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Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells.

Wei Y, Chen L, Chen J, Ge L, He RQ - BMC Cell Biol. (2009)

Bottom Line: 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, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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. weiswallow@sohu.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH
Phase contrast microscope image of cells treated with glycated BSA. SH-SY5Y cells were imaged after incubation with rib-glycated BSA (20 μM, 3-day sample) for 8 h. Normal cells were used as controls. Cells were visualized by inverted contrast microscopy. Statistics of apoptotic cells was also shown. Bar = 25 μm.
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Figure 8: Phase contrast microscope image of cells treated with glycated BSA. SH-SY5Y cells were imaged after incubation with rib-glycated BSA (20 μM, 3-day sample) for 8 h. Normal cells were used as controls. Cells were visualized by inverted contrast microscopy. Statistics of apoptotic cells was also shown. Bar = 25 μm.

Mentions: Having determined that rib-glycated BSA polymers were globular amyloid-like aggregates, we were concerned about the cytotoxicity of these aggregates, and thus tested the cytotoxicity of the glycated protein in cell culture. Morphological evaluation of cell line SH-SY5Y was carried out to investigate effect of rib-glycated BSA on neural cells. As shown in Figure 8, rib-glycated BSA markedly induced axonal atrophy, and cells became spherical with condensed nuclei, as visualized by Hoechst 33258 staining under fluorescence microscopy. BSA alone (or incubated with suc), and glycated BSA with the other reducing monosaccharides, did not show axonal atrophy and cell condensation under the experimental conditions used (data not shown). This suggests that rib-glycated BSA is strongly cytotoxic to neurons in cell culture.


Rapid glycation with D-ribose induces globular amyloid-like aggregations of BSA with high cytotoxicity to SH-SY5Y cells.

Wei Y, Chen L, Chen J, Ge L, He RQ - BMC Cell Biol. (2009)

Phase contrast microscope image of cells treated with glycated BSA. SH-SY5Y cells were imaged after incubation with rib-glycated BSA (20 μM, 3-day sample) for 8 h. Normal cells were used as controls. Cells were visualized by inverted contrast microscopy. Statistics of apoptotic cells was also shown. Bar = 25 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2656460&req=5

Figure 8: Phase contrast microscope image of cells treated with glycated BSA. SH-SY5Y cells were imaged after incubation with rib-glycated BSA (20 μM, 3-day sample) for 8 h. Normal cells were used as controls. Cells were visualized by inverted contrast microscopy. Statistics of apoptotic cells was also shown. Bar = 25 μm.
Mentions: Having determined that rib-glycated BSA polymers were globular amyloid-like aggregates, we were concerned about the cytotoxicity of these aggregates, and thus tested the cytotoxicity of the glycated protein in cell culture. Morphological evaluation of cell line SH-SY5Y was carried out to investigate effect of rib-glycated BSA on neural cells. As shown in Figure 8, rib-glycated BSA markedly induced axonal atrophy, and cells became spherical with condensed nuclei, as visualized by Hoechst 33258 staining under fluorescence microscopy. BSA alone (or incubated with suc), and glycated BSA with the other reducing monosaccharides, did not show axonal atrophy and cell condensation under the experimental conditions used (data not shown). This suggests that rib-glycated BSA is strongly cytotoxic to neurons in cell culture.

Bottom Line: 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, ThT fluorescence did not show the same marked increase in the presence of xylose or glucose.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.

View Article: PubMed Central - HTML - PubMed

Affiliation: 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. weiswallow@sohu.com

ABSTRACT

Background: 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.

Results: 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.

Conclusion: 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.

Show MeSH