State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.
PLoS One. 2010 Feb 4;5(2):e9052. doi: 10.1371/journal.pone.0009052.
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.
α-突触核蛋白（α-Syn）是路易体的主要成分，与帕金森病等几种神经退行性疾病有关。虽然已经研究了与 D-葡萄糖的糖化导致α-Syn 错误折叠和聚集，但尚未研究与 D-核糖的糖化对α-Syn 的影响。
方法/主要发现：在这里，我们表明核糖基化诱导α-Syn 错误折叠，并产生高级糖基化终产物（AGEs），其与高细胞毒性的蛋白质无定形球蛋白形成。来自天然和 SDS-PAGE 的结果表明，D-核糖与α-Syn 快速反应，导致二聚化和聚合。胰蛋白酶消化和测序分析表明，在核糖基化过程中，C 末端区域的赖氨酸残基（K（58）、K（60）、K（80）、K（96）、K（97）和 K（102））比 N 末端区域更快地与 D-核糖反应。使用 Western blotting，在存在 D-核糖的情况下，在 24 小时内观察到α-Syn 糖化产生的 AGEs，但在存在 D-葡萄糖的情况下未观察到。荧光在 410nm 处的变化再次表明，在早期核糖基化过程中形成了 AGEs。在研究蛋白质构象时，CD 光谱法并未清楚地检测到核糖基化α-Syn 二级结构的变化。然而，在存在 D-核糖的情况下，310nm 处的固有荧光显着降低。原子力显微镜观察表明，糖化的α-Syn 的表面形态类似于球状聚集体。无论核糖基化如何，硫黄素 T（ThT）荧光在α-Syn 孵育过程中均增加。随着孵育时间的增加，α-Syn 的核糖基化导致 ANS 荧光发生蓝移（约 100nm）。核糖基化的α-Syn 的光散射强度与天然α-Syn 没有显着差异，这表明核糖基化的α-Syn 以无定形蛋白球蛋白的形式存在。核糖基化产物对 SH-SY5Y 细胞具有高细胞毒性，导致 LDH 释放和活性氧（ROS）水平升高。
结论/意义：在 D-核糖存在下，α-Syn 迅速糖化，生成类似无定形球蛋白的聚集物，导致细胞氧化应激，导致高细胞毒性。
