Ribosylation Rapidly Induces α-Synuclein to Form Highly Cytotoxic Molten Globules of Advanced Glycation End Products

Background

Alpha synuclein (α-Syn) is the main component of Lewy bodies which are associated with several neurodegenerative diseases such as Parkinson''s disease. While the glycation with D-glucose that results in α-Syn misfold and aggregation has been studied, the effects of glycation with D-ribose on α-Syn have not been investigated.

Methodology/Principal Findings

Here, we show that ribosylation induces α-Syn misfolding and generates advanced glycation end products (AGEs) which form protein molten globules with high cytotoxcity. Results from native- and SDS-PAGE showed that D-ribose reacted rapidly with α-Syn, leading to dimerization and polymerization. Trypsin digestion and sequencing analysis revealed that during ribosylation the lysinyl residues (K₅₈, K₆₀, K₈₀, K₉₆, K₉₇ and K₁₀₂) in the C-terminal region reacted more quickly with D-ribose than those of the N-terminal region. Using Western blotting, AGEs resulting from the glycation of α-Syn were observed within 24 h in the presence of D-ribose, but were not observed in the presence of D-glucose. Changes in fluorescence at 410 nm demonstrated again that AGEs were formed during early ribosylation. Changes in the secondary structure of ribosylated α-Syn were not clearly detected by CD spectrometry in studies on protein conformation. However, intrinsic fluorescence at 310 nm decreased markedly in the presence of D-ribose. Observations with atomic force microscopy showed that the surface morphology of glycated α-Syn looked like globular aggregates. thioflavin T (ThT) fluorescence increased during α-Syn incubation regardless of ribosylation. As incubation time increased, ribosylation of α-Syn resulted in a blue-shift (∼100 nm) in the fluorescence of ANS. The light scattering intensity of ribosylated α-Syn was not markedly different from native α-Syn, suggesting that ribosylated α-Syn is present as molten protein globules. Ribosylated products had a high cytotoxicity to SH-SY5Y cells, leading to LDH release and increase in the levels of reactive oxygen species (ROS).

Conclusions/Significance

α-Syn is rapidly glycated in the presence of D-ribose generating molten globule-like aggregations which cause cell oxidative stress and result in high cytotoxicity.