|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.
|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.