Frank I. Comer

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-GlcNAc and the control of gene expression

Many eukaryotic proteins contain O-linked N-acetylglucosamine (O-GlcNAc) on their serine and threonine side chain hydroxyls. In contrast to classical cell surface glycosylation, O-GlcNAc occurs on resident nuclear and cytoplasmic proteins. O-GlcNAc exists as a single monosaccharide residue, showing no evidence of further elongation. Like phosphorylation, O-GlcNAc is highly dynamic, transiently modifying proteins. These post-translational modifications give rise to functionally distinct subsets of a given protein. Furthermore, all known O-GlcNAc proteins are also phosphoproteins that reversibly form multimeric complexes that are sensitive to the state of phosphorylation. This observation implies that O-GlcNAc may work in concert with phosphorylation to mediate regulated protein interactions. The proteins that bear the O-GlcNAc modification are very diverse, including RNA polymerase II and many of its transcription factors, numerous chromatin-associated proteins, nuclear pore proteins, proto-oncogenes, tumor suppressors and proteins involved in translation. Here, we discuss the functional implications of O-GlcNAc-modifications of proteins involved in various aspects of gene expression, beginning with proteins involved in transcription and ending with proteins involved in regulating protein translation.

O-Linked N-Acetylglucosamine: The “Yin-Yang” of Ser/Thr Phosphorylation?

O-linked N-acetylglucosamine (O-GlcNAc) was discovered during studies using bovine milk galactosyltransferase to ‘map’ the surface topography on cells of the murine immune system (Torres and Hart, 1984). Later, O-GlcNAc was shown to reside almost exclusively in the nucleus and cytoplasm (Kearse and Hart, 1991b), and to be present in eukaryotes ranging from trypanosomes, yeast, plants, to man, as well as in viruses (Hart et al. 1989; Hart et al. 1994a; Greis and Hart, 1994). We now know that O-GlcNAc is an exceedingly abundant post-translational modification of specific serine/threonine residues of many important nuclear and cytoplasmic proteins (Haltiwanger et al.1992b). Figure 1 lists the O-GlcNAc-bearing proteins identified to date. O-GlcNAc is attached as a monosaccharide and is generally not further modified. The O-GlcNAc turn-over rates are typically many-times that of the polypeptide backbone on which it is found (Chou et al.1992a; Hart and Roquemore, unpublished). The saccharide is attached at sites similar to those also used by the ‘growth-factor’, proline-directed family of kinases (Roach, 1991; Taylor and Adams, 1992). O-GlcNAc-bearing proteins have a diverse range of functions, but are characterized by several common features: 1) They all are also phosphorylated. 2) They typically form specific and regulated multimeric associations with other polypeptides. 3) In several cases, O-GlcNAcylation and phosphorylation appear to be reciprocal events.

Site-Specific GlcNAcylation of Human Erythrocyte Proteins Potential Biomarker(s) for Diabetes

OBJECTIVE—O-linked N-acetylglucosamine (O-GlcNAc) is upregulated in diabetic tissues and plays a role in insulin resistance and glucose toxicity. Here, we investigated the extent of GlcNAcylation on human erythrocyte proteins and compared site-specific GlcNAcylation on erythrocyte proteins from diabetic and normal individuals. RESEARCH DESIGN AND METHODS—GlcNAcylated erythrocyte proteins or GlcNAcylated peptides were tagged and selectively enriched by a chemoenzymatic approach and identified by mass spectrometry. The enrichment approach was combined with solid-phase chemical derivatization and isotopic labeling to detect O-GlcNAc modification sites and to compare site-specific O-GlcNAc occupancy levels between normal and diabetic erythrocyte proteins. RESULTS—The enzymes that catalyze the cycling (addition and removal) of O-GlcNAc were detected in human erythrocytes. Twenty-five GlcNAcylated erythrocyte proteins were identified. Protein expression levels were compared between diabetic and normal erythrocytes. Thirty-five O-GlcNAc sites were reproducibly identified, and their site-specific O-GlcNAc occupancy ratios were calculated. CONCLUSIONS—GlcNAcylation is differentially regulated at individual sites on erythrocyte proteins in response to glycemic status. These data suggest not only that site-specific O-GlcNAc levels reflect the glycemic status of an individual but also that O-GlcNAc site occupancy on erythrocyte proteins may be eventually useful as a diagnostic tool for the early detection of diabetes.

Localization of the O-GlcNAc transferase and O-GlcNAc-modified proteins in rat cerebellar cortex

O-linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous nucleocytoplasmic protein modification that has a complex interplay with phosphorylation on cytoskeletal proteins, signaling proteins and transcription factors. O-GlcNAc is essential for life at the single cell level, and much indirect evidence suggests it plays an important role in nerve cell biology and neurodegenerative disease. Here we show the localization of O-GlcNAc Transferase (OGTase) mRNA, OGTase protein, and O-GlcNAc-modified proteins in the rat cerebellar cortex. The sites of OGTase mRNA expression were determined by in situ hybridization histochemistry. Intense hybridization signals were present in neurons, especially in the Purkinje cells. Fluorescent-tagged antibody against OGTase stained almost all of the neurons with especially intense reactivity in Purkinje cells, within which the nucleus, perikaryon, and dendrites were most intensely stained. Using immuno-electron microscopic labeling, OGTase was seen to be enriched in euchromatin, in the cytoplasmic matrix, at the nerve terminal, and around microtubules in dendrites. In nerve terminals, immuno-gold labeling was observed around synaptic vesicles, with the enzyme more densely localized in the presynaptic terminals than in the postsynaptic ones. Using an antibody to O-GlcNAc, we found the sugar localizations reflected results seen for OGTase. Collectively, these data support hypothesized roles for O-GlcNAc in key processes of brain cells, including the regulation of transcription, synaptic vesicle secretion, transport, and signal transduction. Thus, by modulating the phosphorylation or protein associations of key regulatory and cytoskeletal proteins, O-GlcNAc is likely important to many functions of the cerebellum.

Glycosylation of Proteins — A Major Challenge in Mass Spectrometry and Proteomics

Although we have known for many years that most cell surface and extracellular proteins are glycosylated, only recently have we come to appreciate that most proteins within the nucleus and cytoplasm are also dynamically modified by the addition and removal of saccharides [1]. Indeed, in eukaryotes most polypeptides are glycosylated. Extracellular protein-bound glycans are generally complex and large, whereas cytosolic and nuclear glycans are often modified by simple monosaccharides [2]. Each unique type of protein glycosylation presents special challenges to the structural analyses or identification of glycoproteins by mass spectrometry (MS)[3–9]. MS analyses of extracellular or cell-surface glycoproteins are complicated by the enormous structural diversity of the glycan side chains, by their large size, by the astonishing site-specific structural variability of glycans, and by the fact that many component monosaccharides have the same mass. Mass spectrometric analysis of O-G1cNAc-bearing nuclear and cytoplasmic glycoproteins is confounded by the highly-dynamic nature of the modification [10], causing sub-stoichiometric levels at single sites, by the inherent insensitivity of the method to glycopeptides as compared to unmodified peptides, and most importantly, by the lability of the saccharide linkage under most MS analytical conditions [11, 12]. Nonetheless, mass spectrometry of all types is the most powerful tool currently available to the glycoscientist interested in the structure/functions of posttranslationally modified proteins as they actually occur in biological systems.