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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
10% SDS-PAGE of the products of BSA incubation with different saccharides. Incubation conditions were as in Figure 1, except that aliquots were taken from incubation mixtures at different time intervals for SDS-PAGE electrophoresis.
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Figure 2: 10% SDS-PAGE of the products of BSA incubation with different saccharides. Incubation conditions were as in Figure 1, except that aliquots were taken from incubation mixtures at different time intervals for SDS-PAGE electrophoresis.

Mentions: To investigate further whether glycation was faster in the presence of rib than other reducing sugars, we incubated BSA with rib and analysed aliquots taken at different time intervals by SDS-PAGE (Figure 2). Retardation of protein bands was observed during the glycation of BSA in the presence of rib. The increasing apparent molecular masses of BSA during glycation probably resulted from bound rib. Analysis using mass spectrometry showed that the difference in the molecular mass between rib-glycated BSA (incubated for 7 days) and native BSA was 5,478 Da, showing ~41 rib (133 Da each) molecules bound to BSA. However, the retardation of BSA bands that had been incubated with other reducing sugars was not so distinct as that of BSA glycated with rib observed under the same conditions. No significant band retardation was exhibited with BSA alone or in the presence of suc which was used as a negative control. These results demonstrate that BSA is more vulnerable to rib glycation than to glycation with the other sugars tested.


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)

10% SDS-PAGE of the products of BSA incubation with different saccharides. Incubation conditions were as in Figure 1, except that aliquots were taken from incubation mixtures at different time intervals for SDS-PAGE electrophoresis.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: 10% SDS-PAGE of the products of BSA incubation with different saccharides. Incubation conditions were as in Figure 1, except that aliquots were taken from incubation mixtures at different time intervals for SDS-PAGE electrophoresis.
Mentions: To investigate further whether glycation was faster in the presence of rib than other reducing sugars, we incubated BSA with rib and analysed aliquots taken at different time intervals by SDS-PAGE (Figure 2). Retardation of protein bands was observed during the glycation of BSA in the presence of rib. The increasing apparent molecular masses of BSA during glycation probably resulted from bound rib. Analysis using mass spectrometry showed that the difference in the molecular mass between rib-glycated BSA (incubated for 7 days) and native BSA was 5,478 Da, showing ~41 rib (133 Da each) molecules bound to BSA. However, the retardation of BSA bands that had been incubated with other reducing sugars was not so distinct as that of BSA glycated with rib observed under the same conditions. No significant band retardation was exhibited with BSA alone or in the presence of suc which was used as a negative control. These results demonstrate that BSA is more vulnerable to rib glycation than to glycation with the other sugars tested.

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