GRP78 is a Major Ribosylated Protein in CHO Cells

Abstract

Over 200 year ago, Chevreul (1786-1889) discovered that the sweetness in the urine of diabetics comes from grape sugar or D-glucose. Glycation with D-glucose leads to the formation of advanced glycation end products (AGEs), which is an important biochemical abnormality that accompanies diabetes mellitus. As one of the most reactive glycation reagents, D-ribose, which can yield large amounts of AGEs in vivo and in vitro, has long been ignored in Diabetes studies. In this laboratory, we have observed abnormally high levels of D-ribose in the urine of diabetic patients, compared with those of age-matched normal control. Our results suggested that diabetes mellitus is not only suffered from dysmetabolism of D-glucose, but also dysmetabolism of D-ribose. Here, we show that treatment with D-ribose in Chinese hamster ovary cells increased expression of cellular AGEs along with the increase of D-ribose concentration and administration time. Mass spectrum analysis of isolated AGE-modified proteins from cell lysates showed glucose-regulated protein 78 kD (GRP78) as a ribosylated protein. Co-immunoprecipitation assay further confirmed interaction between AGEs and GRP78. Immunofluorescence co-localization analysis manifested partial superimposition of AGEs and GRP78. As showed in Western blotting results, changes in GRP78 protein levels in cells exhibited in a time dependent manner in the D-ribose treatment. Inhibition of GRP78 expression with RNAi or function in D-ribose-treated cells both dramatically increased the level of AGEs. The findings from Chinese hamster ovary cells indicated that GRP78 is a major component response to ribosylation playing in protection role for cells in the presence of D-ribose.