O-GlcNAc-Specific Antibody CTD110.6 Cross-Reacts with N-GlcNAc2-Modified Proteins Induced under Glucose Deprivation

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.

Glucose Deprivation-induced Increase in Protein O-GlcNAcylation in Cardiomyocytes Is Calcium-dependent

“…1C). Furthermore, treatment of lysates from C2C12 myocytes following glucose deprivation, with PNGase F to remove N-glycans as previously described (33), resulted in only a modest decrease in intensity of CTD110.6 positive staining (Fig. 1D).…”

“…Evaluation of Glucose Deprivation on N-Glycan Modification-Cells were treated with PNGase F to hydrolyze N-glycans by incubating lysates with 1/15 unit PNGase for 1 h at 37 ºC based on methods similar to those described previously (33). Concanavalin A (ConA) was obtained from GE Healthcare (17-0450-01).…”

“…A recent report suggested that the increase in CTD110.6 immunoreactive bands in response to glucose deprivation was largely due to cross-reactivity with the attenuated N-linked glycan, chitobiose (Asn-GlcNAc-GlcNAc) (33). This increase in chitobiose could be due to incomplete synthesis of N-linked oligosaccharide chains, perhaps the synthesis of N-linked chitobiose without any addition of mannose, or because of excessive trimming of the oligosaccharide core leaving only chitobiose.…”

Metabolically programmed quality control system for dolichol-linked oligosaccharides

“…Using a reconstituted lipid bilayer, it was demonstrated that the DLO flippase enables the bidirectional movement of DLOs with various oligosaccharide structures in the ER (6). This is also true in mammalian cultured cells, because GlcNAc 2 -PP-dolichol synthesized on the cytosolic side of the ER membrane is transferred to proteins (19). Therefore, the degradation of DLO intermediates (13,14) can theoretically occur on either side of the ER membrane.…”

“…Our results suggest that the pyrophosphatase-mediated degradation of premature DLOs functions as a quality control system to avoid abnormal N-glycosylation under conditions that impair efficient DLO biosynthesis. subset of glycoproteins (19). However, it remains largely unknown how glucose availability controls the biosynthesis of DLOs.…”