Gerald W. Hart

Cycling of O-linked |[beta]|-N-acetylglucosamine on nucleocytoplasmic proteins

All animals and plants dynamically attach and remove O-linked β-N-acetylglucosamine (O-GlcNAc) at serine and threonine residues on myriad nuclear and cytoplasmic proteins. O-GlcNAc cycling, which is tightly regulated by the concerted actions of two highly conserved enzymes, serves as a nutrient and stress sensor. On some proteins, O-GlcNAc competes directly with phosphate for serine/threonine residues. Glycosylation with O-GlcNAc modulates signalling, and influences protein expression, degradation and trafficking. Emerging data indicate that O-GlcNAc glycosylation has a role in the aetiology of diabetes and neurodegeneration.

Cross Talk Between O-GlcNAcylation and Phosphorylation: Roles in Signaling, Transcription, and Chronic Disease

O-GlcNAcylation is the addition of β-D-N-acetylglucosamine to serine or threonine residues of nuclear and cytoplasmic proteins. O-linked N-acetylglucosamine (O-GlcNAc) was not discovered until the early 1980s and still remains difficult to detect and quantify. Nonetheless, O-GlcNAc is highly abundant and cycles on proteins with a timescale similar to protein phosphorylation. O-GlcNAc occurs in organisms ranging from some bacteria to protozoans and metazoans, including plants and nematodes up the evolutionary tree to man. O-GlcNAcylation is mostly on nuclear proteins, but it occurs in all intracellular compartments, including mitochondria. Recent glycomic analyses have shown that O-GlcNAcylation has surprisingly extensive cross talk with phosphorylation, where it serves as a nutrient/stress sensor to modulate signaling, transcription, and cytoskeletal functions. Abnormal amounts of O-GlcNAcylation underlie the etiology of insulin resistance and glucose toxicity in diabetes, and this type of modification plays a direct role in neurodegenerative disease. Many oncogenic proteins and tumor suppressor proteins are also regulated by O-GlcNAcylation. Current data justify extensive efforts toward a better understanding of this invisible, yet abundant, modification. As tools for the study of O-GlcNAc become more facile and available, exponential growth in this area of research will eventually take place.

Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDPβ-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3β at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and β-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.

Glycomics Hits the Big Time

Cells run on carbohydrates. Glycans, sequences of carbohydrates conjugated to proteins and lipids, are arguably the most abundant and structurally diverse class of molecules in nature. Recent advances in glycomics reveal the scope and scale of their functional roles and their impact on human disease.

Carbohydrates in Chemistry and Biology

= Abstract Carbohydrate Chemistry and Glycobiology have witnessed a rapid expansion during the last few years with the development of numerous new, imaginative and efficient syntheses which provide further insight into structures and biological interactions of glycoconjugates. Glycosylation reactions are widely used in the synthesis of pharmaceuticals and bio-active compounds. In biology and medicine oligosaccharides play a central role in immunostimulation, cancer or allergic responses. Glycoscience is a very instructive example of how one common topic of interest stimulates both chemistry and biology to collectively open scientific frontiers. This synergy is made visible in this work. Three leading experts in the fields of Glycochemistry and Glycobiology have invited numerous renowned authors to provide a comprehensive overview of the recent advances and findings in Glycoscience. This four-volume handbook presents an integrated and cutting-edge view, and covers all chemical aspects, such as syntheses and analysis of carbohydrates and oligosaccharides, as well as the biological role and activity of oligosaccharides and carbohydrate/protein interactions.

Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications

Identifying sites of post-translational modifications on proteins is a major challenge in proteomics. O-Linked β-N-acetylglucosamine (O-GlcNAc) is a dynamic nucleocytoplasmic modification more analogous to phosphorylation than to classical complex O-glycosylation. We describe a mass spectrometry-based method for the identification of sites modified by O-GlcNAc that relies on mild β-elimination followed by Michael addition with dithiothreitol (BEMAD). Using synthetic peptides, we also show that biotin pentylamine can replace dithiothreitol as the nucleophile. The modified peptides can be efficiently enriched by affinity chromatography, and the sites can be mapped using tandem mass spectrometry. This same methodology can be applied to mapping sites of serine and threonine phosphorylation, and we provide a strategy that uses modification-specific antibodies and enzymes to discriminate between the two post-translational modifications. The BEMAD methodology was validated by mapping three previously identified O-GlcNAc sites, as well as three novel sites, on Synapsin I purified from rat brain. BEMAD was then used on a purified nuclear pore complex preparation to map novel sites of O-GlcNAc modification on the Lamin B receptor and the nucleoporin Nup155. This method is amenable for performing quantitative mass spectrometry and can also be adapted to quantify cysteine residues. In addition, our studies emphasize the importance of distinguishing between O-phosphate versus O-GlcNAc when mapping sites of serine and threonine post-translational modification using β-elimination/Michael addition methods.

REGULATION OF A CYTOSOLIC AND NUCLEAR O-GLCNAC TRANSFERASE: ROLE OF THE TETRATRICOPEPTIDE REPEATS

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 homo- and 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 distinctK 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-glycosylation of nuclear and cytosolic proteins. Dynamic interplay between O-GlcNAc and O-phosphate

Despite the long held view that protein glycosylation occurs exclusively on extracellular or lumenal polypeptides (1), it is clear that many nuclear and cytoplasmic proteins are multiply O-glycosylated at specific serine or threonine hydroxyl groups by single b-N-acetylglucosamine moieties (O-GlcNAc) (2–4). O-GlcNAc modification is common to nearly all eukaryotes, including filamentous fungi, plants, animals, and animal parasites, as well as viruses that infect eukaryotes. Mounting evidence suggests a direct role for O-GlcNAc in cellular regulation. For example, the a-toxin of the gangrene causing bacteria Clostridium novyi is an O-GlcNAc transferase that exerts its toxic effects by the addition of O-GlcNAc to proteins in the Rho subfamily (5). Thus, the disruption of normal O-GlcNAc-regulated pathways may be responsible for the pathology of some bacteria. Moreover, disruption of the gene for OGlcNAc transferase demonstrates that O-GlcNAc modification is essential for life, even at the single cell level (6). O-GlcNAc appears to be both as abundant and as dynamic as protein phosphorylation. In several documented instances, phosphorylation and O-GlcNAc modification are reciprocal, occurring at the same or adjacent hydroxyl moieties (7, 8). Furthermore, all O-GlcNAc-modified proteins identified to date also occur as phosphorylated proteins. Nevertheless, the interrelationship between Ser/Thr O-GlcNAc modification and O-phosphorylation appears to be complex. Although there are examples of mutually exclusive O-GlcNAc modification and O-phosphorylation, it is likely that all possible combinations are represented in the complex environment of a eukaryotic cell (Fig. 1). The specific addition and removal of these two differentially regulated post-translational modifications might allow for nearly infinite modulation of protein function. The immense task of coordinating cellular activities and responding to extracellular cues with both temporal and spatial accuracy is likely to require the concerted action of both of these regulatory modifications. b-O-GlcNAc Is a Ubiquitous and Dynamic Modification Reports that alkali-induced b-elimination of adenovirus fiber proteins releases GlcNAcitol hinted at the existence of O-linked GlcNAc (9). Subsequent analysis confirmed the presence of b-OGlcNAc and suggested that the modification may be involved in adenovirus fiber assembly or stabilization (10). Although another study suggested the existence of O-glycosidically linked GlcNAc on extracellular proteins (11), later structural analyses suggested that these workers were likely observing a-linked GlcNAc, a mucin-like modification common in primitive eukaryotes (12). Another early study showed that the GlcNAc-binding lectin wheat germ agglutinin blocked ATP-dependent RNA nuclear transport (13). The characterization of b-O-GlcNAc in 1984 explained some of these preliminary observations and established O-GlcNAc as a major form of intracellular protein glycosylation (14). The nuclear pore proteins were among the first structurally characterized O-GlcNAc proteins (15, 16). Since then, several laboratories have shown that hundreds, if not thousands, of proteins in the nucleus and cytoplasm are modified with O-GlcNAc (3, 4). Given the broad spectrum of proteins that contain this modification, there are likely to be many different functions for O-GlcNAc. Studies with the transcription factor Sp1 suggest that O-GlcNAc protects the protein from proteasome degradation (17). Recent reports have shown that recognition of O-GlcNAc on peptides constitutes an important feature of major histocompatibility complex Class I antigen presentation (18). Pulse-chase analyses have shown that the O-GlcNAc modification of some proteins is highly transient, with turnover rates similar to phosphorylation (19, 20). Another study found that dynamic changes of O-GlcNAc-modified proteins are associated with lymphocyte activation (21). Several recent reports with phosphatase and kinase inhibitors have provided direct support for a relationship between O-phosphorylation and O-glycosylation of serine or threonine residues of some proteins (4, 22–24). Consistent with the hypothesis that O-GlcNAc has a regulatory role, disruptions of the O-GlcNAc transferase homolog, SPY, in Arabidopsis results in impaired gibberellin signal transduction (25). It is clear that OGlcNAc is involved in very diverse aspects of cellular physiology (Fig. 2). The challenge for the coming years is to determine the precise contribution of O-GlcNAc in the regulation of these systems.

O-Linked β-N-Acetylglucosamine (O-GlcNAc): Extensive Crosstalk with Phosphorylation to Regulate Signaling and Transcription in Response to Nutrients and Stress

BACKGROUND Since its discovery in the early 1980s, O-linked-beta-N-acetylglucosamine (O-GlcNAc), a single sugar modification on the hydroxyl group of serine or threonine residues, has changed our views of protein glycosylation. While other forms of protein glycosylation modify proteins on the cell surface or within luminal compartments of the secretory machinery, O-GlcNAc modifies myriad nucleocytoplasmic proteins. GlcNAcylated proteins are involved in transcription, ubiquitination, cell cycle, and stress responses. GlcNAcylation is similar to protein phosphorylation in terms of stoichiometry, localization and cycling. To date, only two enzymes are known to regulate GlcNAcylation in mammals: O-GlcNAc transferase (OGT), which catalyzes the addition of O-GlcNAc, and beta-N-acetylglucosaminidase (O-GlcNAcase), a neutral hexosaminidase responsible for O-GlcNAc removal. OGT and O-GlcNAcase are regulated by RNA splicing, by nutrients, and by post-translational modifications. Their specificities are controlled by many transiently associated targeting subunits. As methods for detecting O-GlcNAc have improved our understanding of O-GlcNAcs functions has grown rapidly. SCOPE OF REVIEW In this review, the functions of GlcNAcylation in regulating cellular processes, its extensive crosstalk with protein phosphorylation, and regulation of OGT and O-GlcNAcase will be explored. MAJOR CONCLUSIONS GlcNAcylation rivals phosphorylation in terms of its abundance, protein distribution and its cycling on and off of proteins. GlcNAcylation has extensive crosstalk with phosphorylation to regulate signaling, transcription and the cytoskeleton in response to nutrients and stress. GENERAL SIGNIFICANCE Abnormal crosstalk between GlcNAcylation and phosphorylation underlies dysregulation in diabetes, including glucose toxicity, and defective GlcNAcylation is involved in neurodegenerative disease and cancer and most recently in AIDS.

Ogt-Dependent X-Chromosome-Linked Protein Glycosylation Is a Requisite Modification in Somatic Cell Function and Embryo Viability

ABSTRACT The Ogt gene encodes a glycosyltransferase that links N-acetylglucosamine to serine and threonine residues (O-GlcNAc) on nuclear and cytosolic proteins. Efforts to study a mammalian model of Ogt deficiency have been hindered by the requirement for this X-linked gene in embryonic stem cell viability, necessitating the use of conditional mutagenesis in vivo. We have extended these observations by segregating Ogt mutation to distinct somatic cell types, including neurons, thymocytes, and fibroblasts, the latter by an approach developed for inducible Ogt mutagenesis. We show that Ogt mutation results in the loss of O-GlcNAc and causes T-cell apoptosis, neuronal tau hyperphosphorylation, and fibroblast growth arrest with altered expression of c-Fos, c-Jun, c-Myc, Sp1, and p27. We further segregated the mutant Ogt allele to parental gametes by oocyte- and spermatid-specific Cre-loxP mutagenesis. By this we established an in vivo genetic approach that supports the ontogeny of female heterozygotes bearing mutant X-linked genes required during embryogenesis. Successful production and characterization of such female heterozygotes further indicates that mammalian cells commonly require a functional Ogt allele. We find that O-GlcNAc modulates protein phosphorylation and expression among essential and conserved cell signaling pathways.