O-GlcNAc Modification of α-Synuclein Can Alter Monomer Dynamics to Control Aggregation Kinetics

Abstract

The intrinsically disordered protein α-Synuclein is identified as a major toxic aggregate in Parkinson's as well as several other neurodegenerative diseases. Recent work on this protein has focused on the effects of posttranslational modifications on aggregation kinetics. Among them, O-GlcNAcylation of α-Synuclein has been observed to inhibit the aggregation propensity of the protein. Here, we investigate the monomer dynamics of two O-GlcNAcylated α-Synucleins, α-Syn(gT72), and α-Syn(gS87) and correlate them with the aggregation kinetics. We find that, compared to the unmodified protein, glycosylation at T72 makes the protein less compact and more diffusive, while glycosylation at S87 makes the protein more compact and less diffusive. Based on a model of the earliest steps in aggregation, we predict that T72 should aggregate slower than unmodified protein, which is confirmed by ThT fluorescence measurements. In contrast, S87 should aggregate faster, which is not mirrored in ThT kinetics of later fibril formation but does not rule out a higher rate of formation of small oligomers. Together, these results show that posttranslational modifications do not uniformly affect aggregation propensity.

Keywords: Parkinson’s disease; aggregation; glycosylation; intramolecular diffusion; posttranslational modification; α-Synuclein.

Figure 1

O-GlcNAc modified α-Synuclein. a) Synthesis of α-Syn(gT72) with C69 and W94 using expressed protein ligation. b) Synthesis of α-Syn(gS87) with C69 and W94 using expressed protein ligation.

Figure 2

Kinetics of α-Syn fibril formation. The indicated α-Syn proteins bearing C69 and W94 (50 μM) where subjected to aggregation conditions and analysis using ThT fluorescence (λ_ex = 450 nm, λ_em = 482 nm). b) The fibrillization onset-times were calculated by measuring the time required for fluorescence to reach 2.5-times the initial reading. Onset-time results are mean ± SEM of experimental replicates (n = 3). Statistical significance was determined using a two-way, unpaired Student’s t test.

Figure 3

Schematic depicting the Trp (W)-Cys (C) contact quenching model. UV radiation excites W to a triplet state where it diffuses toward C at a rate of k_D+. At close contact W is either quenched at a rate of q or diffuse away at a rate of k_D–. Excitation is marked as *.

Figure 4

Observed decay rates. 1/k_obs plotted against viscosity for α-Syn, α-Syn(gT72), and α-Syn(gS87) at 37 °C. Rates and their standard errors were obtained from 1st order decay fits.

Figure 5

Computed rates and diffusion coefficients for α-Syn, α-Syn(gT72) and α-Syn(gS87) at 37 °C. a) Reaction-limited rates. b) Diffusion-limited rates calculated for η = 0.68 cP (37 °C in water). c) Diffusion coefficients. Triangles indicate the lower or upper limits computed in cases where the 1/k_R and 1/k_D+ values were consistent with zero.

Figure 6

Kinetic model of aggregation. a) Formation of oligomers using Scheme 1 for k₁ = k_–1 = 4.7 × 10⁶ s^–1 (black, unmodified protein), k₁ = k_–1 = 7.8 × 10⁶ s^–1 (dark blue, a-syn(gT72)) and k₁ = k_–1 = 1.6 × 10⁶ s^–1 (light blue, a-syn(gS87)). All other rates are the same for each (k_bi = 9.7 × 10⁴ s^–1, k_olig = 100 s^–1). b) Formation of fibrils and c) formation of oligomers using Scheme 1 and Scheme 2. Rates are the same as for (a) with the addition of k_nuc = 0.001 s^–1, k_f = 100 s^–1, k_sec = 100 s^–1. d) Formation of fibrils and e) formation of oligomers. The rates are same as for (b) and (c) except k_f = 1 s^–1 for a-syn(gT72) and a-syn(gS87).