Study Of Ribosylation Induced Cytotoxicity And Cognitive Dysfunction

Non-enzymatic glycation of proteins by reducing saccharides is the reaction between a free amino group of a protein and a cargonyl group from a reducing sugar, forming an unstable intermediate product Schiff’s base and a relatively stable Amadori rearrangement product. Then the products go through an array of processes such as autooxidation and cross-linking, finally turn to form an inreversible product called advanced glycation end products (AGEs). Non-enzymatic glycation reaction proceeded widely in vivo. Its products are closely related to the structural change and dysfunction of proteins, what’s more, to the aging and pathological changes of tissues and organs. D-ribose, belonging to reducing aldopentoses, is a basic subunit of RNA and a constituent of metabolic molecules such as ATP, ADP and cAMP. D-ribose has drawn more attention in recent years as an active reducing monosaccharide in glycating proteins and forming AGEs. Nevertheless, the mechanisms of which D-ribose induces cellular cytotoxicity and spatial cognition dysfunction in vivo are hitherto unknown.In this study we used the cultured Chinese hamster ovary (CHO) cells as subjects to investigate the target proteins which are glycated by D-ribose. Treatment with D-ribose decreased cell viability by CCK8and MTT assay. Western blotting results showed that the level of cellular AGEs increased compared to control, and the increase is D-ribose concentration and incubation time dependent. Under the same condition, D-glucose could not induce such remarkable changes in cells. Cellular immunofluorescence microscopy manifested that AGEs in D-ribose treated cells is primarily distributed in the cytoplasm. Mass spectrum results of proteins samples which were purified by anti-AGEs antibody revealed glucose-regulated protein78kD (GRP78) and glucose-regulated protein75kD (GRP75) as the main ribosylated proteins in CHO cells. Co-IP experiments of GRP78and GRP75further confirmed that both of them were AGEs linked proteins. Immunofluorescence image showed partial co-localization of AGEs and GRP78. The cellular level of GRP78increased at the early stage of D-ribose treatment, and decreased at later stage. Western blotting results also showed that GRP78level all significantly decreased at the later stage regardless of the concentration of D-ribose used to incubate the cells. Using GRP78specific siRNA to knockdown the protein level of GRP78and using inhibitors to block the function of GRP78both displayed aggravation of cytotoxicity effect of D-ribose compared to D-ribose treated only group. These results demonstrate that D-ribose glycates cellular proteins and form AGEs fast. The target proteins ribosylated by D-ribose are probably restricted, which may lose their normal structure and function after ribosylation, leading to cellular dysfunction.To investigate protein ribosylation effects in the brain, we intraperitoneally injected C57BL/6J male mice with different concentrations of D-ribose for10days. We found that the higher concentrations of D-ribose accerlerated the level of glycated proteins and AGEs both in the blood serum and brain by fructosamine assay and Western blotting. D-ribose injected mice displayed decline of learning and memory ability in Morris water maze test. Immunohistochemical microscopy manifested the activation of astrocytes in hippocampus region of mice brain. Meanwhile, the expression of the receptor for AGEs (RAGE), the proinflammatory nuclear transcription factor kappa B (NF-κB) and tumor necrosis factor alpha (TNF-α) were found to be up-regulated. These data confirm our hypothesis that D-ribose reacts rapidly with proteins in the brain and produces AGEs in vivo, and may lead to injured spatial cognition through activation of astrocytes and proinflammatory response.To further explore the mechanism underlying ribosylation induced decline in spatial cognition, we studied the relationships between inflammation response and ribosylation effects in two kinds of human astroglioma cells U251and U87MG. The same phenomenon that AGEs, RAGE, NF-κB, TNF-α and GFAP level all became higher after D-ribose treatment were observed in both cell lines. Using RAGE specific siRNA to knockdown the protein level of RAGE and using anti-RAGE antibody to block the function of RAGE both played a role in rescuing inflammatory response effects induced by D-ribose compared to D-ribose treated only group. These data indicate that mouse spatial cognitive impairment caused by D-ribose derived AGEs is correlated with activation of an astrocyte-mediated, RAGE-dependent inflammatory response.Above all, we have shown that D-ribose reacts with proteins quickly both in cultured cells and the mouse brain, produces significant amounts of AGEs. In one hand, D-ribose is prone to glycate some certain cellular proteins to change their important function and leads to cell toxicity. In another hand, ribosylation-induced AGEs bind to RAGE to activate inflammation response and astrocytes, results in damage in mouse spatial cognition.