Conformational Effects of the A21G Flemish Mutation on the Aggregation of Amyloid β Peptide

Abstract

Among the various hereditary mutants of amyloid β (Aβ) in familial Alzheimer’s disease (AD), the A21G Flemish-type mutant has unique properties showing a low aggregation propensity but progressive deposition in vascular walls. Moreover, in contrast to other familial AD cases that show extensive Aβ_1–42 deposition in the brain, patients with Flemish AD predominantly exhibit the deposition of the Aβ_1–40 isoform. Here we report the structural characterization of the Flemish-type mutant (A21G) in comparison with the wild-type Aβ_1–40 peptide to examine the possible effects of the A21G mutation on the conformation of the Aβ_1–40 isoform. The kinetic analysis of the aggregation of the peptides monitored by thioflavin T fluorescence measurement indicates that the mutation precludes the initial nucleation process of amyloid fibril formation by Aβ_1–40. Spectroscopic data indicate that the Flemish-type mutant bound to aqueous micelles composed of lyso-GM1, in which the mobile N-terminal segment is tethered through the C-terminal helical segment, has reduced α-helical structure compared to the wild-type peptide. Our findings suggest that the mutational perturbation to the membrane binding properties is coupled with the changes in nucleation behavior of Aβ during its fibril formation.

Alzheimer’s disease (AD) and cerebral amyloid angiopathy are characterized by the assembly and deposition of amyloid β (Aβ) peptides, the most abundant of which contain 40 or 42 amino acid residues and are cleaved from a precursor membrane protein by the action of α- and β-secretases.^1,2) These Aβ peptides form cross-β amyloid fibrils that bind thioflavin T (ThT) and are a major component of senile plaques, which are the hallmark of AD.^3,4) Although the Aβ peptides can undergo spontaneous self-assembly in aqueous solution, growing evidence supports the notion that membrane environments, including the presence of ganglioside components, can be key factors involved in the Aβ assembly process.^5–7) Moreover, it has been demonstrated that membranes stimulate nucleation and subsequent aggregation of other pathogenic proteins such as α-synuclein.^8,9)

Various genetic mutations of the Aβ precursor protein have been identified in familial AD, including those causing a single amino acid substitution in the central tripeptide segment of Aβ: the Flemish (A21G), Arctic (E22G), Dutch (E22Q), Italian (E23K), and Iowa (D23N) mutations.^10–14) These hereditary Aβ mutants exhibit different physicochemical properties, such as aggregation propensities and ganglioside specificities, which are likely to be associated with AD pathogenesis.^15–18) Among the Aβ variants, the Flemish mutant has unique properties, such as the lowest aggregation propensity, and undergoes progressive deposition in vascular walls resulting in strokes and formation of unusually large senile plaque cores.^15,19–21) Moreover, in contrast to other familial AD cases with predominant Aβ_1–42 deposition in the brain, the deposits of patients with Flemish AD contain primarily the Aβ_1–40 isoform.¹⁹⁾ Therefore, detailed structural characterization of the effects of the A21G mutation on the intrinsic physicochemical property of the Aβ peptides is crucial for a detailed understanding of the molecular mechanisms underlying the Aβ assembly process in this form of AD.

Computational simulations have been reported concerning the conformational effects of the A21G mutation on Aβ_1–40 as well as Aβ_1–42.^22,23) However, residue-specific experimental characterization of the effects of this mutation in solution by NMR spectroscopy has thus far been limited to artificial constructs: shorter Aβ fragments containing the mutation site, i.e. Aβ_11–28^24,25) and Aβ_21–30,²⁶⁾ and a 55-residue fragment of the Aβ precursor protein²⁷⁾ containing the Flemish-type Aβ. In view of this situation, we here conduct comparative NMR analyses between the Flemish mutant of Aβ_1–40 and its wild-type (WT) counterpart and also present the effects of the A21G mutation on the aggregation behavior of Aβ_1–40.

RESULTS AND DISCUSSION

We prepared the Flemish Aβ_1–40 mutant along with the WT protein using recombinant expression systems. First, we compared the aggregation behaviors of these two Aβ_1–40 variants by monitoring the time-dependent enhancement of ThT fluorescence during incubation under condition known to stimulate aggregation (Fig. 1). Compared with WT Aβ, the Flemish mutant showed a significantly longer lag phase before the onset of fibril formation. This tendency is qualitatively consistent with previous observations reported for the fully oxidized Aβ_1–40 variants.²⁰⁾ We then applied a recently developed mathematical protocol^28,29) to perform a quantitative analysis of the ThT-fluorescence growth curves of the Flemish mutant and the WT peptide. The results summarized in Fig. 1D indicate that the A21G mutation inhibits the primary nucleation process of Aβ fibrillization.

Fig. 1. Changes in the ThT Fluorescence Signals of a Range of Concentrations of WT (A) and Flemish-Type (B) Aβ_1–40 (Red, 2 µM; Orange, 4 µM; Yellow, 6 µM; Light Green, 8 µM; Green, 10 µM; Cyan, 12 µM; Blue, 15 µM; and Purple, 18 µM); (C) Summary of the Reaction Scheme; The Rate Constants Are k₊ (Elongation at Fibril Ends), k_n (Primary Nucleation in Solution of Order n_c), and k₂ (Secondary Nucleation on the Fibril Surface of Order n₂); (D) Summary of the Combined Rate Constants Obtained from the Global Fit of Experimental Data to an Equation with Three Free Parameters: K_M and the Combinations k₊k_n and k₊k₂ for the Entire Dataset (n₂ and n_c Were Set to 2); Gives the Concentration of Half Saturation of Secondary Nucleation

To examine the possible conformational effects of the A21G mutation, we conducted NMR analyses of the WT and Flemish Aβ_1–40 peptides in their free and micelle-bound forms (Fig. 2). The heteronuclear single quantum coherence (HSQC) spectra of their free forms were quite similar to each other, except for signals originating from the mutation site and residues in its proximity, indicating that the WT and the Flemish Aβ_1–40 are both intrinsically disordered in an aqueous solution. In contrast, these two Aβ_1–40 peptides exhibited marked differences in their HSQC spectral properties when they were bound to lyso-GM1 micelles, which were used as membrane mimics. In a previous study, we have shown that the WT Aβ_1–40 is bound to the micelles at the hydrophilic/hydrophobic interface of gangliosidic clusters, with an up-and-down topology in which two α-helices (His14–Val24 and Ile31–Val36) and the C-terminal dipeptide (Val39–Val40) are in contact with the hydrophobic interior and remaining regions are exposed to the aqueous environment.^30,31) In the HSQC spectrum of the Flemish mutant, the majority peaks originating from the N-terminal Val12–Val24 segment were perturbed, while the WT spectral pattern was preserved for the remaining C-terminal segment. Torsion angle predictions based on the backbone chemical shifts confirmed that the C-terminal segment of the Flemish Aβ_1–40 peptide, Ile31–Val36, forms an α-helical structure upon binding to lyso-GM1 micelles. These data indicate that the N-terminal segment of the Flemish mutant is in intermediate conformational exchange between different conformational states on the NMR time scale, whereas the C-terminal α-helical segment remains tightly bound and acts as a membrane anchor. We also compared circular dichroism (CD) spectral data of the WT and Flemish forms of Aβ_1–40 in the presence and absence of lyso-GM1 micelles (Fig. 3). Consistent with the NMR results, the CD data demonstrated that these Aβ_1–40 peptides both adopt a significantly α-helical conformation upon binding to micelles, but the helical content of the Flemish mutant was considerably lower (11%) than that of the WT (37%). These data reveal that the A21G mutation disrupts the first α-helix, at His14–Val24, in the membrane-bound Aβ_1–40 molecule. This tendency is consistent with the results of previous studies of the Aβ_11–28 fragment in sodium dodecyl sulfate (SDS) micelles.²⁵⁾

Fig. 2. ¹H-¹⁵N HSQC Spectra of WT (A) and Flemish-Type (B) Aβ_1–40 and ¹H–¹⁵N TROSY Spectra of WT (C) and Flemish-Type (D) Aβ_1–40 in the Presence of Lyso-GM1 Micelles; The Asterisk Indicates the Peak Originating from Lyso-GM1

Fig. 3. CD Spectra of WT (A) and Flemish-Type (B) Aβ_1–40 (0.05 mM) Were Recorded in the Absence (Dashed Line) and Presence of Lyso-GM1 (1.5 mM)

In summary, our findings indicate that the glycine residue at position 21 acts as a secondary structure breaker in the Flemish mutant. This finding is consistent with a previously reported molecular dynamics simulation that suggested that this mutation suppresses dimerization of Aβ_1–40, in this case mediated by β-strands.²²⁾ Thus, it appears that the Flemish mutation in the Aβ peptide inhibits secondary structure formation upon membrane interaction and self-assembly, both of which are critical steps in the initial nucleation resulting in amyloid plaques. Other important issues, however, remain to be defined including the observation that the fibril formation of the Flemish mutant appears to be specifically promoted through its interaction with ganglioside GD3, which is abundant in vascular walls,¹⁷⁾ and the fact that the A21G mutation appears to inhibit aggregation of Aβ_1–42 more effectively than that of Aβ_1–40 in the clinical states.¹⁹⁾ Structural analyses of the putative interactions of the Flemish and other Aβ variants with their specific gangliosides, and the conformational effects of the A21G substitution on its different isoforms of Aβ are currently in progress.

MATERIALS AND METHODS

Preparation of Aβ_1–40

Recombinant Aβ_1–40 was expressed and purified as previously described.³²⁾ Flemish Aβ_1–40 (A21G) was constructed by standard polymerase chain reaction and genetic engineering techniques. It was expressed and purified using the protocol used for WT Aβ_1–40. Both Aβ_1–40 peptides were dissolved at an approximate concentration of 2 mM in 0.1% (v/v) ammonia and then collected and stored in aliquots at −80°C until use.

Kinetic Assays

For kinetic experiments, aliquots of purified WT and Flemish-type Aβ_1–40 were subjected to size-exclusion chromatography on a Superdex 75 column (GE Healthcare) in 20 mM potassium phosphate buffer, pH 7.2. The fraction corresponding to the center of the monomer peak was collected and used to prepare, by dilution, a series of samples of concentrations between 2 and 18 µM on ice. Protein concentrations were quantitated using the Bradford assay.³³⁾ All samples contained 70 µM ThT. Each sample (100 µL per well) was then placed into a well of a 96-well half-area plate (Corning 3881). Assays were performed at 37°C under quiescent conditions in a plate reader (Fluostar Omega, BMG Labtech). The ThT fluorescence was measured through the bottom of the plate every 600 s with excitation and emission filters of 440 nm and 480 nm, respectively. The ThT fluorescence was followed for 2–3 repeats of each monomer concentration. The kinetic data were fitted using the online aggregation fitter (http://www.amylofit.ch.cam.ac.uk/), which is based on a basin-hopping algorithm.^28,29)

Preparation of Micelles

Powdered lyso-GM1 (TaKaRa Bio Inc.) was suspended at a concentration of 12 mM in 10 mM potassium phosphate buffer, pH 7.2, and then mixed by vortexing

NMR Measurements

Isotopically-labeled WT and Flemish-type Aβ_1–40 were dissolved at a concentration of 0.2 mM in 10 mM potassium phosphate buffer containing 10% (v/v)²H₂O in the presence and absence of 6 mM lyso-GM1. NMR spectral measurements were made on a JEOL EC-920 spectrometer as well as a Bruker DMX-500 and AVANCE800 spectrometers equipped with a cryogenic probe. For the free form, the probe temperature was set to 5°C in order to prevent possible aggregation. Backbone resonance assignments were obtained using 2D ¹H–¹⁵N HSQC and the following three dimensional measurements: HN(CA)NNH, HNCO, HNCA, and HN(CO)CA. In the presence of lyso-GM1, 2D ¹H–¹⁵N transverse relaxation optimized spectroscopy (TROSY) spectra, TROSY-type HNCO, and TROSY-type HNCA were measured at 37°C. The NMR data were processed and analyzed using NMRPipe and SPARKY software. Secondary structural elements were identified using δ2D³⁴⁾ and TALOS³⁵⁾ software.

CD Measurements

Aβ_1–40 was dissolved at a concentration of 50 µM in 10 mM potassium phosphate buffer, pH 7.2, in the presence and absence of lyso-GM1 micelles. CD spectra were measured at 37°C on a Jasco J-725 spectrometer equipped with a 1.0-mm path length quartz cell. Eight scans were averaged for each sample. The averaged blank spectrum was subtracted from that of each sample. The secondary structural contents were analyzed using the program SELCON 3.³⁶⁾

Acknowledgments

We thank Drs. Tuomas Knowles (University of Cambridge) and Koichi Kato (National Institutes of Natural Sciences) for valuable discussions. We are also grateful to Georg Meisl and Dr. Céline Galvagnion for helpful advice. This work was supported in part by a JSPS/the Ministry of Eduction, Culture, Sports, Science and Technology (MEXT) KAKENHI Grant-in-Aid for Yong Scientists (B) (15K21680 to M.Y.-U.), Research Funding for Longevity Sciences (25-19) from the National Center for Geriatrics and Gerontology, the Nanotechnology Platform Program of MEXT of Japan, and the Okazaki ORION project. M. Y.-U. is a recipient of the Naito Foundation Grant for Studying Overseas.

Conflict of Interest

The authors declare no conflict of interest.

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