Identification of O-linked N-Acetylglucosamine Proteins in Rat Skeletal Muscle Using Two-dimensional Gel Electrophoresis and Mass Spectrometry

Abstract

O-linked N-acetylglucosaminylation (O-GlcNAc) is a regulatory post-translational modification of nucleo-cytoplasmic proteins that has a complex interplay with phosphorylation. O-GlcNAc has been described as a nutritional sensor, the level of UDP-GlcNAc that serves as a donor for the uridine diphospho-N-acetylglucosamine:polypeptide ␤-N-acetyl-glucosaminyltransferase being regulated by the cellular fate of glucose. Because muscular contraction is both dependent on glucose metabolism and is highly regulated by phosphorylation/dephosphorylation processes, we decided to investigate the identification of O-GlcNAc-modified proteins in skeletal muscle using a proteomic approach. Fourteen proteins were identified as being O-GlcNAc modified. These proteins can be classified in three main classes: i) proteins implicated in the signal transduction and in the translocation between the cytoplasm and the nucleus or structural proteins, ii) proteins of the glycolytic pathway and energetic metabolism, and iii) contractile proteins (myosin heavy chain). A decrease in the O-GlcNAc level was measured in the slow postural soleus muscle after 14-day hindlimb unloading, a model of functional atrophy characterized by a decrease in the force of contraction. These results strongly suggest that O-GlcNAc modification may serve as an important regulation system in skeletal muscle physiology.

Exploring the O -GlcNAc proteome: Direct identification of O -GlcNAc-modified proteins from the brain

“…The tag provides both a straightforward means to enrich low-abundance O-GlcNAc peptides from complex mixtures and a unique signature upon MS͞MS for unambiguous identification of the O-GlcNAc-glycosylated species. In contrast to reported antibody, lectin, and metabolic labeling methods (11)(12)(13), the strategy provides direct evidence of OGlcNAc glycosylation and permits mapping of modification sites to short amino acid sequences. The ability to localize O-GlcNAc is essential for surveying its distribution across the proteome and understanding its functional significance on a given protein or family of proteins.…”

“…Proteins have been tritiumlabeled (10), enriched with lectins or antibodies (11,12), or chemically tagged by metabolic labeling or BEMAD (␤-elimination followed by Michael addition with DTT) (12,13). However, none of the existing methods is ideally suited to the direct, highthroughput identification of O-GlcNAc proteins from tissues or cell lysates.…”

Chemical approaches to understanding O-GlcNAc glycosylation in the brain

“…Traditional methods for detecting O-GlcNAc glycosylation include the use of wheat germ agglutinin (WGA) lectin 81 , pan-specific O-GlcNAc antibodies 73,77 or radiolabeling using β-1,4-galactosyltransferase (GalT) 82 Recently, chemical approaches have been developed to tag O-GlcNAc proteins with reporter groups such as biotin to enable more rapid, sensitive detection. Khidekel and co-workers designed an unnatural substrate for GalT containing a ketone moiety at the C2 position of UDP-galactose 83 (Fig.…”

Excessive O‐GlcNAcylation of proteins suppresses spontaneous cardiogenesis in ES cells

“…Excess O-GlcNAcylation has been detected in the pancreas, corneas, coronary endothelial cells, and atherosclerotic plaques of diabetic patients and laboratory animals [8][9][10]. Numerous skeletal muscle proteins have recently been shown to be O-GlcNAcylated, and variations in O-GlcNAc levels are associated with the development of skeletal muscle atrophy [11,12]. Increased O-GlcNAcylation, in particular, is implicated in impaired cardiac myocyte function and the development of diabetic cardiomyopathy, a condition associated with cardiomyocyte loss [3,5,7,13].…”