Sp1 modulates ncOGT activity to alter target recognition and enhanced thermotolerance in E. coli

cDNAs encoding three isoforms of OGT (ncOGT, mOGT, and sOGT) were expressed in Escherichia coli in which the coexpression system of OGT with target substrates was established in vivo. No endogenous bacterial proteins were significantly O-GlcNAcylated by any type of OGT isoform while co-expressed p62 and Sp1 were strongly O-GlcNAcylated by ncOGT. These results suggest that most of bacterial proteins appear not to be recognized as right substrates by mammalian OGT whereas cytosolic environments may supply UDP-GlcNAc enough to proceed to O-GlcNAcylation in E. coli. Under these conditions, sOGT was auto-O-GlcNAcylated whereas ncOGT and mOGT were not. Importantly, we found that when Sp1 was coexpressed, ncOGT can O-GlcNAcylate not only Sp1 but also many bacterial proteins. Our findings suggest that Sp1 may modulate the capability of target recognition of ncOGT by which ncOGT can be led to newly recognize bacterial proteins as target substrates, finally generating the O-glyco-bacteria. Our results demonstrate that the O-glyco-bacteria showed enhanced thermal resistance to allow cell survival at a temperature as high as 52 °C.     

OGT 多克隆抗体的制备及特异性分析

目的:为了探讨O-GlcNAc糖基转移酶OGT的生理和病理作用,需制备能高效特异性检测OGT的抗体。方法:在NCBI数据库中,查找人源OGT基因序列,根据OGT的结构特点,选取OGT的C末端催化结构域中的一段多肽序列(464-949位点氨基酸)做抗原。首先,构建OGT的C末端催化结构域(464-949位点氨基酸)的重组表达载体pET30-a-OGT-C,转化至大肠杆菌BL21(DE3)感受态细胞中,IPTG诱导表达融合His标签的OGT-C蛋白,Ni+珠亲和层析法纯化提取OGT-C蛋白。再以OGT-C重组蛋白作为抗原,免疫Wistar大鼠制备多克隆抗体,并用间接ELISA法检测OGT抗体的效价,Western blotting鉴定抗体特异性。结果:多抗效价达1:80000;在免疫印迹实验中,此多抗可以高效的检测重组抗原,并可以特异性识别培养细胞内源表达的ncOGT和mOGT这2种OGT亚型。结论:实验结果表明,获得高效价、高特异性的OGT多克隆抗体,在OGT的生物学研究中可以用于检测ncOGT和mOGT的表达。     

Microinjection of recombinant O-GlcNAc transferase potentiates Xenopus oocytes M-phase entry

In order to understand the importance of the cytosolic and nuclear-specific O-linked N-acetylglucosaminylation (O-GlcNAc) on cell cycle regulation, we recently reported that inhibition of O-GlcNAc transferase (OGT) delayed or blocked Xenopus laevis oocyte germinal vesicle breakdown (GVBD). Here, we show that increased levels of the long OGT isoform (ncOGT) accelerate X. laevis oocyte GVBD. A N-terminally truncated isoform (sOGT) with a similar in vitro catalytic activity towards a synthetic CKII-derived peptide had no effect, illustrating the important role played by the N-terminal tetratrico-peptide repeats. ncOGT microinjection in the oocytes increases both the speed and extent of O-GlcNAc addition, leads to a quicker activation of the MPF and MAPK pathways and finally results in a faster GVBD. Microinjection of anti-OGT antibodies leads to a delay of the GVBD kinetics. Our results hence demonstrate that OGT is a key molecule for the timely progression of the cell cycle.     

Glucose deprivation stimulates O-GlcNAc modification of proteins through up-regulation of O-linked N-acetylglucosaminyltransferase

O-Linked N-acetylglucosamine (O-GlcNAc) is a post-translational modification of proteins that functions as a nutrient sensing mechanism. Here we report on regulation of O-GlcNAcylation over a broad range of glucose concentrations. We have discovered a significant induction of O-GlcNAc modification of a limited number of proteins under conditions of glucose deprivation. Beginning 12 h after treatment, glucose-deprived human hepatocellular carcinoma (HepG2) cells demonstrate a 7.8-fold increase in total O-GlcNAc modification compared with cells cultured in normal glucose (5 mm; p = 0.008). Some of the targets of glucose deprivation-induced O-GlcNAcylation are distinct from those modified in response to high glucose (20 mm) or glucosamine (10 mm) treatment, suggesting differential targeting with glucose deprivation and glucose excess. O-GlcNAcylation of glycogen synthase is significantly increased with glucose deprivation, and this O-GlcNAc increase contributes to a 60% decrease (p = 0.004) in glycogen synthase activity. Increased O-GlcNAc modification is not mediated by increased UDP-GlcNAc, the rate-limiting substrate for O-GlcNAcylation. Rather, the mRNA for nucleocytoplasmic O-linked N-acetylglucosaminyltransferase (OGT) increases 3.4-fold within 6 h of glucose deprivation (p = 0.006). Within 12 h, OGT protein increases 1.7-fold (p = 0.01) compared with normal glucose-treated cells. In addition, 12-h glucose deprivation leads to a 49% decrease in O-GlcNAcase protein levels (p = 0.03). We conclude that increased O-GlcNAc modification stimulated by glucose deprivation results from increased OGT and decreased O-GlcNAcase levels and that these changes affect cell metabolism, thus inactivating glycogen synthase.     

Alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-D-glucosaminidase

We have previously shown that streptozotocin (STZ) inhibits O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase (O-GlcNAcase), the enzyme that removes O-GlcNAc from proteins. In light of this observation, we explored the possibility that the diabetogenic toxin alloxan, an O-GlcNAc transferase (OGT) inhibitor, might also inhibit O-GlcNAcase. Alloxan inhibited islet O-GlcNAcase with a dose-response much like that of STZ. Similar to STZ, islet O-GlcNAcase was more susceptible to alloxan inhibition than was brain O-GlcNAcase. Alloxan directly inhibited recombinant O-GlcNAcase activity with a dose-response very similar to that of STZ. Subsequent LC/MS/MS analysis revealed that alloxan modified the tryptic digest pattern of the enzyme. One tryptic peptide LGCFEIAK(894-901) was modified by alloxan. Two other tryptic peptides, LDQVSQFGCR(158-167) and SFALLFDDIDHNMCAADK(168-185), both N-terminal active site peptides, were absent after alloxan treatment. Together, these data demonstrate that alloxan is an inhibitor of O-GlcNAc-selective N-acetyl-beta-d-glucosaminidase, with inhibition corresponding to an altered tryptic digest pattern of N-terminal active site peptides.     

A mitotic GlcNAcylation/phosphorylation signaling complex alters the posttranslational state of the cytoskeletal protein vimentin

O-linked β-N-acetylglucosamine (O-GlcNAc) is a highly dynamic intracellular protein modification responsive to stress, hormones, nutrients, and cell cycle stage. Alterations in O-GlcNAc addition or removal (cycling) impair cell cycle progression and cytokinesis, but the mechanisms are not well understood. Here, we demonstrate that the enzymes responsible for O-GlcNAc cycling, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) are in a transient complex at M phase with the mitotic kinase Aurora B and protein phosphatase 1. OGT colocalized to the midbody during telophase with Aurora B. Furthermore, these proteins coprecipitated with each other in a late mitotic extract. The complex was stable under Aurora inhibition; however, the total cellular levels of O-GlcNAc were increased and the localization of OGT was decreased at the midbody after Aurora inhibition. Vimentin, an intermediate filament protein, is an M phase substrate for both Aurora B and OGT. Overexpression of OGT or OGA led to defects in mitotic phosphorylation on multiple sites, whereas OGT overexpression increased mitotic GlcNAcylation of vimentin. OGA inhibition caused a decrease in vimentin late mitotic phosphorylation but increased GlcNAcylation. Together, these data demonstrate that the O-GlcNAc cycling enzymes associate with kinases and phosphatases at M phase to regulate the posttranslational status of vimentin.     

A Modified Coupled Enzyme Method for O-linked GlcNAc Transferase Activity Assay

In order to determine the activity of O-linked GlcNAc transferase (OGT), a modified coupled enzyme method was proposed. This method was based on the measurement of uridine 5′-(trihydrogen diphosphate) (UDP), a product generated in transglycosylation reaction. In the assay, UDP was coupled to the conversion of phosphoenolpyruvate to pyruvate using pyruvate kinase. Using a commercial pyruvate assay kit, the pyruvate was converted to a red terminal product, which could be photometrically measured at 570 nm or fluorometrically measured at 587 nm (E _m = 535 nm) on a microplate reader. Kinetic study of a truncated recombinant mOGT and quantitative analysis of OGT in two biological samples indicated that this method was practical and competitive for quantitative analysis of OGT.     

Developmental Regulation of Protein O-GlcNAcylation, O-GlcNAc Transferase, and O-GlcNAcase in Mammalian Brain

O-GlcNAcylation is a common posttranslational modification of nucleocytoplasmic proteins by β-N-acetylglucosamine (GlcNAc). The dynamic addition and removal of O-GlcNAc groups to and from proteins are catalyzed by O-linked N-acetylglucosamine transferase (O-GlcNAc transferase, OGT) and β-N-acetylglucosaminidase (O-GlcNAcase, OGA), respectively. O-GlcNAcylation often modulates protein phosphorylation and regulates several cellular signaling and functions, especially in the brain. However, its developmental regulation is not well known. Here, we studied protein O-GlcNAcylation, OGT, and OGA in the rat brain at various ages from embryonic day 15 to the age of 2 years. We found a gradual decline of global protein O-GlcNAcylation during developmental stages and adulthood. This decline correlated positively to the total protein phosphorylation at serine residues, but not at threonine residues. The expression of OGT and OGA isoforms was regulated differently at various ages. Immunohistochemical studies revealed ubiquitous distribution of O-GlcNAcylation at all ages. Strong immunostaining of O-GlcNAc, OGT, and OGA was observed mostly in neuronal cell bodies and processes, further suggesting the role of O-GlcNAc modification of neuronal proteins in the brain. These studies provide fundamental knowledge of age-dependent protein modification by O-GlcNAc and will help guide future studies on the role of O-GlcNAcylation in the mammalian brain.     

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.     

O-GlcNAc-specific antibody CTD110.6 cross-reacts with N-GlcNAc2-modified proteins induced under glucose deprivation

Modification of serine and threonine residues in proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is a feature of many cellular responses to the nutritional state and to stress. O-GlcNAc modification is reversibly regulated by O-linked β-N-acetylglucosamine transferase (OGT) and β-D-N-acetylglucosaminase (O-GlcNAcase). O-GlcNAc modification of proteins is dependent on the concentration of uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), which is a substrate of OGT and is synthesized via the hexosamine biosynthetic pathway. Immunoblot analysis using the O-GlcNAc-specific antibody CTD110.6 has indicated that glucose deprivation increases protein O-GlcNAcylation in some cancer cells. The mechanism of this paradoxical phenomenon has remained unclear. Here we show that the increased glycosylation induced by glucose deprivation and detected by CTD110.6 antibodies is actually modification by N-GlcNAc(2), rather than by O-GlcNAc. We found that this induced glycosylation was not regulated by OGT and O-GlcNAcase, unlike typical O-GlcNAcylation, and it was inhibited by treatment with tunicamycin, an N-glycosylation inhibitor. Proteomics analysis showed that proteins modified by this induced glycosylation were N-GlcNAc(2)-modified glycoproteins. Furthermore, CTD110.6 antibodies reacted with N-GlcNAc(2)-modified glycoproteins produced by a yeast strain with a ts-mutant of ALG1 that could not add a mannose residue to dolichol-PP-GlcNAc(2). Our results demonstrated that N-GlcNAc(2)-modified glycoproteins were induced under glucose deprivation and that they cross-reacted with the O-GlcNAc-specific antibody CTD110.6. We therefore propose that the glycosylation status of proteins previously classified as O-GlcNAc-modified proteins according to their reactivity with CTD110.6 antibodies must be re-examined. We also suggest that the repression of mature N-linked glycoproteins due to increased levels of N-GlcNAc(2)-modified proteins is a newly recognized pathway for effective use of sugar under stress and deprivation conditions. Further research is needed to clarify the physiological and pathological roles of N-GlcNAc(2)-modified proteins.     

O-linked beta-N-acetylglucosaminyltransferase substrate specificity is regulated by myosin phosphatase targeting and other interacting proteins

O-GlcNAc-transferase (OGT) substrate specificity is regulated by transiently interacting proteins. To further examine the regulation of OGT, we have identified 27 putative OGT-interacting proteins through a yeast two-hybrid screen. Two of these proteins, Trak1 (OIP106) and O-GlcNAcase, have been shown previously to interact with and regulate OGT. We demonstrate here that MYPT1 and CARM1 also interact with and target OGT. MYPT1 and CARM1 are substrates of OGT in vitro and in vivo. MYPT1 and CARM1 also function to alter OGT substrate specificity in vitro. Furthermore depletion of MYPT1 in Neuro-2a neuroblastoma cells alters GlcNAcylation of several proteins under basal conditions, suggesting that MYPT1 regulates OGT substrate specificity in vivo.     

Functional Heterogeneity of Alternatively Spliced Isoforms of Drosophila Ca2+/Calmodulin-Dependent Protein Kinase II

Abstract: The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian α, β, γ, and δ isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher K _act for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower K _m for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.     

Prediction of structure and functional residues for O-GlcNAcase,a divergent homologue of acetyltransferases

N-Acetyl-beta-D-glucosaminidase (O-GlcNAcase) is a key enzyme in the posttranslational modification of intracellular proteins by O-linked N-acetylglucosamine (O-GlcNAc). Here, we show that this protein contains two catalytic domains, one homologous to bacterial hyaluronidases and one belonging to the GCN5-related family of acetyltransferases (GNATs). Using sequence and structural information, we predict that the GNAT homologous region contains the O-GlcNAcase activity. Thus, O-GlcNAcase is the first member of the GNAT family not involved in transfer of acetyl groups, adding a new mode of evolution to this large protein family. Comparison with solved structures of different GNATs led to a reliable structure prediction and mapping of residues involved in binding of the GlcNAc-modified proteins and catalysis.     

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.     

Ataxin-10 interacts with O-GlcNAc transferase OGT in pancreatic beta cells

Several nuclear and cytoplasmic proteins in metazoans are modified by O-linked N-acetylglucosamine (O-GlcNAc). This modification is dynamic and reversible similar to phosphorylation and is catalyzed by the O-linked GlcNAc transferase (OGT). Hyperglycemia has been shown to increase O-GlcNAc levels in pancreatic beta cells, which appears to interfere with beta-cell function. To obtain a better understanding of the role of O-linked GlcNAc modification in beta cells, we have isolated OGT interacting proteins from a cDNA library made from the mouse insulinoma MIN6 cell line. We describe here the identification of Ataxin-10, encoded by the SCA10 (spinocerebellar ataxia type 10) gene as an OGT interacting protein. Mutations in the SCA10 gene cause progressive cerebellar ataxias and seizures. We demonstrate that SCA10 interacts with OGT in vivo and is modified by O-linked glycosylation in MIN6 cells, suggesting a novel role for the Ataxin-10 protein in pancreatic beta cells.