Differential Detection of O-GlcNAcylated proteins in the heart using antibodies

Bhargavi Narayanan; Fiddia Zahra; Russell A Reeves; Akanksha Aggarwal; Robert N O'Meally; Roger K Henry; Megan Craven; Avital Jacobson; Robert N Cole; Mark J Kohr; Priya Umapathi; Natasha E Zachara

doi:10.1016/j.ab.2023.115262

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies

Anal Biochem. 2023 Oct 1:678:115262. doi: 10.1016/j.ab.2023.115262. Epub 2023 Jul 27.

Authors

Affiliations

¹ The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
² The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Radiology, David Geffen School of Medicine at UCLA, Los Angeles, CA, 90095, USA.
³ The Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁴ The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; Department of Ophthalmology and Visual Sciences at the Rutgers University New Jersey Medical School Newark, NJ, 07103, USA.
⁵ The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; The Mass Spectrometry and Proteomics Facility, The Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁶ The Department of Environmental Health and Engineering, The Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, 21205, USA.
⁷ The Department Medicine at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA.
⁸ The Department of Biological Chemistry at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA; The Department of Oncology at the Johns Hopkins University School of Medicine, Baltimore, MD, 21205, USA. Electronic address: nzachara@jhmi.edu.

PMID: 37507081
PMCID: PMC10523836 (available on 2024-10-01)
DOI: 10.1016/j.ab.2023.115262

Abstract

Thousands of mammalian intracellular proteins are dynamically modified by O-linked β-N-acetylglucosamine (O-GlcNAc). Global changes in O-GlcNAcylation have been associated with the development of cardiomyopathy, heart failure, hypertension, and neurodegenerative disease. Levels of O-GlcNAc in cells and tissues can be detected using numerous approaches; however, immunoblotting using GlcNAc-specific antibodies and lectins is commonplace. The goal of this study was to optimize the detection of O-GlcNAc in heart lysates by immunoblotting. Using a combination of tissue fractionation, immunoblotting, and galactosyltransferase labeling, as well as hearts from wild-type and O-GlcNAc transferase transgenic mice, we demonstrate that contractile proteins in the heart are differentially detected by two commercially available antibodies (CTD110.6 and RL2). As CTD110.6 displays poor reactivity toward contractile proteins, and as these proteins represent a major fraction of the heart proteome, a better assessment of cardiac O-GlcNAcylation is obtained in total tissue lysates with RL2. The data presented highlight tissue lysis approaches that should aid the assessment of the cardiac O-GlcNAcylation by immunoblotting.

Keywords: Antibodies; Click chemistry; Glycosylation; Heart; O-GlcNAc.

Publication types

Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

MeSH terms

Acetylglucosamine
Animals
Antibodies / metabolism
Contractile Proteins / metabolism
Heart
Mammals / metabolism
Mice
Neurodegenerative Diseases*
Protein Processing, Post-Translational
Proteome / metabolism

Differential Detection of O-GlcNAcylated proteins in the heart using antibodies

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