Dynamic O-glycosylation of nuclear and cytosolic proteins: further characterization of the nucleocytoplasmic beta-N-acetylglucosaminidase, O-GlcNAcase.

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic post-translational modification implicated in protein regulation that appears to be functionally more similar to phosphorylation than to classical glycosylation. There are nucleocytoplasmic enzymes for the attachment and removal of O-GlcNAc. Here, we further characterize the recently cloned beta-N-acetylglucosaminidase, O-GlcNAcase. Both recombinant and purified endogenous O-GlcNAcase rapidly release free GlcNAc from O-GlcNAc-modified peptide substrates. The recombinant enzyme functions as a monomer and has kinetic parameters (K(m) = 1.1 mm for paranitrophenyl-GlcNAc, k(cat) = 1 s(-1)) that are similar to those of lysosomal hexosaminidases. The endogenous O-GlcNAcase appears to be in a complex with other proteins and is predominantly localized to the cytosol. Overexpression of the enzyme in living cells results in decreased O-GlcNAc modification of nucleocytoplasmic proteins. Finally, we show that the enzyme is a substrate for caspase-3 but, surprisingly, the cleavage has no effect on in vitro O-GlcNAcase activity. These studies support the identification of this protein as an O-GlcNAcase and identify important interactions and modifications that may regulate the enzyme and O-GlcNAc cycling.     

Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine

beta-O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of resident nuclear and cytoplasmic proteins in eukaryotes. Increasing evidence suggests that O-GlcNAc plays a regulatory role in numerous cellular processes. Here we report on the production and characterization of a highly specific mouse monoclonal antibody, MAb CTD110.6, that specifically reacts with O-GlcNAc. The antibody recognizes O-GlcNAc in beta-O-glycosidic linkage to both serine and threonine. We could detect no cross-reactivity with alpha-linked Ser/Thr-O-GlcNAc, alpha-linked Ser-O-linked N-acetylgalactosamine (O-GalNAc), or N-linked oligosaccharides on ovalbumin and immunoglobulin G. The monosaccharide GlcNAc, but not GalNAc, abolishes immunoreactivity, further demonstrating specificity toward O-GlcNAc. Furthermore, galactose capping of O-GlcNAc sites also inhibits CTD110.6 immunoreactivity. Enrichment of GlcNAc-containing glycoproteins using the lectin wheat germ agglutinin dramatically enriches for CTD110.6-reactive proteins. The antibody reacts with a large number of proteins from cytoplasmic and nuclear extracts and readily detects in vivo changes in O-GlcNAc modification. These studies demonstrate that CTD110.6 is highly specific toward O-GlcNAc, with no cross-reactivity toward similar carbohydrate antigens or toward peptide determinants.     

Vertebrate lens alpha-crystallins are modified by O-linked N-acetylglucosamine.

Crystallins are structural proteins responsible for establishing the remarkable optical properties of the lens. Yet many of these highly conserved proteins are also expressed in nonocular tissues, where they have alternative functions apparently unrelated to their structural role in the lens. Here we report that lens alpha-crystallins, some of which function as heat-shock proteins in other tissues, are modified with O-linked N-acetylglucosamine (O-GlcNAc). An in vitro enzymatic assay that transfers [3H]Gal to terminal GlcNAc moieties labels alpha A and alpha B crystallins in lens homogenates from man, rhesus monkey, rat, cow, and rhea (an ostrich-like bird). O-Linkage of the saccharide is demonstrated by sensitivity to base-catalyzed beta-elimination and resistance to peptide:N-glycosidase F treatment. Chromatographic analyses of the beta-elimination products and fast atom bombardment-mass spectrometry of [3H]Gal-labeled tryptic peptides confirm the saccharide structure. Isoelectric focusing of [3H]Gal-labeled bovine lens proteins reveals the presence of O-GlcNAc on all four alpha-crystallin subunits, A1, A2, B1, and B2. Electrospray mass spectrometry of bovine alpha-crystallin demonstrates the presence of a single O-GlcNAc substitution on alpha A2. Gas-phase protein sequencing and fast atom bombardment-mass spectrometry of the major radiolabeled tryptic peptide from bovine alpha-crystallin reveal that GlcNAc is attached to the alpha A subunits at serine 162. This post-translational modification may play an important role in the molecular organization of lens alpha-crystallin.     

Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase

We have previously shown that diabetogenic antibiotic streptozotocin (STZ), an analog of N-acetylglucosamine (GlcNAc), inhibits the enzyme O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase) which is responsible for the removal of O-GlcNAc from proteins. Alloxan, another beta-cell toxin is a uracil analog. Since the O-GlcNAc transferase (OGT) uses UDP-GlcNAc as a substrate, we investigated whether alloxan might interfere with the process of protein O-glycosylation by blocking OGT, a very abundant enzyme in beta-cells. In isolated pancreatic islets, alloxan almost completely blocked both glucosamine-induced and STZ-induced protein O-GlcNAcylation, suggesting that alloxan indeed was inhibiting (OGT). In order to show definitively that alloxan was inhibiting OGT activity, recombinant OGT was incubated with 0-10 mM alloxan, and OGT activity was measured directly by quantitating UDP-[(3)H]-GlcNAc incorporation into the recombinant protein substrate, nucleoporin p62. Under these conditions, OGT activity was completely inhibited by 1 mM alloxan with half-maximal inhibition achieved at a concentration of 0.1 mM alloxan. Together, these data demonstrate that alloxan is an inhibitor of OGT, and as such, is the first OGT inhibitor described.     

A high-throughput assay for O-GlcNAc transferase detects primary sequence preferences in peptide substrates

O-GlcNAc transferase (OGT) catalyzes the addition of N-acetylglucosamine (O-GlcNAc) onto a diverse array of intracellular proteins. Although hundreds of proteins are known to be modified by O-GlcNAc, a strict amino acid consensus sequence for OGT has not been identified. In this study, we describe the development of a high-throughput assay for OGT and use it to profile the specificity of the enzyme among a panel of peptide substrates.     

Metabolism of Vertebrate Amino Sugars with N-Glycolyl Groups: INTRACELLULAR β-O-LINKED N-GLYCOLYLGLUCOSAMINE (GlcNGc), UDP-GlcNGc, AND THE BIOCHEMICAL AND STRUCTURAL RATIONALE FOR THE SUBSTRATE TOLERANCE OF β-O-LINKED β-N-ACETYLGLUCOSAMINIDASE

The O-GlcNAc modification involves the attachment of single β-O-linked N-acetylglucosamine residues to serine and threonine residues of nucleocytoplasmic proteins. Interestingly, previous biochemical and structural studies have shown that O-GlcNAcase (OGA), the enzyme that removes O-GlcNAc from proteins, has an active site pocket that tolerates various N-acyl groups in addition to the N-acetyl group of GlcNAc. The remarkable sequence and structural conservation of residues comprising this pocket suggest functional importance. We hypothesized this pocket enables processing of metabolic variants of O-GlcNAc that could be formed due to inaccuracy within the metabolic machinery of the hexosamine biosynthetic pathway. In the accompanying paper (Bergfeld, A. K., Pearce, O. M., Diaz, S. L., Pham, T., and Varki, A. (2012) J. Biol. Chem. 287, 28865-28881), N-glycolylglucosamine (GlcNGc) was shown to be a catabolite of NeuNGc. Here, we show that the hexosamine salvage pathway can convert GlcNGc to UDP-GlcNGc, which is then used to modify proteins with O-GlcNGc. The kinetics of incorporation and removal of O-GlcNGc in cells occur in a dynamic manner on a time frame similar to that of O-GlcNAc. Enzymatic activity of O-GlcNAcase (OGA) toward a GlcNGc glycoside reveals OGA can process glycolyl-containing substrates fairly efficiently. A bacterial homolog (BtGH84) of OGA, from a human gut symbiont, also processes O-GlcNGc substrates, and the structure of this enzyme bound to a GlcNGc-derived species reveals the molecular basis for tolerance and binding of GlcNGc. Together, these results demonstrate that analogs of GlcNAc, such as GlcNGc, are metabolically viable species and that the conserved active site pocket of OGA likely evolved to enable processing of mis-incorporated analogs of O-GlcNAc and thereby prevent their accumulation. Such plasticity in carbohydrate processing enzymes may be a general feature arising from inaccuracy in hexosamine metabolic pathways.     

Streptozotocin inhibits O-GlcNAcase via the production of a transition state analog

Streptozotocin (STZ) is a 2-deoxy-d-glucopyranose derivative of a class of drugs known as alkylnitrosoureas, and is an established diabetogenic agent whose cytotoxic affects on pancreatic beta-cells has been partially explained by the presence of its N-methyl-N-nitrosourea side chain, which has the ability to release nitric oxide as well as donate methyl groups to nucleotides in DNA. It has also been observed that STZ administration results in a rise in the level of O-GlcNAcylated proteins within beta-cells. Not coincidentally, STZ has also been shown to directly inhibit the O-GlcNAcase activity of the enzyme NCOAT in vitro, which is the only enzyme that possesses the ability to remove O-GlcNAc modifications on proteins in the nucleus and cytosol. Since O-GlcNAc modification plays a role on a number of proteins in a vast amount of cellular processes, this shift in whole-cell protein O-GlcNAcylation state affords another source of cell death. We set about to find the exact mechanism by which STZ inhibits O-GlcNAcase activity. Inhibition is achievable because the GlcNAc analog STZ targets the active site of the enzyme whereby it is catalyzed. During this process, the enzyme converts STZ to a compound that closely resembles the natural ligand transition state, but is distinctly more stable energetically. As a result, this analog is catalyzed to completion at a much slower rate, thereby out-competing GlcNAc substrate for the active site, and inhibiting the enzyme.     

Insights into O-linked N-acetylglucosamine ([0-9]O-GlcNAc) processing and dynamics through kinetic analysis of O-GlcNAc transferase and O-GlcNAcase activity on protein substrates

Cellular O-linked N-acetylglucosamine (O-GlcNAc) levels are modulated by two enzymes: uridine diphosphate-N-acetyl-D-glucosamine:polypeptidyltransferase (OGT) and O-GlcNAcase (OGA). To quantitatively address the activity of these enzymes on protein substrates, we generated five structurally diverse proteins in both unmodified and O-GlcNAc-modified states. We found a remarkably invariant upper limit for k(cat)/K(m) values for human OGA (hOGA)-catalyzed processing of these modified proteins, which suggests that hOGA processing is driven by the GlcNAc moiety and is independent of the protein. Human OGT (hOGT) activity ranged more widely, by up to 15-fold, suggesting that hOGT is the senior partner in fine tuning protein O-GlcNAc levels. This was supported by the observation that K(m,app) values for UDP-GlcNAc varied considerably (from 1 μM to over 20 μM), depending on the protein substrate, suggesting that some OGT substrates will be nutrient-responsive, whereas others are constitutively modified. The ratios of k(cat)/K(m) values obtained from hOGT and hOGA kinetic studies enable a prediction of the dynamic equilibrium position of O-GlcNAc levels that can be recapitulated in vitro and suggest the relative O-GlcNAc stoichiometries of target proteins in the absence of other factors. We show that changes in the specific activities of hOGT and hOGA measured in vitro on calcium/calmodulin-dependent kinase IV (CaMKIV) and its pseudophosphorylated form can account for previously reported changes in CaMKIV O-GlcNAc levels observed in cells. These studies provide kinetic evidence for the interplay between O-GlcNAc and phosphorylation on proteins and indicate that these effects can be mediated by changes in hOGT and hOGA kinetic activity.     

Modulation of O-GlcNAc glycosylation during Xenopus oocyte maturation

O-linked N-acetylglucosamine (O-GlcNAc) glycosylation is a post-translational modification, which is believed antagonises phosphorylation. We have studied the O-GlcNAc level during Xenopus oocyte meiotic resumption, taking advantage of the high synchrony of this model which is dependent upon a burst of phosphorylation. Stimulation of immature stage VI oocytes using progesterone was followed by a 4.51 +/- 0.32 fold increase in the GlcNAc content, concomitantly to an increase in phosphorylation, notably on two cytoplasmic proteins of 66 and 97 kDa. The increase of O-GlcNAc for the 97 kDa protein, which we identified as beta-catenin was partly related to its accumulation during maturation, as was demonstrated by the use of the protein synthesis inhibitor--cycloheximide. Microinjection of free GlcNAc, which inhibits O-glycosylated proteins-lectins interactions, delayed the progesterone-induced maturation without affecting the O-GlcNAc content. Our results suggest that O-GlcNAc glycosylation could regulate protein-protein interactions required for the cell cycle kinetic.     

Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment

Mass spectrometry-based studies of proteins that are post-translationally modified by O-linked β-N-acetylglucosamine (O-GlcNAc) are challenged in effectively identifying the sites of modification while simultaneously sequencing the peptides. Here we tested the hypothesis that a combination of high-energy C-trap dissociation (HCD) and electron transfer dissociation (ETD) could specifically target the O-GlcNAc modified peptides and elucidate the amino acid sequence while preserving the attached GlcNAc residue for accurate site assignment. By taking advantage of the recently characterized O-GlcNAc-specific IgG monoclonal antibodies and the combination of HCD and ETD fragmentation techniques, O-GlcNAc modified proteins were enriched from HEK293T cells and subsequently characterized using the LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) mass spectrometer. In our data set, 83 sites of O-GlcNAc modification are reported with high confidence confirming that the HCD/ETD combined approach is amenable to the detection and site assignment of O-GlcNAc modified peptides. Realizing HCD triggered ETD fragmentation on a linear ion trap/Orbitrap platform for more in-depth analysis and application of this technique to other post-translationally modified proteins are currently underway. Furthermore, this report illustrates that the O-GlcNAc transferase appears to demonstrate promiscuity with regards to the hydroxyl-containing amino acid modified in short stretches of primary sequence of the glycosylated polypeptides.     

High-sensitivity FAB-MS strategies for O-GlcNAc characterization.

In this paper we report the first application of fast atom bombardment mass spectrometry (FAB-MS) to O-linked N-acetylglucosamine (O-GlcNAc)-bearing glycopeptides. Using N-acetylgalactosamine (GalNAc)- and Gal-GalNAc-containing glycopeptides (isolated from Tn glycophorin and desialylated normal glycophorin, respectively) as readily available model compounds, rapid and sensitive derivatization/FAB-MS strategies applicable to serine/threonine-rich glycopeptides have been devised. Peptides and glycopeptides were propionylated in a 1 min reaction using a mixture of trifluoroacetic anhydride and propionic acid, and the product mixtures were analysed directly by FAB-MS. Glycopeptides and peptides rich in hydroxylated residues afforded characteristic clusters of molecular ions at high sensitivity. Additional sensitivity enhancement was achieved by prior esterification of carboxyl groups. These methods were used in a study of O-GlcNAc glycopeptides produced by purified O-GlcNAc transferase addition of GlcNAc to the synthetic peptides YSDSPSTST and YSGSPSTST in which Y is tyrosine, S is serine, D is aspartic acid, P is proline, T is threonine and G is glycine. The propionyl derivatives afforded high-quality spectra which unequivocally showed that the majority of the glycopeptides were substituted with a single GlcNAc residue. Low pmol quantities of material gave detectable signals. The propionylation/FAB-MS procedure has been combined with gas-phase sequencing strategies and shows promise for defining the sites of glycosylation of O-GlcNAc glycopeptides that are available in limited quantities.     

Erythrocytes contain cytoplasmic glycoproteins. O-linked GlcNAc on Band 4.1

Previously we reported that the novel protein-saccharide linkage, O-linked N-acetylglucosamine (GlcNAc), is found in abundance on proteins associated with the cytoplasmic and nucleoplasmic faces of the nuclear pore complex. Here we demonstrate that O-GlcNAc moieties are also added to human erythrocyte cytoplasmic proteins. Intact or permeabilized erythrocytes, as well as subcellular fractions, were labeled with bovine milk galactosyltransferase and UDP-[3H] galactose. The proportion of the incorporated label found on O-GlcNAc was determined by a variety of chemical and enzymatic techniques. The bulk of the O-GlcNAc residues are found in the cytoplasm of erythrocytes, the majority of which are on an as yet unidentified 65-kDa protein. In addition, we have determined that Band 4.1, a protein which serves as a bridge joining the cytoskeleton to the inner surface of the plasma membrane in erythrocytes, also contains O-GlcNAc moieties. One of the sites of O-GlcNAc addition has been localized to the last 117 amino acids of the carboxy terminus of Band 4.1.     

Identification of an N-acetylglucosaminyltransferase in Dictyostelium discoideum that transfers an "intersecting" N-acetylglucosamine residue to high mannose oligosaccharides

Glycoproteins synthesized by the cellular slime mold Dictyostelium discoideum have been shown to contain asparagine-linked high-mannose oligosaccharides which have an N-acetylglucosamine group in a novel intersecting position (attached beta 1-4 to the mannose linked alpha 1-6 to the core mannose). We have used crude membrane preparations from vegetative D. discoideum (strain M4) to characterize the enzyme activity responsible for catalyzing the transfer of GlcNAc to the intersecting position of high-mannose oligosaccharides. UDP-GlcNAc:oligosaccharide beta-N-acetylglucosaminyltransferase activity in these preparations attaches GlcNAc to the mannose residue-linked alpha 1-6 to the beta-linked core mannose of the following Man9GlcNAc oligosaccharide as shown by the arrow. (formula; see text) It will also attach GlcNAc to the same intersecting position and/or to the bisecting position (beta-linked core mannose) of the following Man5GlcNAc oligosaccharide. (formula; see text) An analysis of the pH profiles, effects of heat denaturation, and substrate inhibitions on the addition of GlcNAc to either the intersecting or bisecting position of this Man5GlcNAc oligosaccharide indicates that a single enzyme activity is responsible for transferring GlcNAc to both positions. Various oligosaccharides were assayed to determine the substrate specificity of the transferase activity. These data indicate that both the mannose-attached alpha 1-3 and the mannose-attached alpha 1-6 to the mannose receiving the GlcNAc play a critical role in substrate suitability; absence of the alpha 1-6 mannose results in at least a 90% decrease in activity, while absence of the alpha 1-3 mannose results in a completely inactive substrate. This suggests that the minimal substrate is the disaccharide Man alpha 1-3Man.     

Cytosolic O-GlcNAc accumulation is not involved in beta-cell death in HIT-T15 or Min6

O-linked N-acetylglucosamine (O-GlcNAc) is attached to and detached from proteins by O-GlcNAc transferase (OGT) and O-GlcNAcase, respectively. It has been proposed that streptozotocin induces pancreatic beta-cell death by blocking O-GlcNAcase and increasing O-GlcNAc. To elucidate the relationship between cytosolic O-GlcNAc accumulation and beta-cell death, we treated beta-cell lines HIT-T15 and Min6 with glucosamine. Glucosamine markedly reduced cell viability in both cell lines only at 10 mM. The measurement of cytosolic O-GlcNAc under glucosamine treatment revealed that O-GlcNAc accumulation was observed even at 2 mM glucosamine and maximized at 5 mM, but did not occur very well at 10 mM. Furthermore, 100 microM PUGNAc, an inhibitor of O-GlcNAcase, increased cytosolic O-GlcNAc but did not induce cell death in these cells. Therefore, no correlation between accumulation of cytosolic O-GlcNAc and beta-cell death was suggested. Alternatively, inosine partially rescued cell death induced by glucosamine in Min6 cells, suggesting that energy depletion partly contributes to beta-cell death by glucosamine.