CALF THYMUS HIGH-MOBILITY GROUP PROTEINS ARE NONENZYMATICALLY GLYCATED BUT NOT SIGNIFICANTLY GLYCOSYLATED

Over the past decade, there have been many reports suggesting the pres ence of complex carbohydrates on nuclear and cytoplasmic proteins in m ammalian cells, Some of the most often cited of these reports deal wit h the glycosylation of the high mobility group (HMG) proteins, These a re relatively abundant chromosomal proteins that are known to be assoc iated with nucleosomes and actively transcribed regions of chromatin, The original report describing HMG protein glycosylation presented sev eral lines of evidence suggesting that these proteins are glycosylated , including carbohydrate compositional analysis and periodic-acid Schi ff staining, We have attempted to repeat these observations with more highly purified protein than was utilized in the original study, Using carbohydrate compositional analysis performed by high pH anion exchan ge chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins, In addition, we found no evidence for the presence of O-GlcNAc, a well known form of nuclear glycosylation, The HMG proteins did react with periodate, s uggesting the presence of a modification containing cis-diols on the p rotein. Several tryptic peptides isolated from HMG 14 and 17 which ret ained the periodate reactivity had in common lysine residues, suggesti ng a potential modification of the epsilon-amino groups of lysines suc h as nonenzymatic glycation, Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.