Ribosylation rapidly induces alpha-synuclein to form highly cytotoxic molten globules of advanced glycation end products

Abstract

Background: Alpha synuclein (alpha-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 alpha-Syn misfold and aggregation has been studied, the effects of glycation with D-ribose on alpha-Syn have not been investigated.

Methodology/principal findings: Here, we show that ribosylation induces alpha-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 alpha-Syn, leading to dimerization and polymerization. Trypsin digestion and sequencing analysis revealed that during ribosylation the lysinyl residues (K(58), K(60), K(80), K(96), K(97) and K(102)) 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 alpha-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 alpha-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 alpha-Syn looked like globular aggregates. thioflavin T (ThT) fluorescence increased during alpha-Syn incubation regardless of ribosylation. As incubation time increased, ribosylation of alpha-Syn resulted in a blue-shift (approximately 100 nm) in the fluorescence of ANS. The light scattering intensity of ribosylated alpha-Syn was not markedly different from native alpha-Syn, suggesting that ribosylated alpha-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: alpha-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.

Figure 1. Ribosylated α-Syn on native-PAGE and SDS-PAGE.

0.7 mM α-Syn was incubated with 1 M D-ribose (A) and 1 M D-glucose (B) for 0–7 days, and then loaded on 15% native-PAGE (left) and 15% SDS-PAGE (right) gels. (C) Native α-Syn alone was used as a control.

Figure 2. Changes in fluorescence during protein riboslyation.

Glycation conditions were the same as those in Fig. 1. (A) Aliquots were taken for measurements of the intrinsic fluorescence at an excitation wavelength of 320 nm after incubation for 3 days. (B) Characteristics of the fluorescence of glycated products. (C) Changes in the maximal fluorescent intensity (λ_ex320 nm; λ_em410 nm) were monitored while α-Syn was incubated with D-ribose, or glucose for different time intervals.

Figure 3. Detection of AGEs by Western blotting and NBT assays.

The glycation conditions of α-Syn were the same as those in Fig. 1. (A) Western blotting of the aliquots (ribosylated α-Syn) with anti-AGEs was performed at different time intervals. (B) Fructosamine was assayed with NBT. (C) Data in panel B were analyzed according to Tsou's method .

Figure 4. Digestion of glycated α-Syn in the presence of trypsin.

(A) Aliquots were digested by trypsin at a mass ratio [α-Syn]/[trypsin] of 20∶1 (37°C, 1 h). (B) Native α-Syn alone was used as a control. (C) α-Syn amino acid sequence. The protein fragments ‘a’ and ‘b’ as shown in panel A were sequenced as described . Lysine residues are shown in green, except those at the cleavage sites (as determined by sequencing) which are shown in red.

Figure 5. Changes in the CD spectra of α-Syn in the presence of D-ribose.

A representative CD spectra of α-Syn incubated with D-ribose (day 3) is shown. Conditions for glycation of α-Syn with D-ribose were the same as those in Fig. 1.

Figure 6. Conformational changes in glycated α-Syn observed by intrinsic fluorescence.

Experimental conditions were the same as those in Fig. 1. (A) Fluorescence spectra of α-Syn glycated with D-ribose at an excitation wavelength of 280 nm. (B) The kinetics of fluorescence in D-ribose glycated with α-Syn was analyzed at different time intervals (λ_ex 280 nm; λ_em 310 nm). (C) The incubation time-dependent increase in non tryptophan fluorescence is shown for 0 to 7 days.

Figure 7. Changes in ANS fluorescence of α-Syn during glycation.

Experimental conditions were the same as those in Fig. 1. (A) ANS (70 µM) was added to samples of ribosylated α-Syn at different time intervals. Fluorescence spectra of ANS were recorded at λex 350 nm. (B) Native α-Syn alone was used as a control.

Figure 8. Glycated α-Syn deposits were imaged by atomic force microscopy.

(A) Aliquots of α-Syn incubated with D-ribose for three days were taken for observation by AFM. (B) Native α-Syn was employed as a control. (C) D-Ribose alone was used as a negative control. (D) Horizontal diameter at mid-height.

Figure 9. Changes in the fluorescence of thioflavin T and light scattering in the presence of ribosylated α-Syn at different time intervals.

(A) ThT (30 µM) was added to samples of α-Syn in different concentrations of D-ribose for different time intervals. The intensity of ThT fluorescence was recorded (λ_ex 450 nm; λ_em 485 nm). The kinetics of the increase in the fluorescence emission of ThT with glycated α-Syn is shown. (B) Aliquots were taken for measurements of the intensity of scattered light (λ_ex 480 nm; λ_em 480 nm) under the same conditions. Native α-Syn alone was used as a control.

Figure 10. Cell viability was measured with the MTT assay.

Cell viability of SHSY5Y cells was measured after adding 3.5 µM, 35 µM and 140 µM ribosylated α-Syn at 24 h (A), 48 h (B) and 72 h (C).

Figure 11. Ribosylated α-Syn induces SH-SY5Y cell apoptosis.

SH-SY5Y cells were treated with ribosylated α-Syn for 8 h and then cultured for 24 h, before analysis by flow cytometry (D). Normal cells were used as controls (A), cells incubated with D-ribose (B) and α-Syn (C) are shown.

Figure 12. LDH assay and measurement of reactive oxygen species in SH-SY5Y cells.

(A) LDH activity was measured using a cytotoxicity detection kit (Roche, Switzerland). (B) The levels of reactive oxygen species in cells treated with glycated α-Syn for 24 h were measured. Cells treated with α-Syn, D-ribose and a blank were used as controls.

Figure 13. A putative scheme for the aggregation of ribosylated α-Syn protein into highly cytotoxic molten globules.