Abstract
BackgroundD-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.ResultsHere, 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.ConclusionGlycation with D-ribose induces BSA to misfold rapidly and form globular amyloid-like aggregations which play an important role in cytotoxicity to neural cells.
Highlights
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
bovine serum albumin (BSA) is rapidly glycated in the presence of ribose To investigate whether glycation of a protein with rib is a rapid process, we incubated BSA with rib, glc, xylose, fructose or sucrose, and monitored changes in the intensity of the fluorescence that is commonly used to detect the formation of glycated products [28,29,30,31]
BSA alone, or in the presence of suc as a negative control, showed no significant changes in fluorescence. This suggests that furanose, especially rib, reacts much more rapidly with BSA than pyranose in the production of glycated products
Summary
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. The mechanism by which D-ribose-glycated proteins induce cell death is still unclear. Non-enzymatic glycation of proteins by reducing saccharides such as glucose (glc) and ribose (rib) leads to the formation of fructosamine [1] and advanced glycation end products (AGEs) [2,3]. The role of glc in the glycation of proteins has been widely studied, and is implicated in diabetes [4], cataracts [5], renal failure [6], and other disorders [7]. Protein crosslinking by glycation results in the formation of detergentinsoluble and protease-resistant aggregates. The study of AGEs has become one of the most important areas of biomedical research today
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