Abstract
Modification of serine and threonine residues in proteins by O-linked β-N-acetylgulcosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylgulcosamine transferase (OGT) and β-D-N-acetylgulcosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5′-diphospho-N-acetylgulcosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc2, rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc2-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc2-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc2. Our results demonstrated that N-GlcNAc2-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc2-modifed proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc2-modifed proteins.
Highlights
We previously screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and health tissues and identified some proteins, calreticulin et al, as markers [1,2,3]
We examined the effects of treating the cells with small interference RNA (siRNA) specific for O-linked b-N-acetylgulcosamine transferase (OGT) and with PUGNAc, an inhibitor of O-GlcNAcase, on expression of proteins that reacted with CTD110.6 antibodies under glucose deprivation (Figure 1c)
Our work has demonstrated that production of N-GlcNAc2-modifed proteins will induced by inhibiting the addition of a mannose residue to dolichol-PPGlcNAc2 using the yeast Alg1 ts-mutant
Summary
We previously screened proteins as tumor markers for bladder cancer by proteomic analysis of cancerous and health tissues and identified some proteins, calreticulin et al, as markers [1,2,3]. We attempted to screen for proteins that were key molecules in metastasis and invasiveness by proteomic analysis of bladder carcinoma cell line T24 cells. Application of focused proteomics involves screening of minor key proteins. We examined changes in the O-linked bN-acetylgulcosamine (O-GlcNAc)-glycosylated proteins in T24 cells under glucose deprivation. OGlcNAc glycosylated proteins can be reversiblely deglycosylated by b-D-N-acetylgulcosaminase (O-GlcNAcase) These reversible O-GlcNAc glycosylations are distinct from stable, complex N-linked glycosylations of membrane or secreted proteins, which takes place in the lumen of the endoplasmic reticulum and in the Golgi apparatus. We attempted to identify and characterize the proteins in T24 cells that reacted with the O-GlcNAc-specific antibody CTD110.6 under glucose deprivation. Our results demonstrated that N-GlcNAc2-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6
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