Wei Yan, Chen Lan, Chen Ji, Ge Lin, He Rong Qiao
State Key Laboratory of Brain and Cognitive Sciences, Institute of Biophysics, Chinese Academy of Sciences, 15 Da Tun Road, Chaoyang District, Beijing 100101, PR China.
BMC Cell Biol. 2009 Feb 13;10:10. doi: 10.1186/1471-2121-10-10.
D-ribose in cells and human serum participates in glycation of proteins resulting in advanced glycation end products (AGEs) that affect cell metabolism and induce cell death. However, the mechanism by which D-ribose-glycated proteins induce cell death is still unclear.
Here, we incubated D-ribose with bovine serum albumin (BSA) and observed changes in the intensity of fluorescence at 410 nm and 425 nm to monitor the formation of D-ribose-glycated BSA. Comparing glycation of BSA with xylose (a control for furanose), glucose and fructose (controls for pyranose), the rate of glycation with D-ribose was the most rapid. Protein intrinsic fluorescence (335 nm), Nitroblue tetrazolium (NBT) assays and Western blotting with anti-AGEs showed that glycation of BSA incubated with D-ribose occurred faster than for the other reducing sugars. Protein intrinsic fluorescence showed marked conformational changes when BSA was incubated with D-ribose. Importantly, observations with atomic force microscopy showed that D-ribose-glycated BSA appeared in globular polymers. Furthermore, a fluorescent assay with Thioflavin T (ThT) showed a remarkable increase in fluorescence at 485 nm in the presence of D-ribose-glycated BSA. However, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose. This suggests that glycation with D-ribose induced BSA to aggregate into globular amyloid-like deposits. As observed by Hoechst 33258 staining, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) activity assay, flow cytometry using Annexin V and Propidium Iodide staining and reactive oxygen species (ROS) measurements, the amyloid-like aggregation of glycated BSA induced apoptosis in the neurotypic cell line SH-SY5Y.
Glycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells.
细胞和人血清中的D-核糖参与蛋白质糖基化，产生晚期糖基化终产物（AGEs），影响细胞代谢并诱导细胞死亡。然而，D-核糖糖化蛋白诱导细胞死亡的机制仍不清楚。
在此，我们将D-核糖与牛血清白蛋白（BSA）孵育，观察410nm和425nm处荧光强度的变化，以监测D-核糖糖化BSA的形成。将BSA与木糖（呋喃糖对照）、葡萄糖和果糖（吡喃糖对照）的糖基化进行比较，D-核糖的糖基化速率最快。蛋白质固有荧光（335nm）、硝基蓝四氮唑（NBT）测定以及用抗AGEs进行的蛋白质印迹显示，与D-核糖孵育的BSA糖基化比其他还原糖更快。当BSA与D-核糖孵育时，蛋白质固有荧光显示出明显的构象变化。重要的是，原子力显微镜观察显示，D-核糖糖化BSA呈球形聚合物出现。此外，用硫黄素T（ThT）进行的荧光测定显示，在存在D-核糖糖化BSA的情况下，485nm处的荧光显著增加。然而，在存在木糖或葡萄糖的情况下，ThT荧光没有显示出相同的显著增加。这表明D-核糖糖基化诱导BSA聚集形成球形淀粉样沉积物。通过Hoechst 33258染色、3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四氮唑（MTT）和细胞计数试剂盒-8（CCK-8）测定、乳酸脱氢酶（LDH）活性测定、使用膜联蛋白V和碘化丙啶染色的流式细胞术以及活性氧（ROS）测量观察到，糖化BSA的淀粉样聚集诱导神经型细胞系SH-SY5Y发生凋亡。
D-核糖糖基化诱导BSA迅速错误折叠并形成球形淀粉样聚集物，这在对神经细胞的细胞毒性中起重要作用。
