Regulation of a Cytosolic and Nuclear O-GlcNAc Transferase

Abstract

The O-GlcNAc transferase (OGT) is a unique nuclear and cytosolic glycosyltransferase that contains multiple tetratricopeptide repeats. We have begun to characterize the mechanisms regulating OGT using a combination of deletion analysis and kinetic studies. Here we show that the p110 subunit of the enzyme forms both homoand heterotrimers that appear to have different binding affinities for UDP-GlcNAc. The multimerization domain of OGT lies within the tetratricopeptide repeat domain and is not necessary for activity. Kinetic analyses of the full-length trimer and the truncated monomer forms of OGT suggest that both forms function through a random bi-bi kinetic mechanism. Both the monomer and trimer have similar specific activities and similar K m values for peptide substrates. However, they differ in their binding affinities for UDP-GlcNAc, indicating that subunit interactions affect enzyme activity. The findings that recombinant OGT has three distinct K m values for UDP-GlcNAc and that UDP-GlcNAc concentrations modulates the affinity of OGT for peptides suggest that OGT is exquisitely regulated by the levels of UDP-GlcNAc within the nucleus and cytoplasm.O-GlcNAc is an abundant intracellular posttranslational modification consisting of a single N-acetylglucosamine O-linked to serine/threonine residues. Unlike other carbohydrate modifications, O-GlcNAc is not further modified and is found almost exclusively in the nucleus and cytoplasm. Since it was first described in lymphocytes (1) O-GlcNAc has been found on an ever increasing number of proteins, including RNA polymerase II and its transcription factors, nuclear pore proteins, tumor suppressor proteins, intermediate filaments, viral proteins, and oncoproteins (reviewed in Refs. 2-5). O-GlcNAc is an abundant and dynamic modification exhibiting properties more like phosphorylation than typical N-and O-linked glycosylation (2, 3). The O-GlcNAc modification (termed O-GlcNAcylation) has been suggested to play a direct role in regulating a number of cellular functions including protein synthesis (6, 7), neurofilament assembly (8), and transcription (9 -11). In our laboratory, we have purified and characterized both a UDP-Nacetylglucosamine:peptide N-acetylglucosaminyl-transferase (O-GlcNAc transferase) (12) specific for the attachment of OGlcNAc to proteins and a soluble N-acetyl-␤-D-glucosaminidase (O-GlcNAcase) (13) specific for the removal of O-GlcNAc from proteins. These enzymes may work together to regulate the attachment and removal of O-GlcNAc in response to cellular signals in much the same way that kinases and phosphatases regulate protein phosphorylation.Although significant progress has been made in our understanding of the distribution of O-GlcNAc in the cell, little is known about how the attachment of O-GlcNAc to proteins is regulated. The problem is significant because numerous proteins are O-GlcNAcylated, many at more then one site. A further complexity is added by the lack of a canonical consensus site for the attachment of O-GlcNAc to proteins (4, 5)...

Global Analysis of O-GlcNAc Glycoproteins in Activated Human T Cells

“…Although the expression of full-length ncOGT was not significantly altered in activated T cells, we noted a striking difference in the expression levels of sOGT and possibly mOGT. Because OGT is thought to function as a heterotrimer composed of two ncOGT subunits and one sOGT subunit (73), changes in the relative expression of these subunits may influence substrate specificity or enzymatic activity. Studies with recombinant OGT showed that certain substrates are only modified by specific isoforms (74).…”

Dynamic O-GlcNAcylation and its roles in the cellular stress response and homeostasis

“…Recently, the structure of OGT and the TPR domain have been solved (Lazarus et al 2011;Martinez-Fleites et al 2008;Jinek et al 2004). The TPR domain forms an extended alpha-helix similar to importin-α (Jinek et al 2004), and is important for mediating protein-protein interactions and enzyme activity (Kreppel and Hart 1999;Lubas and Hanover 2000). The catalytic domain contains two Rossman folds, separated by an intervening sequence.…”

Effects of exercise on the hexosamine biosynthetic pathway and glycosylation

“…Furthermore, insulin stimulation recruits OGT from the nucleus to the plasma membrane, wherein OGT catalyzes O-GlcNAcylation on some insulin signaling proteins and attenuates its signal transduction by altering their phosphorylation status, and hepatic overexpression of OGT impairs the transcription of insulin-responsive genes and causes the perturbation of insulin-induced inhibition of gluconeotransfer of a single GlcNAc from UDP-GlcNAc to serine/threonine residues of substrate proteins 25,26) ; whereas OGA catalyzes the removal of the GlcNAc from such residues 27,28) . Since the affinity of OGT for substrate proteins is highly sensitive to the intracellular UDP-GlcNAc level 29) , glucose flux via the hexosamine biosynthetic pathway directly influences O-GlcNAc levels. Indeed, hyperglycemia 30) , glucosamine infusion 30) , GLUT overexpression 31) , and GFAT overexpression 32) , all of which increase flux via the hexosamine biosynthetic pathway, result in increased intracellular UDP-GlcNAc and OGlcNAc levels.…”

“…OGT catalyzes the transfer of a single GlcNAc from UDP-GlcNAc to serine/threonine residues of substrate proteins 25,26) , while OGA catalyzes the removal of the GlcNAc from such residues 27,28) . The affinity of OGT for substrate proteins is highly sensitive to the intracellular UDP-GlcNAc level 29) . …”