The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny

Abstract

Nuclear and cytoplasmic protein glycosylation is a widespread and reversible posttranslational modification in eukaryotic cells. Intracellular glycosylation by the addition of N-acetylglucosamine (GlcNAc) to serine and threonine is catalyzed by the O-GlcNAc transferase (OGT). This ''O-GlcNAcylation'' of intracellular proteins can occur on phosphorylation sites, and has been implicated in controlling gene transcription, neurofilament assembly, and the emergence of diabetes and neurologic disease. To study OGT function in vivo, we have used gene-targeting approaches in male embryonic stem cells. We find that OGT mutagenesis requires a strategy that retains an intact OGT gene as accomplished by using Cre-loxP recombination, because a deletion in the OGT gene results in loss of embryonic stem cell viability. A single copy of the OGT gene is present in the male genome and resides on the X chromosome near the centromere in region D in the mouse spanning markers DxMit41 and DxMit95, and in humans at Xq13, a region associated with neurologic disease. OGT RNA expression in mice is comparably high among most cell types, with lower levels in the pancreas. Segregation of OGT alleles in the mouse germ line with ZP3-Cre recombination in oocytes reveals that intact OGT alleles are required for completion of embryogenesis. These studies illustrate the necessity of conditional gene-targeting approaches in the mutagenesis and study of essential sex-linked genes, and indicate that OGT participation in intracellular glycosylation is essential for embryonic stem cell viability and for mouse ontogeny.I ntracellular protein glycosylation is ubiquitous in eukaryotic cells yet is less studied than other types of posttranslational modifications such as phosphorylation. This is, in part, because of the relatively recent discovery that serine and threonine residues of many cytoplasmic and nuclear proteins are modified by the addition of an O-linked N-acetylglucosamine (O-GlcNAc) (1, 2). Moreover, this modification originally was difficult to detect before the development of new approaches. O-GlcNAc formation, also termed O-GlcNAcylation, has been found on nuclear pore proteins, RNA polymerase II, and many transcription factors, including Sp1, serum response factor, and the estrogen receptors. In addition, cytoskeletal proteins such as Tau, vinculin, talin, ankyrin, neurofilaments, cytokeratins, and clathrin assembly protein AP-3; and viral or oncogene proteins, such as the cytomegalovirus UL32 protein, myc, fos, jun, simian virus 40 T-antigen, and p53 are also .UDP-N-acetyl-D-glucosamine:protein ␤-N-acetyl-D-glucosaminyltransferase (OGT) (EC 2.4.1.94) catalyzes the addition of O-GlcNAc to the polypeptide chain. All higher eukaryotic cell types studied contain OGT activity. Purified rat liver OGT is a heterotrimer with two catalytic subunits of 110 kDa and a 78-kDa subunit of unknown function (22). The 78-kDa form is structurally and immunologically related to the 110-kDa protein and may result from an internal translation start site or fro...

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

“…Ultimately, these signal-transduction cascades dictate cellular behavior, a prime example of which is the activation of T cells during an adaptive immune response to a pathogen. Compared with other types of posttranslational modifications, the glycosylation of intracellular proteins by O-GlcNAc in T cells remains largely unexplored, despite several studies indicating that it possesses functional significance (23,25,(37)(38)(39).…”

“…Mounting evidence suggests that O-GlcNAc functions in the regulation of protein activity much like phosphorylation and other modifications. Although the molecular mechanisms remain mostly unresolved, O-GlcNAc has clear biological significance because deletion of the OGT or OGA gene in mice leads to embryonic lethality (23) or perinatal death (24), respectively. Furthermore, conditional deletion of OGT results in cellular senescence and apoptosis in numerous cell types, including T cells (25).…”

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

“…OGT is essential for embryonic stem cell viability and somatic cell function (O'Donnell et al 2004;Shafi et al 2000). Deletion of OGT in Arabidopsis (Jacobsen et al 1996), Drosophila (Sinclair et al 2009;Gambetta et al 2009), and mice is lethal (Shafi et al 2000), and leads to dauer phenotypes in Caenorhabditis elegans (Hanover et al 2005), suggesting that O-GlcNAc is essential for cellular homeostasis.…”

“…1). Highlighting the importance of O-GlcNAc in cellular homeostasis, deletion of OGT, OGlcNAcase, and other key enzymes in the hexosamine biosynthetic pathway (HBP) is lethal in mammals (Mio et al 1999;Greig et al 2007;Boehmelt et al 2000a, b;Shafi et al 2000;Forsythe et al 2006;Yang et al 2012).…”

The clinical relevance of glycobiology

“…These structures vary significantly between cell types and at different stages of mammalian development and probably play important roles in the interaction of a cell with its cellular and fluid environment (3)(4)(5). Glycoproteins and proteoglycans are essential for normal development in mice (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16), Drosophila melanogaster (17)(18)(19)(20), and Caenorhabditis elegans (18,(21)(22)(23)(24)(25)(26). Table 1 lists mice with null mutations in genes required for glycosylation; other null mutant mice are described in reviews by Stanley (9) and Varki and Marth (11).…”