Glycosylation produces an abundant, diverse, and highly regulated repertoire of cellular glycans that are frequently attached to proteins and lipids. The past decade of research on glycan function has revealed that the enzymes responsible for glycosylation-the glycosyltransferases and glycosidases-are essential in the development and physiology of living organisms. Glycans participate in many key biological processes including cell adhesion, molecular trafficking and clearance, receptor activation, signal transduction, and endocytosis. This review discusses the increasingly sophisticated molecular mechanisms being discovered by which mammalian glycosylation governs physiology and contributes to disease.

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Deletion of the promoter and the first exon of the DNA polymerase beta gene (pol beta) in the mouse germ line results in a lethal phenotype. With the use of the bacteriophage-derived, site-specific recombinase Cre in a transgenic approach, the same mutation can be selectively introduced into a particular cellular compartment-in this case, T cells. The impact of the mutation on those cells can then be analyzed because the mutant animals are viable.

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...

The glycan symbol nomenclature proposed by Harvey et al. in these pages has relative advantages and disadvantages. The use of symbols to depict glycans originated from Kornfeld in 1978, was systematized in the First Edition of “Essentials of Glycobiology” and updated for the second edition, with input from relevant organizations such as the Consortium for Functional Glycomics. We also note that >200 illustrations in the second edition have already been published using our nomenclature and are available for download at PubMed.

Neurofibromatosis type 1 (NF1) is a prevalent genetic disorder that affects growth properties of neural-crest-derived cell populations. In addition, approximately one-half of NF1 patients exhibit learning disabilities. To characterize NF1 function both in vitro and in vivo, we circumvent the embryonic lethality of NF1 null mouse embryos by generating a conditional mutation in the NF1 gene using Cre/loxP technology. Introduction of a Synapsin I promoter driven Cre transgenic mouse strain into the conditional NF1 background has ablated NF1 function in most differentiated neuronal populations. These mice have abnormal development of the cerebral cortex, which suggests that NF1 has an indispensable role in this aspect of CNS development. Furthermore, although they are tumor free, these mice display extensive astrogliosis in the absence of conspicuous neurodegeneration or microgliosis. These results indicate that NF1-deficient neurons are capable of inducing reactive astrogliosis via a non-cell autonomous mechanism.

. O-GlcNAc is thought to act as a modulator of protein function, in a manner analogous to protein phosphorylation; the addition of O-GlcNAc to the protein backbone is dynamic and responds to morphogens, the cell cycle, and changes in glucose metabolism (1). The mechanisms by which O-GlcNAc act are complex, and changes in O-GlcNAc levels have been shown to alter the behavior of specific proteins by modulating the following: 1) the half-life and proteolytic processing of proteins (2-7); 2) subcellular localization (8 -14); 3) protein-protein interactions (6, 15, 16); 4) DNA binding (17); and 5) enzyme activity or regulation (18 -20). One mechanism by which O-GlcNAc may mediate these events is by altering protein phosphorylation. Notably, phosphorylation and O-GlcNAc are reciprocal on some well studied proteins, which include the C-terminal domain of the large subunit of RNA polymerase (21, 22), the c-myc protooncogene (23-25), SV40 large T-antigen (26), estrogen receptor-␤ (7), and endothelial nitric-oxide synthase (18). These observations suggest that O-GlcNAc and phosphorylation may modulate each other (27-29).Increasing extracellular glucose concentrations affects the functioning of key cellular proteins in an O-GlcNAc-dependent manner, including endothelial nitric-oxide synthase (18), mSin3a (30), the transcription factors, YY1 (31), Sp1 (5, 32-34), CREB (35), and the 26 S proteosomal complex (36, 37). UDPGlcNAc:polypeptide O-␤-N-acetylglucosaminyltransferase (OGT; EC 2.4.1.94), the enzyme that adds O-GlcNAc, is responsive across the physiological range of UDP-GlcNAc. Moreover, the substrate specificity of OGT changes at different UDPGlcNAc concentrations (38). Both in vitro and in vivo data support a model where increased UDP-GlcNAc levels, due to hyperglycemia, result in increased O-GlcNAc levels, leading to insulin resistance, a hallmark of type II diabetes (1, 39). These data and others have led researchers to propose that O-GlcNAc is a nutritional sensor (1, 39 -41).In response to multiple forms of stress, cells rapidly increase glucose uptake. The ability of cells to transport glucose has been linked to the capacity of cells to respond and survive deleterious cellular conditions (42)(43)(44)(45)(46)(47)(48)(49)(50)(51)(52)(53)(54)(55)(56). In many studies, blocking both glycolysis (48,51,57) and the hexosamine biosynthetic pathway (58 -61) results in decreased survival of cells. In some instances, alternative energy sources have been provided suggesting that depletion of ATP levels does NOT explain the decrease in survival (48,51,57). Several insulin-resistant models, including the long lived Caenorhabditis elegans Daf-2 knockout, have an increased stress tolerance to a variety of agents (62)(63)(64). Based upon these data, and recent observations suggesting that heat shock protein (HSP) 70 may act as an O-GlcNAc lectin (65), we investigated the possible link between stress tolerance and O-GlcNAc. We demonstrate that in response to all forms of cellular stress tested, multiple cell lines

Glycosylation produces a diverse and abundant repertoire of glycans, which are collectively known as the glycome. Glycans are one of the four fundamental macromolecular components of all cells, and are highly regulated in the immune system. Their diversity reflects their multiple biological functions that encompass ligands for proteinaceous of receptors known as lectins. Since the discovery that selectins and their glycan ligands are important for the regulation of leukocyte trafficking, it has been shown that additional features of the vertebrate immune system are also controlled by endogenous cellular glycosylation. This Review focuses on the emerging immunological roles of the mammalian glycome.Glycosylation is the enzymatic process that produces glycosidic linkages of saccharides to other saccharides, proteins and lipids, and is probably as ancient as life itself. Unicellular and multicellular organisms depend on glycosylation to produce monomeric and multimeric glycan linkages that are essential for cell viability and normal function [1][2][3][4] . The resulting glycome encompasses a diverse and abundant repertoire of glycans, which are one of the four fundamental macromolecular components of all cells (together with nucleic acids, proteins and lipids) (FIG. 1). Glycans have important biological functions in protein maturation and turnover, cell adhesion and trafficking, and receptor binding and activation [5][6][7][8] .Glycosylation is prominent in the lumen of the endoplasmic reticulum (ER) and in the Golgi apparatus. The cellular repertoire of glycans that are produced by glycosylation in these organelles of the secretory pathway reflects the combinatorial expression of subsets of glycosyltransferase and glycosidase enzymes, of which there are more than 200 in the mammalian genome. The formation and breakdown of glycans are regulated at several levels in the cell. One of the mechanisms involves transcriptional regulation of the genes that encode these enzymes, but others include access to substrates and molecular interactions that alter enzyme localization in the lumen of the ER and Golgi 2,9,10 . Changes in the glycome can occur in response to environmental and genetic stimuli, and are frequently associated with the acquisition of altered cellular phenotypes [1][2][3][4][5][6][7][8][9][10] .Glycosylation also occurs among proteins in the cytoplasm and nucleus through the actions of the Ogt glycosyltransferase, which produces a reversible O-linked β-N-acetylglucosamine NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript physiological processes and disease. In contrast to this intracellular glycosidic bond that is formed by Ogt, secretory glycosylation in the ER and Golgi produces a large structural repertoire, including oligomeric glycan linkages that are presented at the cell surface and in extracellular compartments. Intracellular glycosylation has been reviewed elsewhere and is not discussed further in this Review 12 .Glycosylation can substantially modify the structure ...