilotriacetic acid affinity column (GE excess conditions. In this state, Aβ(1–40) shows an up- Healthcare). Recombinant glutathione S-transferase- (GST- and-down topological mode in which the two α-helices ) tagged yeast ubiquitin hydrolase-1 (YUH-1) was grown 14 24 31 36 at segments His -Val and Ile -Val and the C-terminal until the absorbance reached 0.8 at 600 nm and then induced 39 40 Val -Val dipeptide segment are in contact with the to expressbyIPTG. Cell pelletsweredissolved in buffer hydrophobic interior of the micelles, whereas the remaining B (50 mM Tris-HCl, 1 mM EDTA, 1 mM DTT, pH 8.5) regions are exposed to the aqueous environment. A similar and disrupted by sonication. GST-YUH-1 was purified by tendency of Aβ(1–40) has been observed using excess a glutathione affinity column (GE Healthcare). Aβ(1–40) amounts of GM1, which forms micelles with an approximate protein was enzymatically cleaved from His -Ub by incuba- molecular mass of 140 kDa [15, 17]. These findings indicate tion with GST-YUH-1 for 1 h at 37 Catamolarratio of that ganglioside clusters offer unique platforms at their His -Ub-Aβ(1–40): GST-YUH1 = 10 : 1. The cleaved Aβ(1– hydrophobic/hydrophilic interfaces for binding coupled with 40) was purified by reverse-phase chromatography using an α-helix formation of Aβ molecules. octadecylsilane column (TSKgel ODS-80T , TOSOH) with To gain further insights into the underlying mechanisms a linear gradient of acetonitrile. The fraction containing of the amyloid formation of Aβ, it is necessary to char- Aβ(1–40) was collected and lyophilized. 15 39 acterize the conformational transition from α-helices to β- Synthetic Aβ(1–40) labelled with N selectively at Val structures on the ganglioside clusters. On the basis of the or Val was purchased from AnyGen Co. Both of recombi- circular dichroism (CD) data, Kakio et al. demonstrated nant and synthetic Aβ(1–40) proteins were dissolved at an that Aβ/GM1 ratios influence the secondary structure of approximate concentration of 2 mM in 0.1% (v/v) ammonia Aβ(1–40) on the raft-like lipid bilayers composed of GM1, solution then collected and stored in aliquots at −80 C until cholesterol, and sphingomyelin [18, 19]. Namely, Aβ adopts use. an α-helical structure at lower Aβ/GM1 ratios (≤0.025), 2.2. Preparation of Micelles. Powdered lyso-GM1 and GM1 while it assumes a β-sheet-rich structure at higher ratios were purchased from Takara Bio Inc. and Sigma-Aldrich, (≥0.05). Although more detailed structural information on respectively. These gangliosides were dissolved in methanol. Aβ bound to the GM1 cluster is highly desirable, the small Subsequently, the solvent was removed by evaporation. The unilamellar vesicles used for the CD measurements are still residual ganglioside was suspended at a concentration of too large to investigate with solution NMR techniques. 12 mM in 10 mM potassium phosphate buffer (pH 7.2) and In the present study, we attempt to characterize con- formational states of Aβ(1–40) in the presence of vary- then mixed by vortexing. Micelle sizes were determined by dynamic light scattering using a DynaPro Titan (Wyatt ing amounts of GM1 aqueous micelles using stable- technology). isotope-assisted NMR spectroscopy in conjunction with synchrotron-radiation vacuum-ultraviolet CD (VUVCD) 2.3. Thioflavin T (ThT) Assay. Aβ(1–40) was dissolved at spectroscopy. We found that GM1 micelles also induce a concentration of 0.2 mM in 10 mM potassium phosphate distinct secondary structures of Aβ(1–40) depending on the buffer (pH 7.2) in the absence or presence of 0.4–9 mM Aβ/GM1 ratios. On the basis of the spectroscopic data, we GM1 or lyso-GM1. The samples were kept on ice before will discuss Aβ behaviours on the ganglioside clusters from a measurements. 980 μLof5 μMThT (Sigma)solutionin structural point of view. 50 mM glycine-NaOH buffer (pH 8.5) was added to an aliquot of 20 μL of each sample. Fluorescence was measured 2. Materials and Methods immediately after mixing at the excitation and emission wavelengths of 446 and 490 nm, respectively, [20] using 2.1. Preparation of Aβ(1–40). Recombinant Aβ(1–40) was spectrofluorophotometer (Hitachi F-4500) at 37 C. expressed and purified as a ubiquitin extension. The plasmid vector encoding Aβ(1–40) was constructed and cloned as a fusion protein with hexahistidine-tagged ubiquitin (His - 2.4. VUVCD Measurements. Aβ(1–40) was dissolved at a Ub) using the pET28a(+) vector (Novagene), subsequently concentration of 0.2 mM in 10 mM potassium phosphate transformed into Escherichia coli strain BL21-CodonPlus buffer (pH 7.2). The CD spectra of Aβ(1–40) in the (Stratagene) [15]. Transformed bacteria were grown at presence or absence of GM1 were measured from 265 ◦ -4 ◦ 37 C in LB media containing 15 μg/mL of kanamycin. For to 175 nm under a high vacuum (10 Pa) at 37 C using the production of isotopically labelled Aβ(1–40) protein, the VUVCD spectrophotometer constructed at beamline International Journal of Alzheimer’s Disease 3 15 (0.7 GeV) of the Hiroshima Synchrotron Radiation Center (HiSOR). Details of the spectrophotometer and optical cell were described previously [21, 22]. Thepathlengthof the CaF cell was adjusted with a Teflon spacer to 50 μm or 100 μm for measurements. The VUVCD spectra were recorded with a 1.0-mm slit, a 16-s time constant, a 4- -1 nm min scan speed, and nine accumulations. The molar ellipticities of Aβ(1–40) were calculated with the average residue weight of 107.5. The secondary structure contents of Aβ(1–40) were analysed using the modified SELCON3 program [23] and the VUVCD spectra down to 160 nm for 31 reference proteins with known X-ray structures [24, 0 5 10 15 20 25 30 35 40 45 25]. The secondary structures of these proteins in crystal GM1 or lyso-GM1/Aβ(1–40) form were assigned into four classes (α-helices, β-strandes, Figure 1: ThT fluorescence enhancement by Aβ(1–40) in the turns, and unordered structures) using the DSSP program presence of varying concentrations of GM1 (open circle) or lyso- [26] based on the hydrogen bonds between adjacent amide GM1 (closed circle). Each intensity value indicates the average of groups. In this analysis, the 3 -helix was classified as an four values ± S.D. unordered structure. The root-mean-square deviation (δ) and the Pearson correlation coefficient (r) between the X-ray and VUVCD estimates of the secondary structure contents ×10 of the reference proteins were 0.058 and 0.85, respectively, confirming the high accuracy of the VUVCD estimation [27]. 2.5. NMR Measurements. NMR spectral measurements were made on a Bruker DMX-500 spectrometer equipped with a cryogenic probe as well as a Bruker AVANCE III-400 spectrometer. The probe temperature was set to 37 C. Iso- topically labelled Aβ(1–40) was dissolved at a concentration of 0.2 mM in 10 mM potassium phosphate buffer (pH 7.2) containing 10% (v/v) H O in the presence or absence of 1 15 GM1. For H- N heteronuclear single-quantum correlation (HSQC) measurements, the spectra were recorded using −10 Aβ(1–40) labelled with Nuniformlyorselectively at the 39 40 1 amide group of Val or Val at a Hobservation frequency of 500 MHz with 128 (t ) × 1024 (t ) complex points and 1 2 −20 256 scans per t increment. The spectral width was 1720 Hz 1 180 200 220 240 15 1 for the N dimension and 6000 Hz for the H dimension. Wavelength (nm) One-dimensional carbonyl C spectra were recorded 13 15 1 Figure 2: VUVCD spectra of 0.2 mM Aβ(1–40) in the absence or using uniformly C- and N-labelled Aβ(1–40) at a H presence of GM1. Aβ/GM1 molar ratios were 1 : 0 (black), 1 : 15 observation frequency of 400 MHz with a spectral width (blue), and 1 : 30 (red). of 22,000 Hz. In these experiments, 32,768 data points for acquisition and 16,384 scans were acquired. NMR spectra were processed and analysed with the program nmrPipe/Sparky. observed fluorescence intensity remained almost constant up to 12 h. These data indicated that GM1 micelles at appro- priate Aβ/GM1 ratios promote some Aβ–Aβ interaction 3. Results with formation of their β-sheet-like conformation, which, however, does not result in irreversible fibril formation. 3.1. ThT Fluorescence Enhancement. We examined whether ThT fluorescence is enhanced by Aβ(1–40) in the presence of varying concentrations of GM1 or lyso-GM1. As shown 3.2. Secondary Structure Transition. We characterized the in Figure 1, GM1 exhibited a bell-shaped dependence on conformational transition of Aβ depending on Aβ/GM1 Aβ/GM1 ratios regarding ThT fluorescence enhancement, ratios by CD measurements. The short-wavelength limit of while lyso-GM1 showed virtually no enhancement. Max- CD spectroscopy can be successfully extended using syn- imum enhancement was observed at a 1:15 molar ratio chrotron radiation as a high-flux source of photons, which of Aβ(1–40) to GM1. The dynamic light scattering data yields much more accurate data than those obtained with a confirmed that the GM1 and lyso-GM1 micelles exhibited conventional CD spectrophotometer [28, 29]. The spectral an approximate hydrodynamic radius of 6 nm and 4 nm, data indicated that Aβ(1–40) undergoes conformational respectively, irrespective of the Aβ/ganglioside ratios. The transitions depending on GM1 to Aβ(1–40) ratios (Figure 2). ThT fluorescence intensity (a.u.) (θ)(degcm /dmol) 4 International Journal of Alzheimer’s Disease consistent with the VUVCD data as well as the results of the ThT assay. 3.3. Local Structure of the C-Terminus of Aβ(1–40). To provide more detailed information on the conformational (a) transition of Aβ(1–40) on GM1 micelles, we observed H- N HSQC spectral changes of Aβ(1–40) upon titration with GM1. Interestingly, at an Aβ/GM1 molar ratio of 1 : 15, Aβ(1–40) exhibited HSQC peaks that were not observed in the spectra of free or fully micelle-bound forms (b) (Supplementary Figure 1). By using site-specifically N- labelled Aβ, these extra peaks were assigned to Val and Val (Figure 4 and Supplementary Figure 1 available online at doi:10.4061/2011/925073). Namely, the amide groups of these C-terminal residues of the micelle-bound Aβ species show double HSQC peaks under the condition where 188 186 184 182 180 178 176 174 172 170 168 13 Aβ/GM1 ratio is relatively high. More interestingly, these C (ppm) double peaks were perturbed upon the addition of ThT, (c) while the corresponding peaks originating from the free and fully micelle-bound forms showed little or no change 13 13 Figure 3: Carbonyl C spectra of uniformly C-labelled Aβ(1–40). 1 15 (Figure 4). On the other hand, many of the H- NHSQC Spectral data were obtained using 0.2 mM Aβ(1–40) titrated with 39 40 peaks from Aβ(1–40), including Val and Val ,werenot GM1 micelles at Aβ/GM1 molar ratios of (a) 1 : 0, (b) 1 : 15, and observed at an Aβ/lyso-GM1 molar ratio of 1 : 15 due to (c) 1 : 30. In (b), the spectra measured in the presence of ThT are displayed at Aβ/ThT molar ratios of 1 : 0 (black), 1 : 1 (red), and 1 : 2 intermediate chemical exchange between free and micelle- (blue). bound states of Aβ(1–40) (data not shown). 4. Discussion Table 1: Secondary structure contents (%) of Aβ(1–40) from VUVCD spectra obtained in the presence of varying concentrations Accumulating evidence, including our previous reports, of GM1. indicates that the interaction of Aβ with GM1 involves Aβ :GM1 α-Helix β-Strand Turn Unordered structure multiple steps including the initial encounter complex formation and the accommodating process on the 1 : 0 15.9 17.8 26.3 39.0 hydrophilic/hydrophobic interface of the ganglioside 1 : 15 23.6 23.6 21.6 29.3 clusters [15–17, 30]. NMRspectraldataofAβ(1–40) titrated 1 : 30 40.0 18.3 14.5 27.9 with GM1 micelles under Aβ-excess conditions indicated that they form a weak complex presumably through an interaction between the N-terminal segment of Aβ(1–40) The secondary structure contents of Aβ(1–40) at Aβ/GM1 and the outer carbohydrate branch of GM1 [15, 30]. molar ratios of 1 : 0, 1 : 15, and 1 : 30 were estimated on the Thus, it is conceivable that the outer-branch structures basis of the spectral data (Table 1). The α-helix content of of the carbohydrate moieties of gangliosides influence the Aβ(1–40) in the presence of GM1 at an Aβ/GM1 molar ratio association phase of the interaction and thereby determine of 1:30 was calculated to be 40.0%, which is consistent with the ganglioside specificities of Aβ. Nongangliosidic micelles our previous estimation based on the backbone chemical and vesicles are barely or not capable of trapping Aβ(1– shift data of lyso-GM1 [15], thus confirming close similarity 40) effectively [15, 18, 31, 32]. On the other hand, the of the binding modes of Aβ(1–40) between GM1 and lyso- α-helical conformation of Aβ(1–40) accommodated on GM1micelles. At an Aβ/GM1 molar ratio of 1:15, where sugar-lipid interface of the GM1 and lyso-GM1 micelles the maximum ThT fluorescence enhancement was observed, have been characterized by NMR under ganglioside-excess the CD data consistently indicated a significantly increased conditions (Aβ/ganglioside molar ratio of 1 : 30) [15]. content of β-strands. Because the structure of the inner part is common among The conformation of Aβ(1–40) in the presence of varying the gangliosides, non-GM1 ganglioside, for example, GM2, amounts of GM1 micelles was further characterized by can accommodate Aβ and induce its α-helical conformation 13 13 C NMR spectroscopy. The carbonyl CNMR spectral [16]. Thus, the spectroscopic characterization of the data of uniformly C-labelled Aβ(1–40) indicated that the interactions of Aβ with gangliosidic micelles has so far peaks shifted upfield, roughly corresponding to β-structures, been performed only under the extreme conditions of the are more populated at an Aβ/GM1 molar ration of 1 : 15 Aβ/ganglioside ratios. The present study attempts to bridge in comparison with the GM1-excess conditions (Figure 3). the gap in our understanding of Aβ behavior on GM1 Intriguingly, intensities of these peaks were selectively micelles by carrying out spectroscopic analyses of Aβ in the reduced upon the addition of ThT. These NMR data are again presence of varying amounts of GM1 micelles. International Journal of Alzheimer’s Disease 5 39 39 39 Val Val Val 1:15 1:30 1:0 117 117 117 118 118 118 119 119 120 120 120 8.18 7.97.8 8.18 7.97.8 8.18 7.97.8 1 1 1 H (ppm) H (ppm) H (ppm) (a) (b) (c) 40 40 40 Val Val Val 120 120 120 1:0 1:15 1:30 122 122 122 124 124 124 126 126 126 128 128 128 130 130 130 7.87.67.4 7.87.67.4 7.87.67.4 1 1 1 H (ppm) H (ppm) H (ppm) (d) (e) (f ) 1 15 39 40 Figure 4: H- N HSQC peak originating from Val (upper) and Val (lower) of Aβ(1–40) in the presence or absence of GM1 micelles and ThT. Site specifically N-labelled Aβ(1–40) proteins (0.2 mM each) were titrated with GM1 at Aβ/GM1 molar ratios of 1 : 0 (a, d), 1 : 15 (b, e), and 1 : 30 (c, f ). The spectra measured in the absence (black) and presence (red) of 0.4 mM ThT are overlaid. The peak indicated by asterisk originated from GM1. The present data all indicated that β-structure is more follows. At an extremely low concentration of GM1, most populated in micelle-bound Aβ(1–40) under the condition of Aβ(1–40) exists as a free form, which is an unstructured where the Aβ/GM1 ratio is higher. It is intriguing that the monomer and therefore is not reactive with ThT. Fraction increased β-structure is reactive with ThT. Although the of the micelle-bound form of Aβ(1–40) increases with binding mode of ThT to amyloid fibrils has yet to be fully increase of the GM1 amounts. To some extent, the micelles elucidated, it has been suggested that ThT is more likely promote intermolecular interaction of Aβ(1–40), giving rise to bind perpendicularly to parallel β-strands in a β-sheet to the ThT-reactive Aβ(1–40) species. Under GM1-excess [33–35]. In addition, recently reported solid-state NMR data conditions, however, Aβ(1–40) molecules are presumably indicate that a ThT-reactive, neurotoxic amyloid intermedi- relatively isolated from one another and therefore are not ate of Aβ(1–40) is composed of parallel β-structures [36]. capable of forming an intermolecular β-structure. The These data suggest that formation of parallel β-strands is the Aβ/GM1 molar ratio, where the maximum enhancement was minimum prerequisite for ThT fluorescence enhancement. observed, was 1 : 15, which corresponds to average number of With this in mind, the bell-shape dependence of ThT Aβ/micelle of 11.2 with the assumption of the micellar GM1 fluorescence enhancement (Figure 1) can be interpreted as aggregation number of 168 ± 4[37]. Thus, the Aβ density on N (ppm) N (ppm) N (ppm) N (ppm) N (ppm) N (ppm) 6 International Journal of Alzheimer’s Disease GM1 micelles is a crucial factor determining the occurrence tural insights into the mechanisms underlying the α-to-β of the ThT-reactive Aβ species. conformational transition of Aβ on GM1 clusters, which is Under the circumstance where the Aβ(1-40) density on associated with the nucleation process in the Aβ aggregation. GM1 micelles is high, the C-terminal dipeptide of Aβ(1– 40) shows, at least, two distinct conformational states that Abbreviations are reactive with ThT. In a previous paper, we demonstrated 39 40 Aβ:Amyloid β that the C-terminal Val -Val dipeptide is inserted into the AD: Alzheimer’s disease hydrophobic interior of the gangliosidic micelles [15]. This CD: Circular dichroism C-terminal segment is involved in the parallel β-structure GST: Glutathione S-transferase in the amyloid fibril and intermediate [36, 38]. On the His -Ub: Hexahistidine-tagged ubiquitin basis of these data, we suggest that GM1 clusters pro- HSQC: Heteronuclear single-quantum correlation mote intermolecular Aβ-Aβ interactions coupled with the IPTG: Isopropyl-β- -thiogalactopyranoside conformational transition of their C-terminal hydrophobic NMR: Nuclear magnetic resonance anchors into the ThT-reactive parallel β-structure, in which SDS: Sodium dodecyl sulfate the local chemical environments of the C-terminal segments ThT: Thioflavin T are different in different β-strands. This may account for VUV: Vacuum-ultraviolet the multiple HSQC peaks originating from the C-terminal YUH-1: Yeast ubiquitin hydrolase-1. segments (Figure 4). It has been reported that Aβ exhibits ThT-reactive β- sheet-rich aggregates in the presence of sodium dodecyl Acknowledgments sulfate (SDS) at submicellar concentrations [39, 40]. Under The authors wish to acknowledge Dr. Yoshiki Yamaguchi these conditions, all the amide peaks of Aβ(1–40) disap- 1 15 (RIKEN) for his useful discussions on the NMR analyses. peared from the H- N HSQC spectrum because of the This work was supported in part by the Nanotechnology formation of large aggregates, except for those from the Network Project and Grants-in-Aid for Scientific Research C-terminal residues that should still be mobile in this (nos. 20023033 and 20107004) from the Ministry of Edu- assembly state. On the basis of the NMR data obtained using cation, Culture, Sports, Science, and Technology of Japan, paramagnetic probes, the C-terminal segment of Aβ(1–40) the CREST project from the Japan Science and Technology bound to SDS micelles has shown to be exposed to aqueous Agency, and the Research Funding for Longevity Sciences environment, exhibiting higher mobility [41]. Taking into (22–14) from National Center for Geriatrics and Gerontol- account these data in conjunction with our present data, ogy, Japan. M. Yagi- Utsumi is a recipient of a Japan Society we suggest that different β-like structures of Aβ(1–40) for the Promotion of Science Research Fellowship for Young are induced by GM1 aqueous micelles and submicellar Scientists. concentrations of SDS. Lyso-GM1 micelles could not induce the formation of the ThT-reactive β-structure of Aβ(1–40) although the micelle- References interacting modes of Aβ(1–40) are almost identical between [1] F. Chiti and C. M. Dobson, “Protein misfolding, functional GM1 and lyso-GM1 micelles under ganglioside-excess con- amyloid, and human disease,” Annual Review of Biochemistry, ditions [15]. By inspection of the dynamic light scattering vol. 75, pp. 333–366, 2006. data on an assumption of their globular shapes, the diameters [2] G.B.Irvine,O.M.El-Agnaf, G. M. 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