Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress

Russell A. Reeves, Albert Lee, Roger Henry, Natasha E Zachara

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)8-18
Number of pages11
JournalAnalytical Biochemistry
Volume457
DOIs
StatePublished - Jul 15 2014

Fingerprint

Starvation
Workflow
Antibodies
Glucose
Phosphorylation
Endoplasmic Reticulum Stress
Oxidative stress
Mitochondrial Proteins
Glucosamine
Osmotic Pressure
Medical problems
Nuclear Proteins
Type 2 Diabetes Mellitus
DNA Damage
Shock
Cell Cycle
Proteins
Oxidative Stress
Hot Temperature
Adenosine Triphosphate

Keywords

  • CTD110.6
  • Nutrient deprivation
  • O-GlcNAc
  • Specificity
  • Stress response

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology
  • Cell Biology
  • Medicine(all)

Cite this

Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress. / Reeves, Russell A.; Lee, Albert; Henry, Roger; Zachara, Natasha E.

In: Analytical Biochemistry, Vol. 457, 15.07.2014, p. 8-18.

Research output: Contribution to journalArticle

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