Discovery and Confirmation of O-GlcNAcylated Proteins in Rat Liver Mitochondria by Combination of Mass Spectrometry and Immunological Methods

Abstract

O-linked β-N-acetylglucosamine (O-GlcNAc) is an important post-translational modification (PTM) consisting of a single N-acetylglucosamine moiety attached via an O-β-glycosidic linkage to serine and threonine residues. Glycosylation with O-GlcNAc occurs on myriad nuclear and cytosolic proteins from almost all functional classes. However, with respect to O-GlcNAcylated proteins special in mitochondria, little attention has been paid. In this study, we combined mass spectrometry and immunological methods to perform global exploration of O-GlcNAcylated proteins specific in mitochondria of rat liver. First, highly purified mitochondrial proteins were obviously shown to be O-GlcNAcylated by immunoblot profiling. Then, β-elimination followed by Michael Addition with Dithiothreitol (BEMAD) treatment and LC-MS/MS were performed to enrich and identify O-GlcNAcylated mitochondrial proteins, resulting in an unambiguous assignment of 14 O-GlcNAcylation sites, mapping to 11 O-GlcNAcylated proteins. Furthermore, the identified O-GlcNAcylated mitochondrial proteins were fully validated by both electron transfer dissociation tandem mass spectrometry (ETD/MS/MS) and western blot. Thus, for the first time, our study definitely not only identified but also validated that some mitochondrial proteins in rat liver are O-GlcNAcylated. Interestingly, all of these O-GlcNAcylated mitochondrial proteins are enzymes, the majority of which are involved in a wide variety of biological processes, such as urea cycle, tricarboxylic acid cycle and lipid metabolism, indicating a role for protein O-GlcNAcylation in mitochondrial function.

O-GlcNAcomic Profiling Identifies Widespread O-Linked β-N-Acetylglucosamine Modification (O-GlcNAcylation) in Oxidative Phosphorylation System Regulating Cardiac Mitochondrial Function

“…brain synaptosomes and liver) using different approaches (11,33,34). Moreover, three O-GlcNAc sites on these proteins have also been unambiguously detected (supplemental Table 2) 11, 33, 34).…”

“…Very recently, several mitochondrial proteins have been identified from different tissues of rodents (i.e. heart (11), liver (34), and brain synaptosomes (33)) by advanced mass spectrometry techniques.…”

The mitochondrial O-linked N-acetylglucosamine transferase (mOGT) in the diabetic patient could be the initial trigger to develop Alzheimer disease

“…However, O-GlcNAcylation might protect against oxidative stress and promote mitochondrial respiration in the aged retina. In a recent and interesting report, Cao et al (2013) confirmed the significance of O-GlcNAcylation in mitochondrial functionality. The authors demonstrated that mitochondrial proteins in the rat liver are O-GlcNAcylated, and that most of these enzymes are involved in the urea cycle, tricarboxylic acid cycle, and lipid metabolism.…”

Discovery of a nucleocytoplasmic O-mannose glycoproteome in yeast

“…Analysis of the subset of proteins for which mammalian orthologs could be reliably established (79/160) revealed that 24% of these have also been reported to be O-GlcNAcylated in rodents or humans (Table S1) (24)(25)(26)(27). This analysis is likely biased by the limited depth of the O-glycoproteome data available currently, and we expect the overlap to increase with deeper characterization of the two glycoproteomes.…”