POGLUT1 biallelic mutations cause myopathy with reduced satellite cells, α-dystroglycan hypoglycosylation and a distinctive radiological pattern.

Protein O-glucosyltransferase 1 (POGLUT1) activity is critical for the Notch signaling pathway, being one of the main enzymes responsible for the glycosylation of the extracellular domain of Notch receptors. A biallelic mutation in the POGLUT1 gene has been reported in one family as the cause of an adult-onset limb-girdle muscular dystrophy (LGMD R21; OMIM# 617232). As the result of a collaborative international effort, we have identified the first cohort of 15 patients with LGMD R21, from nine unrelated families coming from different countries, providing a reliable phenotype-genotype and mechanistic insight. Patients carrying novel mutations in POGLUT1 all displayed a clinical picture of limb-girdle muscle weakness. However, the age at onset was broadened from adult to congenital and infantile onset. Moreover, we now report that the unique muscle imaging pattern of "inside-to-outside" fatty degeneration observed in the original cases is indeed a defining feature of POGLUT1 muscular dystrophy. Experiments on muscle biopsies from patients revealed a remarkable and consistent decrease in the level of the NOTCH1 intracellular domain, reduction of the pool of satellite cells (SC), and evidence of α-dystroglycan hypoglycosylation. In vitro biochemical and cell-based assays suggested a pathogenic role of the novel POGLUT1 mutations, leading to reduced enzymatic activity and/or protein stability. The association between the POGLUT1 variants and the muscular phenotype was established by in vivo experiments analyzing the indirect flight muscle development in transgenic Drosophila, showing that the human POGLUT1 mutations reduced its myogenic activity. In line with the well-known role of the Notch pathway in the homeostasis of SC and muscle regeneration, SC-derived myoblasts from patients' muscle samples showed decreased proliferation and facilitated differentiation. Together, these observations suggest that alterations in SC biology caused by reduced Notch1 signaling result in muscular dystrophy in LGMD R21 patients, likely with additional contribution from α-dystroglycan hypoglycosylation. This study settles the muscular clinical phenotype linked to POGLUT1 mutations and establishes the pathogenic mechanism underlying this muscle disorder. The description of a specific imaging pattern of fatty degeneration and muscle pathology with a decrease of α-dystroglycan glycosylation provides excellent tools which will help diagnose and follow up LGMD R21 patients.

Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine.

A novel form of protein-saccharide linkage consisting of single N-acetylglucosamine (GlcNAc) residues attached in O-linkages directly to the polypeptide backbone has been described (Holt, G. D., and G. W. Hart, 1986, J. Biol. Chem., 261:8049-8057). This modification was found on proteins distributed throughout the cell, although proteins bearing O-linked GlcNAc moieties were particularly abundant in the cytosolic and nuclear envelope fractions of rat liver. In the accompanying article (Snow, C. M., A. Senior, and L. Gerace, 1987, J. Cell. Biol., 104: 1143-1156), the authors describe monoclonal antibodies directed against eight proteins localized to the nuclear pore complex. These proteins occur on the cytoplasmic and nucleoplasmic (but not lumenal) sides of nuclear membranes. In this report, we demonstrate that all members of this group of pore complex proteins bear multiple O-linked GlcNAc residues. Further, we show that the O-linked GlcNAc moieties are linked via serine (and possibly threonine) side chains to these proteins. Perturbing the O-linked GlcNAc residues either by covalently attaching galactose to them or by releasing them with beta-N-acetylglucosaminidase strongly diminishes the immunoreactivity of the proteins with all of the monoclonal antibodies. However, the O-linked GlcNAc moieties are only part of the epitopes recognized, since O-GlcNAc-containing limit pronase fragments of nuclear pore complex proteins cannot be immunoprecipitated by these antibodies. These findings, taken together with those in the accompanying article, are a direct demonstration that proteins of the cytoplasm and nucleoplasm bear O-linked GlcNAc residues.

Synergy between trehalose and Hsp104 for thermotolerance in Saccharomyces cerevisiae.

We isolated a mutant strain unable to acquire heat shock resistance in stationary phase. Two mutations contributed to this phenotype. One mutation was at the TPS2 locus, which encodes trehalose-6-phosphate phosphatase. The mutant fails to make trehalose and accumulates trehalose-6-phosphate. The other mutation was at the HSP104 locus. Gene disruptions showed that tps2 and hsp104 null mutants each produced moderate heat shock sensitivity in stationary phase cells. The two mutations were synergistic and the double mutant had little or no stationary phase-induced heat shock resistance. The same effect was seen in the tps1 (trehalose-6-phosphate synthase) hsp104 double mutant, suggesting that the extreme heat shock sensitivity was due mainly to a lack of trehalose rather than to the presence of trehalose-6-phosphate. However, accumulation of trehalose-6-phosphate did cause some phenotypes in the tps2 mutant, such as temperature sensitivity for growth. Finally, we isolated a high copy number suppressor of the temperature sensitivity of tps2, which we call PMU1, which reduced the levels of trehalose-6-phosphate in tps2 mutants. The encoded protein has a region homologous to the active site of phosphomutases.

Molecular evolution of protein O-fucosyltransferase genes and splice variants.

O-Fucose has been described on both epidermal growth factor-like (EGF-like) repeats and Thrombospondin type 1 repeats (TSRs). The enzyme adding fucose to EGF-like repeats, protein O-fucosyltransferase 1 (Pofut1), is a soluble protein located in the lumen of endoplasmic reticulum (ER). A second protein O-fucosyltransferase, Pofut2, quite divergent from its homolog Pofut1, has recently been shown to O-fucosylate TSRs but not EGF-like repeats. To date, Pofut1 genes have only been characterized in human, mouse, and fly, and Pofut2 in mouse, fly, and partially in the nematode Caenorhabditis elegans. Here, we report cDNA sequences and genomic structures of bovine Pofut1 and Pofut2 genes and describe for the first time five alternative spliced transcripts for each gene. Only one transcript for both Pofut1 and Pofut2 encodes an active bovine O-fucosyltransferase. Variant transcript distribution was examined in 13 bovine tissues. Transcripts encoding active forms are ubiquitous, whereas other forms possess a more restricted tissue-expression profile. Sequence comparison and phylogenetic analyses revealed that both Pofut genes are present as a single copy in animal genomes, and their exon-intron organizations are conserved among vertebrates. The last common ancestor of all analyzed bilaterian species would be predicted to possess polyexonic Pofut genes in their genome.

The ligand binding specificity and tissue localization of a rat alveolar macrophage lectin.

The parameters of the reaction between a rat alveolar macrophage lectin (Mr = 180,000) and its ligands have been examined. The reaction is dependent on Ca2+ over the optimal pH range for binding. The apparent dissociation constant for fucosyl bovine serum albumin, the standard ligand used in these studies, is 1.4 X 10(-10) M. The ligand binding specificity was determined by measurement of the inhibition of binding of fucosyl bovine serum albumin by various glycoproteins and saccharides. D-Mannose, L-fucose, and N-acetyl-D-glucosamine were the most effective inhibitors, and D-galactose was much poorer. The equatorial hydroxyl groups on the C-3 and C-4 of the mannose ring are important in the lectin-ligand interaction, and the axial hydroxyl group on the C-2 contributes to a lesser extent. Immunocytological studies revealed that the lectin isolated from alveolar macrophages is widely distributed in other rat tissues. Hepatocytes are devoid of the lectin, but hepatic Kupffer cells and endothelial cells contain significant amounts. This was confirmed by isolation of the lectin from liver. Spleen and skeletal muscle also contain lectin, but much smaller amounts were found in brain, kidney, and heart muscle.

The isolation of a rat alveolar macrophage lectin.

A lectin in rat alveolar macrophage membranes with a high affinity for binding ligands containing L-fucose and N-acetyl-D-glucosamine has been isolated by affinity chromatography on Fuc-BSA-Sepharose (where Fuc is fucosyl and BSA is bovine serum albumin). The lectin was extracted from rat lung homogenates with Triton X-100, absorbed from the extract onto Fuc-BSA-Sepharose in the presence of Ca2+ and eluted by removal of Ca2+. After a second adsorption to and elution from Fuc-BSA-Sepharose, three protein species were detected electrophoretically in fractions that bind Fuc-BSA. One, which was the mannose/N-acetylglucosamine lectin (Mr = 32,000) found earlier in hepatocytes, was removed by adsorption on anti-lectin IgG-Sepharose. Another (Mr = 46,000) was removed by adsorption to Fuc-BSA-Sepharose and elution with galactose. The remaining lectin (Mr = 180,000) bound fucose and N-acetylglucosamine but not galactose. Binding was maximal between pH 6.5 and 9.0 and dependent on Ca2+. Immunocytological analysis with rabbit anti-lectin IgG and fluorescein-labeled goat anti-rabbit IgG revealed the lectin to be in rat alveolar macrophages and nonparenchymal cells of liver. Thus, the lectin appears to be present in macrophages and is likely involved in receptor-mediated endocytosis. It is distinctly different structurally from the hepatocyte lectin with a similar ligand-binding specificity.

Mitotic arrest with nocodazole induces selective changes in the level of O-linked N-acetylglucosamine and accumulation of incompletely processed N-glycans on proteins from HT29 cells.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant protein modification found on nuclear and cytoplasmic proteins. Several lines of evidence suggest that it is a highly dynamic modification and that the levels of this sugar on proteins may be regulated. Previous workers (Chou, C. F., and Omary, M. B. (1993) J. Biol. Chem. 268, 4465-4472) have shown that mitotic arrest with microtubule-destabilizing agents such as nocodazole causes an increase in the O-GlcNAc levels on keratins in the human colon cancer cell line HT29. We have sought to determine whether this increase in glycosylation is a general (i.e. occurring on many proteins) or a limited (i.e. occurring only on the keratins) process. A general increase would suggest that the microtubule-destabilizing agents were somehow affecting the enzymes responsible for addition and/or removal of O-GlcNAc. Our results suggest that the changes in O-GlcNAc induced by nocodazole are selective for the keratins. The levels of O-GlcNAc on other proteins, including the nuclear pore protein p62 and the transcription factor Sp1, are not significantly affected by this treatment. In agreement with these findings, nocodazole treatment caused no change in the activity of the enzymes responsible for addition or removal of O-GlcNAc as determined by direct in vitro assay. Interestingly, nocodazole treatment did cause a dramatic increase in modification of N-glycans with terminal GlcNAc residues on numerous proteins. Potential mechanisms for this and the change in glycosylation of the keratins are discussed.

Calf thymus high mobility group proteins are nonenzymatically glycated but not significantly glycosylated.

Over the past decade, there have been many reports suggesting the presence of complex carbohydrates on nuclear and cytoplasmic proteins in mammalian cells. Some of the most often cited of these reports deal with the glycosylation of the high mobility group (HMG) proteins. These are relatively abundant chromosomal proteins that are known to be associated with nucleosomes and actively transcribed regions of chromatin. The original report describing HMG protein glycosylation presented several lines of evidence suggesting that these proteins are glycosylated, including carbohydrate compositional analysis and periodic-acid Schiff staining. We have attempted to repeat these observations with more highly purified protein than was utilized in the original study. Using carbohydrate compositional analysis performed by high pH anion exchange chromatography coupled to pulsed-amperometric detection, we saw no evidence for significant glycosylation of these proteins. In addition, we found no evidence for the presence of O-GlcNAc, a well known form of nuclear glycosylation. The HMG proteins did react with periodate, suggesting the presence of a modification containing cis-diols on the protein. Several tryptic peptides isolated from HMG 14 and 17 which retained the periodate reactivity had in common lysine residues, suggesting a potential modification of the straightepsilon-amino groups of lysines such as nonenzymatic glycation. Western blot analysis of the HMG proteins using anti-advanced glycation endproducts (AGE) antibodies confirmed the presence of glycation products on the HMG proteins.

O-fucose modifications of epidermal growth factor-like repeats and thrombospondin type 1 repeats: unusual modifications in unusual places.

Recent discoveries revealing that carbohydrate modifications play critical roles in a wide variety of biological processes have brought wide recognition to the field of glycobiology. Growing attention has focused on the function of unusual O-linked carbohydrate modifications such as O-fucose. O-fucose modifications have been described in several different protein contexts, including epidermal growth factor-like repeats and thrombospondin type 1 repeats. The O-fucose modifications on thrombospondin type 1 repeats have only recently been described, but the site of modification occurs in a region proposed to play a role in cell adhesion. O-fucose modifications on epidermal growth factor-like repeats have been described as important players in several signal transduction systems. For instance, Notch, a cell-surface signaling receptor required for many developmental events, bears multiple O-fucose saccharides on the epidermal growth factor-like repeat of its extracellular domain. The O-fucose moieties serve as a substrate for the beta1,3 N-acetylglucosaminyltransferase activity of Fringe, a known modifier of Notch function. The alteration of O-fucose structures by Fringe influences the ability of Notch ligands to activate the receptor and provides a means to regulate Notch signaling. Thus, O-fucose and Fringe provide a clear example of how carbohydrate modifications can have direct functional consequences on the proteins they modify.

The O-linked fucose glycosylation pathway: identification and characterization of a uridine diphosphoglucose: fucose-beta1,3-glucosyltransferase activity from Chinese hamster ovary cells.

O-Linked fucose is an unusual carbohydrate modification in which fucose is linked directly to the hydroxyl groups of serines or threonines. It has been found on the epidermal growth factor-like modules of several secreted proteins involved in blood coagulation and fibrinolysis. We have recently reported the existence of an elongated form of O-linked fucose in Chinese hamster ovary cells consisting of a glucose linked to the 3'-hydroxyl of fucose (Glcbeta1,3Fuc- O-Ser/Thr). This structure is highly unusual for two reasons. First, in mammalian systems fucose is usually a terminal modification of N - and O-linked oligosaccharides. Here the fucose is internal. Secondly, terminal beta-linked glucose is extremely rare on mammalian glycoconjugates. Thus, the Glcbeta1,3Fuc structure is a very unique mammalian carbohydrate structure. Here we report the identification and initial characterization of a novel enzyme activity capable of forming this unique linkage: UDP-glucose: O-linked fucose beta1,3 glucosyltransferase. The enzyme utilizes UDP-glucose as the high energy donor and transfers glucose to alpha-linked fucose residues. The activity is linearly dependent on time, enzyme, and substrate concentrations and is enhanced in the presence of manganese ions. Activity is present in extracts of cultured cells from a variety of species (hamster, human, mouse, rat, chicken) and is enriched in brain and spleen of a normal adult rat. Thus, while this glycosyltransferase appears to be widespread in biology, it forms a very unique linkage, and it represents the first mammalian enzyme identified capable of elongating fucose.

Enzymatic addition of O-GlcNAc to nuclear and cytoplasmic proteins. Identification of a uridine diphospho-N-acetylglucosamine:peptide beta-N-acetylglucosaminyltransferase.

An assay for the enzyme responsible for the addition of O-linked N-acetylglucosamine (O-GlcNAc) to proteins, a UDP-N-acetylglucosamine:peptide N-acetylglucosaminyltransferase, is reported using the synthetic peptide YSDSPSTST as the acceptor substrate. The activity is linearly dependent on time, enzyme, and substrate concentration. Replacement of the proline with a glycine in the peptide renders it ineffective as a substrate, whereas changing of the aspartic acid to a glycine has no effect. Product characterization of the glycosylated peptide demonstrates that the monosaccharide covalently attached to the peptide is N-acetylglucosamine (GlcNAc) and has not been epimerized to N-acetylgalactosamine. Mild base-catalyzed beta-elimination of the in vitro glycosylated peptide quantitatively yields GlcNAcitol, indicating that the GlcNAc is attached via an O-linkage. The transferase activity is strongly inhibited by UDP but is unaffected by GlcNAc or tunicamycin. Interestingly, EDTA only slightly inhibits activity, suggesting that the enzyme may not require divalent cations. The majority of the activity is soluble, and the remainder is lost from membranes after extracting with high salt and EDTA. Consistent with the subcellular localization of most proteins bearing O-GlcNAc, the activity appears to reside in the cytosolic portion of the cell when compared to two lumenal marker enzymes, galactosyltransferase and mannose-6-phosphatase.

Core fucosylation of high-mannose-type oligosaccharides in GlcNAc transferase I-deficient (Lec1) CHO cells.

During studies on the fucosylation of endogenous proteins in parental (Pro5) and N-acetyl-D-glucosamine (GlcNAc) transferase I-deficient (Lec1) Chinese hamster ovary (CHO) cells, we observed that Lec1 cells incorporate approximately 10-fold less [3H]fucose into macromolecules than Pro5 cells. Interestingly, most of the labelled oligosaccharides from both cell types could be released from the macromolecules by digestion with peptide N-glycosidase F (PNGase F). This was unexpected for Lec1 cells because they do not synthesize complex- or hybrid-type N-glycans. Structural analyses of the fucosylated oligosaccharides from Lec1 cells showed the fucose to be in an alpha 1,6 linkage to the core GlcNAc of relatively small oligomannose N-glycans (Man4GlcNAc2 and Man5GlcNAc2, where Man is D-mannose). Comparing the sizes of oligomannose N-glycans from Pro5 and Lec1 cells demonstrated a much higher proportion of the small (Man4GlcNAc2 and Man5GlcNAc2) oligomannose species in Lec1 cells. These results suggest that the core alpha 1,6 fucosyltransferase will fucosylate small (Man4-Man5GlcNAc2), but not large (Man8-Man9GlcNAc2) oligomannose N-glycans.

The binding of fucose-containing glycoproteins by hepatic lectins. The binding specificity of the rat liver fucose lectin.

The parameters that affect the interaction of ligands with a fucose-binding lectin from rat liver have been examined. 125I-Fucosyl-bovine serum albumin (Fuc-BSA) containing 50 residues of fucose/molecule was used as the standard ligand. At low initial concentrations of ligand (10 ng/ml) and lectin (140 ng/ml), the reaction reaches equilibrium at pH 7.8, 23 degrees C, within 40 min. The binding of ligands is Ca2+ dependent with half-maximal binding occurring at 54 microM Ca2+; of several metal ions tested, only Sr2+ partially replaced Ca2+. Binding was maximal between pH 7.6 and 8.6, fell slightly up to pH 10, but fell markedly below pH 7. The lectin-ligand complexes dissociated at low pH, on removal of Ca2+, or in the presence of a large excess of competing ligand. The apparent association constant (Ka) for Fuc-BSA was 1.75 X 10(8) M-1. The fucose content of the Fuc-BSA also influenced binding, with little apparent binding below 24 fucose residues/molecule and maximal binding from 40 to 50 fucose residues/molecule. With knowledge of the parameters influencing binding, sensitive reproducible assays for the lectin were developed. The binding specificity of the lectin was examined by measuring the inhibition of 125I-Fuc-BSA binding by neoglycoproteins, monosaccharides, and glycosides or by direct binding of neoglycoproteins. Galactosides and beta-linked fucosides were the best ligands among the neoglycoproteins, with much weaker binding by mannosyl- or N-acetylglucosaminyl-BSA. On the basis of the pattern of inhibition of Fuc-BSA binding by various monosaccharides and glycosides, it is possible to propose the conformations of saccharides that best fit the lectin-binding site. The C1 conformation of N-acetyl-D-galactosamine fits best, although other not obviously related monosaccharides such as L-fucose, L-arabinose, and D-mannose can also assume conformations that permit them to be effective inhibitors. The pattern of binding of neoglycoproteins to the lectin differs from that of other pure hepatic lectins. Thus, the fucose lectin has a high affinity for Fuc-BSA and galactosyl-BSA but a low affinity for N-acetylglucosaminyl-BSA. The galactose lectin binds only galactosyl-BSA and shows little binding with either N-acetylglucosaminyl-BSA or Fuc-BSA. In contrast, the mannose/N-acetylglucosamine lectin binds N-acetylglucosaminyl-BSA and Fuc-BSA but not galactosyl-BSA.

Glycosylation of nuclear and cytoplasmic proteins. Purification and characterization of a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase.

Using a combination of conventional and affinity chromatographic techniques, we have purified a uridine diphospho-N-acetylglucosamine:polypeptide beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) over 30,000-fold from rat liver cytosol. The transferase is soluble and very large, migrating with an apparent molecular weight of 340,000 on molecular sieve chromatography. Analysis of the purified enzyme on sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals two protein species migrating at 110 (alpha subunit) and 78 (beta subunit) kDa in approximately a two-to-one ratio. Thus, the enzyme likely exists as a heterotrimer complex with two subunits of 110 kDa and one of 78 kDa (alpha 2 beta). The alpha subunit appears to contain the enzyme's active site since it is selectively radiolabeled by a specific photoaffinity probe (4-[beta-32P]thiouridine diphosphate). Photoinactivation and photolabeling of the enzyme are dependent on time and long wavelength ultraviolet light. Photolabeling of the alpha subunit is specifically blocked by UDP. The enzyme has an extremely high affinity for UDP-GlcNAc (Km = 545 nM). This unusually high affinity for the sugar nucleotide donor probably provides the enzyme an advantage over the nucleotide transporters in the endoplasmic reticulum and Golgi apparatus which compete for available cytoplasmic UDP-GlcNAc. The multimeric state and large size of the O-GlcNAc transferase imply that its activity may be highly regulated within the cell.

Synapsins contain O-linked N-acetylglucosamine.

The neuron-specific synaptic vesicle-associated phosphoproteins synapsin I and synapsin II were shown to contain terminal N-acetylglucosamine (GlcNAc) residues as determined by specific labeling with bovine galactosyltransferase and UDP-[3H]galactose. The beta-elimination of galactosyltransferase radiolabeled synapsin I and subsequent analysis of released saccharide on high-voltage paper electrophoresis confirmed the presence of monosaccharidic GlcNAc moieties in O-linkage to the protein. Partial cleavage of synapsin I by collagenase, 2-nitro-5-thiocyanobenzoic acid, and Staphylococcus aureus V8 protease suggests that at least three glycosylation sites exist along the molecule. Taken together these data present the first evidence that a neuron-specific protein contains O-glycosidically bound GlcNAc.

The O-linked fucose glycosylation pathway. Evidence for protein-specific elongation of o-linked fucose in Chinese hamster ovary cells.

O-Linked fucose is an unusual form of glycosylation recently shown to modify the hydroxyls of serine or threonine residues at a strict consensus site within epidermal growth factor-like domains of several serum proteins. Here we demonstrate that Chinese hamster ovary cells modify numerous proteins with O-linked fucose and that the fucose is elongated on specific proteins. We have identified at least two forms of O-linked fucose elongation in Chinese hamster ovary cells: a disaccharide (Glcbeta1,3Fuc) and a larger oligosaccharide of indeterminate structure. Interestingly, it appears that the level of monosaccharide accumulates in the cells over time whereas the disaccharide does not. Analysis of the O-linked fucose-containing saccharides on individual proteins revealed that some proteins are modified with the monosaccharide only, whereas others are modified with monosaccharide and disaccharide, or monosaccharide and oligosaccharide. These results suggest that elongation of the O-linked fucose monosaccharide is a protein-specific phenomena. The presence of elongated O-linked fucose moieties suggests that a novel glycosylation pathway exists in mammalian cells with O-linked fucose as the core.

Modulation of O-linked N-acetylglucosamine levels on nuclear and cytoplasmic proteins in vivo using the peptide O-GlcNAc-beta-N-acetylglucosaminidase inhibitor O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate.

O-Linked N-acetylglucosamine (O-GlcNAc) is a ubiquitous and abundant post-translational modification found on nuclear and cytoplasmic proteins and is thought to be a dynamically regulated modification much like phosphorylation. In this study we have demonstrated that O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbama te (PUGNAc), a potent in vitro inhibitor of the enzyme responsible for the removal of O-GlcNAc from proteins (peptide O-GlcNAc-beta-N-acetylglucosaminidase), can be used to increase O-GlcNAc levels on nuclear and cytoplasmic proteins in vivo. Overall, PUGNAc caused approximately a 2-fold increase in O-GlcNAc levels in the human colon cancer cells, HT29, although the effects on individual proteins varied. The increase appeared to be the result of the direct inhibition of the peptide O-GlcNAc-beta-N-acetylglucosaminidase since neither the O-GlcNAc transferase nor UDP-GlcNAc levels were affected by the treatment. O-GlcNAc levels in other cell lines tested (NIH 3T3, CV-1, and HeLa) were also affected by PUGNAc, although the effects on HeLa cells were minimal. At the concentrations tested, PUGNAc was non-toxic and had no affect on the growth rate of any of the cell lines examined. Interestingly, we demonstrated that an increase in O-GlcNAc levels on the transcription factor Sp1 resulted in a reciprocal decrease in its level of phosphorylation, supporting the hypothesis that O-GlcNAc competes with phosphate on some proteins. These studies demonstrate that PUGNAc is an effective inhibitor of O-GlcNAc turnover within cells and can be used to selectively alter the extent of O-GlcNAc on cellular proteins.