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.     

Molecular mechanisms of O-GlcNAcylation

Protein glycosylation with O-linked N-acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of serines/threonines on metazoan proteins and occurring with similar time scales, dynamics and stoichiometry as protein phosphorylation. Levels of this modification are regulated by two enzymes-O-GlcNAc transferase (OGT) and O-GlcNAc hydrolase (OGA). Although the biochemistry of these enzymes and functional implications of O-GlcNAc have been studied extensively, until recently the structures and molecular mechanisms of OGT/OGA were not understood. This review covers a body of recent work that has led to an understanding of the structure of OGA, its catalytic mechanism and the development of a plethora of different inhibitors that are finding their use in cell biological studies towards the functional implications of O-GlcNAc. Furthermore, the very recent structure determination of a bacterial OGT orthologue has given the first insights into the contribution of the tetratricopeptide repeats (TPRs) to the active site and the role of some residues in catalysis and substrate binding.     

Functional analysis of recombinant human and <Emphasis Type="Italic">Yarrowia lipolytica</Emphasis> <Emphasis Type="Italic">O</Emphasis>-GlcNAc transferases expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

O-linked β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important post-translational modification in many cellular processes. It is mediated by O-GlcNAc transferases (OGTs), which catalyze the addition of O-GlcNAc to serine or threonine residues of the target proteins. In this study, we expressed a putative Yarrowia lipolytica OGT (YlOGT), the only homolog identified in the subphylum Saccharomycotina through bioinformatics analysis, and the human OGT (hOGT) as recombinant proteins in Saccharomyces cerevisiae, and performed their functional characterization. Immunoblotting assays using antibody against O-GlcNAc revealed that recombinant hOGT (rhOGT), but not the recombinant YlOGT (rYlOGT), undergoes auto-O-GlcNAcylation in the heterologous host S. cerevisiae. Moreover, the rhOGT expressed in S. cerevisiae showed a catalytic activity during in vitro assays using casein kinase II substrates, whereas no such activity was obtained in rYlOGT. However, the chimeric human-Y. lipolytica OGT, carrying the human tetratricopeptide repeat (TPR) domain along with the Y. lipolytica catalytic domain (CTD), mediated the transfer of O-GlcNAc moiety during the in vitro assays. Although the overexpression of full-length OGTs inhibited the growth of S. cerevisiae, no such inhibition was obtained upon overexpression of only the CTD fragment, indicating the role of TPR domain in growth inhibition. This is the first report on the functional analysis of the fungal OGT, indicating that the Y. lipolytica OGT retains its catalytic activity, although the physiological role and substrates of YlOGT remain to be elucidated.     

Disease related single point mutations alter the global dynamics of a tetratricopeptide (TPR) α-solenoid domain

Tetratricopeptide repeat (TPR) proteins belong to the class of α-solenoid proteins, in which repetitive units of α-helical hairpin motifs stack to form superhelical, often highly flexible structures. TPR domains occur in a wide variety of proteins, and perform key functional roles including protein folding, protein trafficking, cell cycle control and post-translational modification. Here, we look at the TPR domain of the enzyme O-linked GlcNAc-transferase (OGT), which catalyses O–GlcNAcylation of a broad range of substrate proteins. A number of single-point mutations in the TPR domain of human OGT have been associated with the disease Intellectual Disability (ID). By extended steered and equilibrium atomistic simulations, we show that the OGT-TPR domain acts as an elastic nanospring, and that each of the ID-related local mutations substantially affect the global dynamics of the TPR domain. Since the nanospring character of the OGT-TPR domain is key to its function in binding and releasing OGT substrates, these changes of its biomechanics likely lead to defective substrate interaction. We find that neutral mutations in the human population, selected by analysis of the gnomAD database, do not incur these changes. Our findings may not only help to explain the ID phenotype of the mutants, but also aid the design of TPR proteins with tailored biomechanical properties.     

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.     

Structural insights into mechanism and specificity of O-GlcNAc transferase

Post-translational modification of protein serines/threonines with N-acetylglucosamine (O-GlcNAc) is dynamic, inducible and abundant, regulating many cellular processes by interfering with protein phosphorylation. O-GlcNAcylation is regulated by O-GlcNAc transferase (OGT) and O-GlcNAcase, both encoded by single, essential, genes in metazoan genomes. It is not understood how OGT recognises its sugar nucleotide donor and performs O-GlcNAc transfer onto proteins/peptides, and how the enzyme recognises specific cellular protein substrates. Here, we show, by X-ray crystallography and mutagenesis, that OGT adopts the (metal-independent) GT-B fold and binds a UDP-GlcNAc analogue at the bottom of a highly conserved putative peptide-binding groove, covered by a mobile loop. Strikingly, the tetratricopeptide repeats (TPRs) tightly interact with the active site to form a continuous 120 Å putative interaction surface, whereas the previously predicted phosphatidylinositide-binding site locates to the opposite end of the catalytic domain. On the basis of the structure, we identify truncation/point mutants of the TPRs that have differential effects on activity towards proteins/peptides, giving first insights into how OGT may recognise its substrates.     

The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha

Addition of N-acetylglucosamine (GlcNAc) is a ubiquitous form of intracellular glycosylation catalyzed by the conserved O-linked GlcNAc transferase (OGT). OGT contains an N-terminal domain of tetratricopeptide (TPR) repeats that mediates the recognition of a broad range of target proteins. Components of the nuclear pore complex are major OGT targets, as OGT depletion by RNA interference (RNAi) results in the loss of GlcNAc modification at the nuclear envelope. To gain insight into the mechanism of target recognition, we solved the crystal structure of the homodimeric TPR domain of human OGT, which contains 11.5 TPR repeats. The repeats form an elongated superhelix. The concave surface of the superhelix is lined by absolutely conserved asparagines, in a manner reminiscent of the peptide-binding site of importin alpha. Based on this structural similarity, we propose that OGT uses an analogous molecular mechanism to recognize its targets.     

Functional analysis of SPINDLY in gibberellin signaling in Arabidopsis

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-delta17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-delta17 phenotype but does not reduce rga-delta17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification.     

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.     

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.     

Mutational analysis of the catalytic domain of O-linked N-acetylglucosaminyl transferase

O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades.     

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.     

New ELISA-based method for the detection of O-GlcNAc transferase activity in vitro

O-GlcNAcylation is a dynamic, reversible, post-translational modification that regulates many cellular processes. O-GlcNAc transferase (OGT) is the sole enzyme transferring N-acetylglucosamine from uridine diphosphate (UDP)-GlcNAc to selected serine/threonine residues of cytoplasm and nucleus proteins. Aberrant of OGT activity is associated with several diseases, suggesting OGT as a novel therapeutic target. In this study, we created a new enzyme linked immunosorbent assays (ELISA)-based method for detection of OGT activity. First, casein kinase II (CKII), a well-known OGT substrate, was coated onto ELISA plate. Second, the GlcNAc transferred by OGT from UDP-GlcNAc to CKII was detected using an antibody to O-GlcNAc and then the horseradish peroxidase (HRP)-labeled secondary antibody. At last, 3,3′,5,5′-tetramethylbenzidine (TMB), the substrate of HRP, was used to detect the O-GlcNAcylation level of CKII which reflected the activity of OGT. Based on a series of optimization experiments, the RL2 antibody was selected for O-GlcNAc detection and the concentrations of CKII, OGT, and UDP-GlcNAc were determined in this study. ST045849, a commercial OGT inhibitor, was used to verify the functionality of the system. Altogether, this study showed a method that could be applied to detect OGT activity and screen OGT inhibitors.     

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 distinct K(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.