title: Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress.
abstract: 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.
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA.
authors.forename: Russell A
authors.initials: RA
authors.lastname: Reeves
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA.
authors.forename: Albert
authors.initials: A
authors.lastname: Lee
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA.
authors.forename: Roger
authors.initials: R
authors.lastname: Henry
authors.affiliation: Department of Biological Chemistry, The Johns Hopkins University School of Medicine, 725 N. Wolfe Street, Baltimore, MD 21205-2185, USA. Electronic address: nzachara@jhmi.edu.
authors.forename: Natasha E
authors.initials: NE
authors.lastname: Zachara
mesh_terms: D000117:Acetylglucosamine / Q000737:chemistry / Q000378:metabolism*; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D000918:Antibody Specificity; D045744:Cell Line, Tumor; D003470:Culture Media / Q000737:chemistry; D005947:Glucose / Q000172:deficiency / Q000494:pharmacology; D006801:Humans; D012516:Osteosarcoma / Q000378:metabolism*; D011134:Polysaccharides / Q000737:chemistry; D013312:Stress, Physiological
chemical_list: D000911:Antibodies, Monoclonal; D003470:Culture Media; D011134:Polysaccharides; D005947:Glucose; D000117:Acetylglucosamine
keywords: CTD110.6; Nutrient deprivation; O-GlcNAc; Specificity; Stress response
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