Demonstration of three dopamine molecules bound to α-Synuclein: Implication of oligomerization at the initial stage

Sakurako Shimotakahara, Yuuki Shiroyama, Takashi Fujimoto, Mai Akai, Takaya Onoue, Hiroko Seki, Sayaka Kado, Tomoya Machinami, Yoichi Shibusawa, Kenji Uéda, Mitsuru Tashiro

Chemical Analysis Center, Chiba University, Chiba, Japan.
Department of Chemistry, School of Sciences and Engineering, Meisei University, Tokyo, Japan.
Department of Dementia and Higher Brain Function, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan.
School of Life Science, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.
School of Pharmacy, Tokyo University of Pharmacy and Life Science, Tokyo, Japan.

DOI: 10.4236/jbpc.2012.32017                

Abstract

α-Synuclein is the major component of the filamentous Lewy bodies and Lewy neurites that define neuropathological features and dementia with Lewy bodies. To investigate the role of dopamine (DA) in α-synuclein fibrillation, the structural propensities to form oligomers at the initial stage fibrillation were studied using size exclusion chromatography and various biophysical techniques. Interactions with DA were observed for wild-type α-synuclein and its mutants, A30P, E46K and A53T, using electrospray ionization mass spectrometry (ESI-MS). The results of ESI-MS indicate that an intact α-synuclein, which was not oxidized, had an ability to bind with three molecules of DA at the initial stage. Furthermore, upon binding to DA, α-synuclein oligomerizes to higher molecular weight species. These oligomers are structurally different from amyloid fibrils, as confirmed by thioflavin T and CD analysis.

Keywords

α-Synuclein; Dopamine; Fibrillation; Oligomerization; Mass Spectrometry

1. INTRODUCTION

α-Synuclein is a 140-amino acid protein characterized by an acidic C-terminal region and seven imperfect repeats (consensus KTKEGV) distributed throughout most of the N-terminal half of the polypeptide [1]. α-Synuclein is a highly soluble, heat-stable and natively unfolded protein [2,3] predominantly expressed in the neurons of the central nervous system. It is localized at presynaptic terminals in close proximity to synaptic vesicles [1,4-6], as well as in the neuronal nuclei [7]. Three different missense mutations in the α-synuclein gene, A53T [8], A30P [9], and E46K [10], were identified in the confirmed familial cases of autosomal-dominantly inherited, early-onset Parkinson’s disease (PD) and dementia with Lewy bodies. It has been observed that α-synuclein accumulated in Lewy bodies and Lewy-related neurites mainly forms fibrils in PD [11]. The previous studies [12-17] indicated that the hydrophobic repeat region between residues 65 and 95 within the non-Aβ component of Alzheimer’s disease amyloid (NAC) region and the negative charges at the C-terminal region played a key role in forming fibril structures. Molecular species that were relatively compact but still unfolded were also found to be involved in the fibril formation.

Recently, interactions between α-synuclein and dopamine (DA) have been vigorously studied [18-22]. These studies suggest that distinctly reactive intermediates of α-synuclein interacting with DA induce its oligomerization [20]. Although it has been proposed that the formation of DA-interacting α-synuclein provides an explanation for the dopaminergic pathway of α-synuclein-associated neurotoxicity in PD [18], the details of interactions between α-synuclein and DA are yet to be elucidated. Our group has been engaging in the study of fibrillation process of α-synuclein at the initial stage. The singular value decomposition analysis using the time-dependent CD spectra revealed that five or nine intermediates were formed at the early stage of fibrillation [23]. In our timedependent small angle X-ray scattering (SAXS) measurements, formation of oligomers comprising heptamer was suggested [24]. In order to investigate the detailed oligomer association mechanism of α-synuclein at the initial stage, interactions between α-synuclein and DA have been studied using various biochemical and biophysical techniques. The measurements using an electrospray ionization mass spectrometry (ESI-MS), size exclusion chromatography, fluorescence and CD have been carried out for the wild-type α-synuclein and three mutants, A30P, E46K and A53T, to evaluate the effect of these mutations in oligomerization. Although mechanisms that govern the formation of α-synuclein aggregates and oligomers are not well understood, they are considered to be a central event in the pathogenesis of PD. Moreover, we consider DA to be a one of the critical factors for aggregation or oligomerization event. Hence, elucidation of structural features of α-synuclein oligomerization with DA is expected to provide significantly important clues for the molecular mechanism of PD.

2. MATERIALS AND METHODS

2.1. Preparation of Oligomers and ThT Measurements

The wild-type α-synuclein and three mutants, A30P, E46K and A53T, were cloned, expressed and purified as described previously [23]. α-Synuclein (ca. 2.0 mg/ml) was assembled into oligomers and/or fibrils in the absence and presence of DA (0.1 - 10 mM) by incubating in 10 mM ammonium acetate (pH 7.5) at 30˚C with continuous stirring at 450 rpm. Thioflavin-T (ThT) fluorescence intensities were measured every 15 min at 482 nm with excitation at 450 nm and 100 scans on a microplate reader SH-9000Lab (Corona Electric).

2.2. CD Spectroscopy

CD spectra were recorded using a 1-mm cuvette, scanning from 190 to 250 nm with a step size of 0.1 nm and scanning speed of 50 nm/min on a Jasco J-720 spectrophotometer. For all spectra, an average of eight scans was obtained every 24 hours. CD samples were prepared by diluting 10 μL of the above protein sample (2.0 mg/ml) at various incubation times into 400 μL CD buffer (10 mM sodium phosphate, pH 7.5), resulting in a final protein concentration of 0.05 mg/ml.

2.3. MS Spectrometry

Measurements of FT-MS were carried out for A30P using a LTQ-Orbitrap XL FT-mass spectrometer (Thermo Scientific). The experimental conditions were as follows: desolvation temperature, 200˚C; sample flow rate, 20 μL/min; R = 15000. ESI-MS measurements were carried out for the wild-type, E46K and A53T α-synucleins using a time-of-flight mass spectrometer (JEOL JMS-T100).

The experimental conditions were as follows: acceleration voltage, 2.0 kV; needle voltage, 2600 V; orifice voltage, 65 V; desolvation temperature, 100˚C; sample flow rate, 5.0 μL/min. All spectra were obtained via infusion of the sample solutions in the positive ionization mode.

2.4. Size Exclusion Chromatography

The incubated sample solutions were filtrated through a membrane filter Dismic-03 (pore size, 0.45 μm; Toyo Roshi), and then applied onto a TSKgel G3000SW_XL gel filtration column (7.8 mm I.D. × 300 mm; Tosoh Bioscience) with 50 mM sodium phosphate buffer, pH 7.5, containing 100 mM NaCl. The flow rate was 0.4 mL/min.

3. RESULTS AND DISCUSSION

3.1. Effects of Dopamine in Amyloid Fibril Formation

In order to observe the effects of DA in amyloid fibril formation of α-synucleins, an accumulation of amyloid was monitored by fluorescence spectroscopy using a ThT binding assay. This method is commonly used to observe an amyloid fibril formation of proteins. In general, an increase in ThT fluorescence intensity is considered to be a good indication of filament accumulations [25]. The time-dependent changes of ThT fluorescence intensity in the presence and absence of DA are shown in Figure 1. The striking decreases of ThT fluorescence intensities in the presence of 1 mM DA were observed for the wildtype, A30P and A53T α-synucleins. The ThT fluorescence intensities of E46K in the presence of 1 mM DA were decreased more than 50% of those in the absence of DA. These results indicate that DA significantly inhibited the amyloid fibril formation of the wild-type α-synuclein and its three mutants. Almost no fibril was observed in the wild-type and A30P α-synucleins up to the incubation time of 100 hours (Figure 1(B)).

3.2. β-Sheet Contents; Characterization by CD

Figure 2 shows the time courses of changes in molar ellipticities at 218 nm for the wild-type α-synuclein and its mutants. The amounts of β-sheet structure can be estimated by the negative ellipticity at 218 nm. The overall time dependent changes of ellipticities in the presence and absence of DA were similar for the wild-type, E46K and A53T α-synucleins, and the values of ellipticity was slightly higher for A30P in the presence of 1 mM DA. The results of CD spectra indicate that β-sheet contents were almost unaffected by the presence of DA for the wildtype, E46K and A53T α-synucleins, whereas slight decrease was observed for A30P in the presence of 1 mM DA. Because fibril formation of all mutants were suppressed

Conflicts of Interest

The authors declare no conflicts of interest.

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