|Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress.|
Reeves, RA; Lee, A; Henry, R; Zachara, NE
The dynamic modification of nuclear, cytoplasmic, and mitochondrial proteins by O-linked β-N-acetyl-D-glucosamine (O-GlcNAc) has been shown to regulate over 3000 proteins in a manner analogous to protein phosphorylation. O-GlcNAcylation regulates the cellular stress response and the cell cycle, and is implicated in the etiology of neurodegeneration, type II diabetes, and cancer. The antibody CTD110.6 is often used to detect changes in the O-GlcNAc modification. Recently, it has been demonstrated that CTD110.6 recognizes N-linked N,N'-diacetylchitobiose, which is thought to accumulate in cells experiencing severe glucose deprivation. In this study, we have addressed two questions: (1) Which other antibodies used to detect O-GlcNAc cross-react with N-linked N,N'-diacetylchitobiose? (2) Does N-linked N,N'-diacetylchitobiose accumulate in response to other cellular stressors? To delineate between O-GlcNAc and N-linked N,N'-diacetylchitobiose, we developed a workflow that has been used to confirm the specificity of a variety of O-GlcNAc-specific antibodies. Using this workflow we demonstrated that heat shock, osmotic stress, endoplasmic reticulum stress, oxidative stress, DNA damage, proteasomal inhibition, and ATP depletion induce O-GlcNAcylation but not N-linked N,N'-diacetylchitobiose. Moreover, we demonstrated that while glucose deprivation results in an induction in both O-GlcNAc and N-linked N,N'-diacetylchitobiose, the induction of N-linked N,N'-diacetylchitobiose is exacerbated by the removal of fetal bovine serum.
|Antibodies that Detect O-GlcNAc on the Extracellular Domain of Cell Surface Glycoproteins.|
Tashima, Yuko and Stanley, Pamela
J. Biol. Chem., (2014)
The transfer of N-acetylglucosamine (GlcNAc) to Ser or Thr in cytoplasmic and nuclear proteins is a well-known post-translational modification that is catalyzed by the O-GlcNAc transferase OGT. A more recently identified O-GlcNAc transferase, EOGT, functions in the secretory pathway, and transfers O-GlcNAc to proteins with epidermal growth factor-like (EGF) repeats. A number of antibodies that detect O-GlcNAc in cytosolic and nuclear extracts have been previously described. Here we compare seven of these antibodies (CTD110.6, 10D8, RL2, HGAC85, 18B10.C7 (#3), 9D1.E4 (#10) and 1F5.D6 (#14) for detection of the O-GlcNAc modification on extracellular domains of membrane or secreted glycoproteins that may also carry various N- and O-glycans. We found that CTD110.6 binds not only to O-GlcNAc on proteins but also to terminal β-GlcNAc on the complex N-glycans of Lec8 Chinese hamster ovary (CHO) cells that lack UDP-Gal transporter activity and express GlcNAc-terminating, complex N-glycans. We show that CTD110.6, #3 and #10 antibodies can be used to detect cell surface glycoproteins bearing O-GlcNAc. Cell surface glycoproteins recognized by CTD110.6 antibody included NOTCH1 that possesses many EGF repeats with a consensus site for EOGT. Knockdown of CHO Eogt reduced binding of CTD110.6 to Lec1 CHO cells, and expression of a human EOGT cDNA increased the O-GlcNAc signal on Lec1 cells and the extracellular domain of NOTCH1. Thus, with careful controls, antibodies CTD110.6 (IgM), #3 (IgG) and #10 (IgG) can be used to detect membrane and secreted proteins modified by O-GlcNAc on EGF repeats.
|Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.|
Zou, L; Zhu-Mauldin, X; Marchase, RB; Paterson, AJ; Liu, J; Yang, Q; Chatham, JC
The Journal of biological chemistry
The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca(2+) with EGTA or blocking store operated Ca(2+) entry with SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.
|Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc.|
Teo, Chin Fen, et al.
Nat. Chem. Biol., 6: 338-43 (2010)
Studies of post-translational modification by beta-N-acetyl-D-glucosamine (O-GlcNAc) are hampered by a lack of efficient tools such as O-GlcNAc-specific antibodies that can be used for detection, isolation and site localization. We have obtained a large panel of O-GlcNAc-specific IgG monoclonal antibodies having a broad spectrum of binding partners by combining three-component immunogen methodology with hybridoma technology. Immunoprecipitation followed by large-scale shotgun proteomics led to the identification of more than 200 mammalian O-GlcNAc-modified proteins, including a large number of new glycoproteins. A substantial number of the glycoproteins were enriched by only one of the antibodies. This observation, combined with the results of inhibition ELISAs, suggests that the antibodies, in addition to their O-GlcNAc dependence, also appear to have different but overlapping local peptide determinants. The monoclonal antibodies made it possible to delineate differentially modified proteins of liver in response to trauma-hemorrhage and resuscitation in a rat model.