Demystifying O-GlcNAcylation: hints from peptide substrates

Demystifying O-GlcNAcylation: hints from peptide substrates Abstract O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors. O-GlcNAc transferase, O-GlcNAcase, O-linked N-acetylglucosamine modification, peptide substrates, post-translational modification Introduction O-GlcNAcylation is a ubiquitous post-translational modification (PTM) of serine or threonine residues with a single O-linked β-N-acetylglucosamine (O-GlcNAc) on numerous proteins. In contrast to complex glycans that typically function on the cell surface by mediating numerous ligand–receptor interactions (Gambaryan et al. 1995; Gallegos et al. 2012; Torreno-Pina et al. 2014), O-GlcNAc modifications of proteins ubiquitously occur inside the cells where they regulate many basic cellular processes. Proteomics studies estimated that ~19% of the O-GlcNAcylated proteins, including NF-κB, p53 and STAT, are involved in regulation of gene expression (Hahne et al. 2013). O-GlcNAcylation regulates protein activity and integrates cellular signaling transduction by itself or in combination with intensive cross-talk with other PTMs (Guinez et al. 2008), especially phosphorylation (Wang et al. 2008; Ande et al. 2009; Zhong et al. 2015). Histone proteins are also targets of O-GlcNAcylation, which is suggested to control the cell cycle and cell survival (Zhang et al. 2011; Fong et al. 2012). In addition, O-GlcNAc is also involved in the regulation of proteasome function by modifying the Rpt2 subunit (Zhang et al. 2003). To date, ca. 1492 O-GlcNAcylation sites have been recorded in the PhosphoSitePlus® database (www.phosphosite.org) (Hornbeck et al. 2015) and aberrant regulation of O-GlcNAcylation has been linked to chronic diseases such as type 2 diabetes, cancer and Alzheimer’s disease (AD; Liu et al. 2004; Banerjee et al. 2015). In the case of cancer, modulation of O-GlcNAcylation by inhibitors showed an anticancer effect by inhibiting breast tumor growth and invasion, suggesting that O-GlcNAcylation is a potential therapeutic target for cancer treatment (Caldwell et al. 2010). Like protein phosphorylation, the O-GlcNAc modification of proteins is also reversible, but it is under the control of only a pair of antagonistic enzymes. O-GlcNAc transferase (OGT) catalyzes the addition of GlcNAc onto the protein using uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as the sugar donor, while O-GlcNAcase (OGA) removes the O-GlcNAc from the modified protein. Remarkably, OGT activity is largely influenced by the intracellular concentration of UDP-GlcNAc which is the metabolic end product of the hexosamine biosynthetic pathway (HBP; Kreppel and Hart 1999; Swamy et al. 2016). Since the HBP integrates metabolisms of glucose, acids (acetate), amino acids (glutamine) and nucleotides (UTP) to generate UDP-GlcNAc, O-GlcNAcylation is considered as a nutrient sensitive PTM (Hawkins et al. 1997; Liu et al. 2007; Singleton and Wischmeyer 2008; Onodera et al. 2014; Durning et al. 2016). The gene encoding OGT is localized on the X-chromosome in human and mouse and is considered to be essential in the mammalian development (Shafi et al. 2000). Although only one gene is responsible for OGT, it can generate three separate transcripts that encode three different isoforms of the enzyme. All OGT isoforms consist of a tetratricopeptide repeats (TPR) domain, a linker region and a C-terminal catalytic domain. In humans, all three OGT isoforms have an identical catalytic domain but differ in the number of TPR repeats, each of which includes ca. 34 amino acids (Lubas et al. 1997; Hanover et al. 2003). The longest one contains 13.5 TPR repeats (Jinek et al. 2004), is localized in both the nucleus and the cytoplasm and has been termed as nc-OGT. The second longest one has 9.5 TPR repeats, contains a mitochondrial targeting sequence and has been termed m-OGT. The shortest one has only 2.5 TPR repeats, is expressed in both the nucleus and the cytoplasm and is termed s-OGT, referring to the short nature of the TPR tail. Crystal structures of the OGT catalytic pocket with bound substrates have revealed that an ordered bi-bi mechanism was involved in the glycosylation reaction where the sugar donor UDP-GlcNAc binds first and facilitates the binding of the acceptor peptide (Lazarus et al. 2011). UDP-GlcNAc is bound in the OGT catalytic site by evolutionarily conserved residues important for catalytic activity. The peptide is bound in the OGT catalytic site by its amide backbone interaction with OGT side chains and the UDP moiety with an extended conformation. In the transfer of GlcNAc, the α-phosphate of UDP-GlcNAc is possibly used as a catalytic base, together with catalytical residues of OGT such as Lys842 (Schimpl, Zheng et al. 2012), although the identity of the base has not been firmly established (Withers and Davies 2012). Surprisingly, OGT was also shown to have a proteolytic activity in the maturation process of the cell cycle host cell factor 1 (HCF1) and the cleavage of HCF1 occurs within the OGT catalytic domain in a UDP-GlcNAc dependent manner (Capotosti et al. 2011; Lazarus et al. 2013). The underlying mechanism for this unexpected activity of OGT seems to involve glycosylation of the HCF1 glutamate side chain and conversion of this glycosylation product to an internal pyroglutamate which undergoes backbone cleavage (Janetzko et al. 2016). Studies of TPR repeat structures and interactors have demonstrated that TPR repeats form important protein–protein interactions which might contribute to the substrate specificity of OGT (Iyer and Hart 2003; Iyer et al. 2003; Jinek et al. 2004; Lazarus et al. 2013). Recent studies further suggested that OGT recognizes the protein substrate TAB1 through extensive interactions with the TPR domain, suggesting an essential role of TPR in OGT recognition of physiological substrates (Rafie et al. 2017). In addition, it was proposed that transient interactions of OGT and its partner interactors may form holoenzymes which might be substrate-specific (Deng et al. 2014). While aspects of TPR recognition and holoenzymes formation may play significant roles, all OGT substrates will additionally have to obey the specificity constraints imposed upon them especially from the −3 to +3 positions around the modification sites by the characteristics of the OGT active site, which can be efficiently studied with short peptide OGT substrates (Lazarus et al. 2012, 2013; Schimpl, Zheng et al. 2012). The human OGA gene is localized on chromosome 10q24 and it has been implicated in AD (Kim et al. 2006). It has been reported that humans have two OGA splicing transcripts which produce the long OGA (OGA-L or OGA-FL) and the small nuclear variant of OGA (OGA-S or OGA-NV), respectively (Li et al. 2010). OGA-L is a 103 kDa protein that typically has an N-terminal catalytic domain, a stalk domain and a C-terminal histone acetyltransferase (HAT) like domain. OGA-S (76 kDa) does not have the HAT like domain and it shows a 400-fold lower hydrolase efficiency than OGA-L, although they have similar catalytic properties (Kim et al. 2006; Macauley and Vocadlo 2009; Li et al. 2010). Despite ample progress in the investigation of human OGA, its mechanisms and inhibitors have been mostly derived from studying the bacterial homologs of OGA. In the hydrolysis reaction by bacterial CpOGA, Asp401 (Asp175 in human OGA) forms major hydrogen bonds with the sugar portion of the substrate, while Asp298 (Asp285 in human OGA) protonates the glycosidic bond (Rao et al. 2006; Selvan et al. 2017). As a boost for research on the human OGA, it should be noted that crystal structures of human OGA became available recently (Elsen et al. 2017; Li et al. 2017; Roth et al. 2017). In agreement with bacterial OGA studies, it was found that human OGA mainly uses Asp285 to anchor the GlcNAc moiety of the substrate through hydrogen bonds and uses Asp174 as a catalytic base and Asp175 as a catalytic acid (Cetinbas et al. 2006; Li et al. 2017). Recent research work on demystifying O-GlcNAc processing enzymes, using short peptide substrates, has seen progress and further advanced our understanding of this essential PTM. This motivated us to summarize the usefulness of peptide substrates to gain insights into the properties of OGT and OGA. This review covers five aspects of the use of peptides: (1) it describes peptide substrate-based methods for the study of O-GlcNAcylation; (2) it summarizes the OGT and OGA substrate preferences; (3) it discusses the incorporation of O-GlcNAc into peptides as well as proteins by chemical synthesis; (4) it summarizes the generation of site-specific O-GlcNAc antibodies using glycopeptides and (5) it discusses the early development of OGT inhibitors utilizing the peptide substrate information. Identifying activities of O-GlcNAc processing enzymes using peptide-based approaches OGT activity profiling using peptide substrates The relevance of the use of short peptides, as substrates, to profile activity and acquire substrate specificity for kinases and phosphatases has been shown on numerous occasions for kinases and phosphatases, which has lead to great successes in the field of phosphorylation (Jarboe et al. 2012; Deng Y et al. 2014; Stanford et al. 2014; Hovestad-Bijl et al. 2016; Miller and Turk 2016). By taking advantage of their flexibility and availability, peptide substrates of OGT have become efficient tools to predict OGT activity (Lubas and Hanover 2000; Shen et al. 2017) as well as a means to study aspects of binding and catalysis in crystal structures of complexes (Lazarus et al. 2011; Schimpl, Borodkin et al. 2012). One commonly used peptide substrate of OGT is the CKII_340_352 peptide derived from the α-subunit of casein kinase II which is O-GlcNAcylated at residue Ser347 in vitro and in vivo (Kreppel and Hart 1999; Zachara et al. 2011; Tarrant et al. 2012). Several excellent OGT peptide substrates have also been discovered through different means including RBL2_410_422 (Pathak et al. 2015; Shi et al. 2016), TAB1_389_401 (Pathak et al. 2012; Schimpl, Borodkin et al. 2012) and ZO-3_357_371 (Table I) (Shi et al. 2017). All these peptide substrates have individual protein origins, and therefore provide entries into the study of the biology of the parent proteins. Table I. A list of peptides mentioned in this review Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Table I. A list of peptides mentioned in this review Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  In recent years, protein and peptides arrays of different formats have been applied to identify O-GlcNAcylation and the study of OGT activities (Deng RP et al. 2014; Ortiz-Meoz et al. 2014; Pathak et al. 2015; Shi et al. 2016, 2017). A human protein microarray containing 8000 unique proteins has been successfully used to study OGT activity and hundreds of novel proteins have been identified as OGT substrates with diverse functions, including kinases, transcription factors and apoptosis-related proteins (Ortiz-Meoz et al. 2014). However, the protein array cannot be used to generate straightforward sequence information. While it has its own valuable uses, the use of small peptides focuses more readily on the specificity of the enzyme. The use of a peptide microarray to study OGT offers advantages in generating inherent sequence-specificity information. As such, the method is complementary to the protein array where additional protein–protein interactions may additionally determine the specificity. The first example of a peptide microarray study described a library of 720 synthesized biotinylated peptides with sequences derived from the human proteome, using a scintillation proximity assay (Pathak et al. 2015). In this assay (Figure 1A), the OGT reaction was carried out in 384-well polypropylene plates, with one peptide in each well, in the presence of radio labeled UDP-[3H]GlcNAc. Then, the O-GlcNAcylated peptides were immobilized on a streptavidin coated scintillant flashplate, which allowed the direct quantification of radio labeled peptide by using scintillation counting. This approach revealed comprehensive sequence information which is discussed in Section 3. Disadvantages include the fact that this approach involved radio labeling which requires time for signal development and is therefore less convenient for real time kinetic analysis. Fig. 1. View largeDownload slide Schematic depiction of high-throughput assays for activity of OGT and OGA against peptide substrates. (A) Scintillation proximity-based OGT assay. (B) Peptide microarray-based real time OGT assay. (C) Glycopeptide microarray-based OGA assay. Fig. 1. View largeDownload slide Schematic depiction of high-throughput assays for activity of OGT and OGA against peptide substrates. (A) Scintillation proximity-based OGT assay. (B) Peptide microarray-based real time OGT assay. (C) Glycopeptide microarray-based OGA assay. A different peptide microarray with kinetics monitoring was described for OGT activity by our group (Figure 1B; Shi et al. 2016). This dynamic peptide microarray technology was initially developed for kinase activity profiling (Lemeer et al. 2007; Anderson et al. 2015). In the kinase field, the use of immobilized peptide sequences derived from knowledge of protein phosphorylation sites has proven to be highly valuable for the study of kinase activity (Hilhorst et al. 2009). In this array, each peptide was prepared with additional Cys-Gly residues at the N-terminus, which allows immobilization of a maleimide functionalized porous membrane surface. However, the nature of this immobilization chemistry also makes the array results less quantifiable when studying peptides with multiple cysteines, due to additional non-terminal attachment modes. The OGT reaction was performed in the presence of a fluorescently labeled detection antibody. The reaction mixture can be pumped up and down periodically through the membrane during the entire reaction time and the fluorescent signal of only the bound fluorescent antibody is acquired at regular intervals. This is possible by temporarily placing the solution with unbound fluorescent antibody below the chip, outside the detector range (Figure 1B). As a result, this enables a kinetic reading of the enzymatic modification of each peptide on the array without removing the unbound antibody by extra washing. O-GlcNAcylation of peptides with this array can be achieved not only by using purified OGT enzyme but also by using a cell lysate. In addition, this array-based identification of OGT substrates from peptides known to contain phosphorylation sites, also made it possible to study cross-talk between these two modifications (Shi et al. 2017), which will be discussed in Section 4. Kinase activity profiling with such arrays has been demonstrated to be of relevance for cancer diagnosis and help in the prediction of efficacy of cancer drugs for a specific patient (Folkvord et al. 2010), so ultimately O-GlcNAcylation on peptide arrays may exhibit a similar medicinal relevance. Although these peptide-based techniques provide a tool for OGT substrate identification, these hits were not validated at the protein level. Detection of OGA activities with peptide substrates In vitro OGA activity is normally detected by using the color producing compound p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside (pNPGlcNAc) and the fluorogenic compounds 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside (MuGlcNAc) and fluorescein di(N-acetyl-beta-d-glucosamine) (FDGlcNAc) as substrates (Dong and Hart 1994; Macauley et al. 2005; Kim et al. 2006). However, these compounds are not natural substrates of OGA and cannot fully reflect OGA activity with a natural glycopeptidic substrate. Similarly to the OGT array assay, a fluorescent method for the detection of OGT activity was used to study the removal of GlcNAc from a more natural substrate (Figure 1C). This peptide microarray-based approach was recently described to measure OGA activity from both purified OGA as well as OGA present in cell lysates derived from several cancer cell lines (Sharif et al. 2017). A series of glycopeptides as OGA substrates was prepared based on peptides which are known OGT targets and these were immobilized on the array. The array was subjected to purified OGA or cell lysates, followed by detection of the remaining O-GlcNAc on each peptide with a fluorescent antibody. In addition, a more comprehensive version of this glycopeptide microarray with more diversity has to be used to further validate its potential to profile OGA catalytic properties. Essential role of amino acids around the O-GlcNAc site in substrate recognition OGT substrate recognition It was recently reported that OGT has a peptide sequence preference around its modification site on short peptide substrates (Jochmann et al. 2014; Liu et al. 2014; Pathak et al. 2015). A high density peptide microarray is an ideal platform to study such enzymatic specificities (Lai et al. 2016). In an array analysis of 720 Ser/Thr peptides for OGT activity, ~10% of these peptides were identified as OGT substrates (Pathak et al. 2015), suggesting that OGT has the ability to specifically recognize a substrate by its primary sequence among hundreds of peptides (Shi et al. 2016, 2017). With the sequence information of these identified peptides, an unbiased OGT substrate motif was obtained as [TS][PT][VT]S/T[RLV][ASY] (from −3 to +2 with respect to O-GlcNAcylation site) which was also verified in select cases by crystal structures (Pathak et al. 2015). This motif is generally in line with the motif PPV[S/T]SATT (Liu et al. 2014) and PPV[S/T]TA (Wu et al. 2014) obtained by frequency analysis of known OGT substrates. The clear preference of Pro at the −2 site by OGT enables the peptide substrate to display an extended conformation, which facilitates its binding to the OGT active site (Lazarus et al. 2011; Pathak et al. 2015). Interestingly, it was also reported that OGT has a strictly negative preference for Pro at the +1 subsite, which could prevent O-GlcNAcylation at a Ser/Thr/Pro rich region even if other sites match with the substrate motif (Leney et al. 2017). Despite this progress, the current motifs are not able to accurately predict OGT peptide substrates. We have found that only a few of our experimentally identified novel substrates matched with these reported motifs and some matched peptides in our experiments were not experimentally identified as a substrate (Shi et al. 2016, 2017). Nevertheless, for the best discovered hit peptide from our experiments the motif successfully identified the modification site. This site was confirmed through mutation experiments by making a series of peptides (Shi et al. 2017). Regardless of predictable accuracy, all these findings strongly suggest that the primary sequence is a very important factor in determining the substrate specificity of OGT. It should be noted, also, that no obvious consensus motif has been found by proteomic studies of O-GlcNAc proteins from murine synaptosome although moderate preferences for Pro (−2 and −3 positions) and Val (−1 or −3 positions) by OGT have been observed (Trinidad et al. 2012, 2013; Vosseller et al. 2006). There is a possibility that in vivo OGT might achieve substrate specificity following a less stringent motif and that other determinants such as the TPR repeats and holoenzymes are involved as discussed in Section 1. Regardless of this, it should still be possible to generate a more complete and comprehensive OGT substrate motif, since short peptides only interact with OGT at the active site, which would be a very valuable tool for OGT protein substrate prediction and identification. OGA substrate recognition The investigation of human OGA specificity was primarily limited by the lack of available OGA crystal structures with bound substrate. Hence, a bacterial OGA orthologue such as CpOGA was widely used as a model to explore human OGA catalytic properties (Rao et al. 2006; Schimpl, Borodkin et al. 2012). The crystal structure of CpOGA in complex with synthetic glycopeptides derived from p53 (Yang et al. 2006), TAB1 and OGA (Table I) was used to gain insights into the human OGA specificity (Schimpl, Borodkin et al. 2012). In this structure, extensive hydrogen bonds were observed between the GlcNAc moiety and CpOGA (Schimpl, Borodkin et al. 2012), suggesting an important role of GlcNAc for OGA substrate binding and recognition. Interestingly, although no peptide side chain was found to be involved in direct interaction with the enzyme, a combination of intramolecular hydrogen bonds and hydrophobic interactions between the peptide backbone and CpOGA forced the glycopeptide into a conserved “V-shape” conformation that makes it possible to access the CpOGA active site (Schimpl, Borodkin et al. 2012). In addition, kinetic studies showed that human OGA has similar catalytic parameters for five O-GlcNAcylated proteins and a glycopeptide derived from a protein substrate is a better substrate than the protein itself. These data further suggest that GlcNAc is the primary determinant in OGA catalysis but amino acid residues may hinder (Shen et al. 2012). Now that crystal structures of human OGA have recently become available (Elsen et al. 2017; Li et al. 2017; Roth et al. 2017) with, e.g., the complexed O-GlcNAcylated p53_144-154 peptide, again an essential role of GlcNAc for OGA substrates recognition is indicated (Li et al. 2017). However, in this case also the peptide side chains were involved in hydrophobic interactions with OGA. Substitution of substrate peptide residues and OGA residues involved in the interaction, both lead to a reduction of substrate binding (Li et al. 2017). Although O-GlcNAc is the dominant structural feature for OGA substrate recognition, additional specific interactions involving substrate amino acids might exist, which could lead to differential preferences of substrates by OGA. Whether OGA requires a distinct substrate motif could be further studied, e.g., by using an array of synthetic glycopeptides. Based on the above fact that the sugar moiety is the major determinant for OGA activity, a hydrolytically stable sulfur-linked glycopeptide (S-GlcNAc) was synthesized and incorporated into the protein α-synuclein (De Leon et al. 2017) where indeed stability against OGA was observed. Global regulation of O-GlcNAc cycling While several comprehensive reviews have discussed the regulatory mechanisms of O-GlcNAc cycling (Love et al. 2010; Bond and Hanover 2013), recent findings add to this picture. O-GlcNAcylation cycling is critically controlled by many factors (Figure 2). OGT activity is greatly dependent on the intracellular concentration of UDP-GlcNAc, which is a metabolic product of the HBP together with fatty acid metabolism, amino acid metabolism and nucleotide metabolism (Hawkins et al. 1997; Liu et al. 2007; Singleton and Wischmeyer 2008; Onodera et al. 2014). The altered levels of OGT and OGA in cancer cells suggests the involvement of regulation in the enzyme expression levels (Krzeslak et al. 2012; Lynch et al. 2012; Sharif et al. 2017). UDP, a product of the O-GlcNAcylation reaction, might act as a feedback inhibitory regulator of OGT, considering its strong binding to the enzyme of ca. 2 μM (Dorfmueller et al. 2011). The extensive cross-talk of phosphorylation and O-GlcNAcylation represent novel regulatory mechanisms from the substrate side, which is slowly becoming more clear (Dias et al. 2009; Wang et al. 2010; Leney et al. 2017; Shi et al. 2017). Furthermore, the inherent specificity of especially OGT (Shi et al. 2016) and now seemingly also of OGA, enables their ability to selectively act on substrate proteins based on their amino acid sequence (Li et al. 2017). Finally, the role of OGT’s TPR repeats have been implicated in the substrate preference based on protein–protein interactions with the substrate protein (Rafie et al. 2017). Globally, it seems that to maintain a normal O-GlcNAcylation status, the addition of O-GlcNAc involves stringent and multiple levels of regulation, while for the removal of O-GlcNAc this seems to be less so. Nevertheless, additional OGT/OGA sequence specificities and cross-talk mechanisms will likely be uncovered in the future. Fig. 2. View largeDownload slide The global regulation of O-GlcNAc cycling. The cellular O-GlcNAcylation of proteins can be controlled by factors including OGT expression, inherent specificity (TPR and consensus sequence), nutrient availability, UDP-GlcNAc availability, UDP feedback inhibition, cross-talk with phosphorylation, OGA expression and OGA’s robust hydrolysis ability. Fig. 2. View largeDownload slide The global regulation of O-GlcNAc cycling. The cellular O-GlcNAcylation of proteins can be controlled by factors including OGT expression, inherent specificity (TPR and consensus sequence), nutrient availability, UDP-GlcNAc availability, UDP feedback inhibition, cross-talk with phosphorylation, OGA expression and OGA’s robust hydrolysis ability. Direct role of O-GlcNAcylation from synthetic glycopeptide to glycoprotein Elucidating the physiological function of a specific O-GlcNAcylation event on a specific protein is a challenging task because of the complex natural context of proteins within the cell. Removing of a putative modification site by gene mutation has been widely performed to indirectly observe the effect of a specific PTM in both in vitro and in vivo situations, resulting often in unconfirmed hypotheses. Using synthetic peptides is an efficient way to unambiguously introduce specific PTMs in a peptide which can subsequently be incorporated into a protein using advanced chemical ligation protocols. This then enables the direct study of the role of that modification. The advantage of peptide chemistry in studying a specific PTM has been well-demonstrated in the study of the Tau protein, which is linked to assembly and stabilization of microtubules for the proper function of neurons (Himmler et al. 1989; Smet-Nocca et al. 2011). Misfolding and aggregation of Tau has been implicated in AD and other neurodegenerative disorders, which is mainly caused by abnormal PTMs including phosphorylation and O-GlcNAcylation (Yuzwa et al. 2012; Morris et al. 2015). Using a synthetic phosphorylated Tau peptide as an OGT substrate or an O-GlcNAcylated Tau peptide as a kinase substrate, mass spectrometry experiments revealed a reciprocal relationship between phosphorylation (on Serines 396 and 404) and O-GlcNAcylation (on serine 400) in peptide Tau-392_411 (Smet-Nocca et al. 2011). The phosphate group at Ser400 completely prevents O-GlcNAcylation of this peptide by direct occupation of the O-GlcNAcylation site, and the phosphate groups at Ser396 and Ser404 led to a decreased O-GlcNAcylation level at Ser400. In the other direction, O-GlcNAcylation of Ser400 reduced the phosphorylation of Ser404 mediated by the Cdk2 kinase complex and completely suppressed the phosphorylation of Ser396/400/404 mediated by GSK3β (Figure 3A). Fig. 3. View largeDownload slide Study of O-GlcNAcylation and its cross-talk in a direct manner using synthetic peptide and synthetic proteins. Mass spectrometry-based study of cross-talk on Tau_392-411 (A) and peptide microarray-based study of cross-talk on ZO-3_357-371 (B) with synthetic glycopeptide and phosphopeptide. *This kinetic peptide microarray assay is described in Figure 1B. (C) Functional study of O-GlcNAc modification on CK2α and α-synuclein using synthetic glycoprotein. Fig. 3. View largeDownload slide Study of O-GlcNAcylation and its cross-talk in a direct manner using synthetic peptide and synthetic proteins. Mass spectrometry-based study of cross-talk on Tau_392-411 (A) and peptide microarray-based study of cross-talk on ZO-3_357-371 (B) with synthetic glycopeptide and phosphopeptide. *This kinetic peptide microarray assay is described in Figure 1B. (C) Functional study of O-GlcNAc modification on CK2α and α-synuclein using synthetic glycoprotein. Recently, a dynamic peptide microarray approach has been described to study the interplay between O-GlcNAc and tyrosine phosphorylation in a direct and real time manner (Shi et al. 2017). In this work, dual substrates of OGT and the Jak2 tyrosine kinase were identified from an array of peptides derived from known tyrosine kinase protein substrates. With these dual substrates, the effect of prior O-GlcNAcylation by OGT on the subsequent tyrosine phosphorylation by Jak2 was determined, and vice versa. This approach revealed that tyrosine phosphorylation of six peptides prevented their subsequent O-GlcNAcylation, whereas no correlation was found in the other direction. Peptides derived from the ZO-3 protein were synthesized with a phosphate group on Tyr364 and a GlcNAc group on Ser369, respectively. They were immobilized on the microarray to further validate the identified cross-talk kinetically (Figure 3B). Besides using purified enzymes, this cross-talk was also observed using a HeLa cell lysate as the source of both OGT and the kinase. Although useful kinetics information was achieved in this case, future studies should also confirm these observations with in vivo experiments. This type of cross-talk, might prove to be more general with additional studies (Leney et al. 2017). A modified peptide can be employed to synthetically access a modified full-length OGT protein substrate through a semi-synthetic strategy using native chemical ligation (Dawson et al. 1994; Smet-Nocca et al. 2011). This enables validation of its physiological functions at the protein level. Recently, a synthesis of O-GlcNAcylated α-synuclein by ligating synthesized peptide and expressed protein portions, has indicated that O-GlcNAcylation of this protein has an inhibitory effect on its aggregation, suggesting a crucial role of O-GlcNAcylation in the progression of AD and Parkinson’s disease (Marotta et al. 2015). In this study, the O-GlcNAc modified peptide was prepared by an Fmoc-based solid phase peptide synthesis strategy, resulting in a C-terminal thioester and an N-terminal thioproline-protected cysteine. Through an expressed protein ligation strategy (Figure 3C), the N-terminal cysteine of a recombinant protein segment was trans-esterified with the C-terminus of the above peptide and then rapidly rearranged to form a stable peptide bond. Similarly, the ligation of the newly formed construct and a recombinant protein segment with a C-terminal thioesters was carried out. After radical-based desulfurization of the introduced extra cysteine, a full-length α-synuclein containing an O-GlcNAc modification was obtained. The synthetic protein showed secondary structure characteristics that were the same as those derived from the unmodified protein. With this synthetic O-GlcNAc modified α-synuclein, it was demonstrated that a single O-GlcNAcylation at Thr72 has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, suggesting that modulating a specific O-GlcNAcylation is perhaps a viable therapeutic strategy for PD. In addition, O-GlcNAcylation of this protein at Thr72 was shown to be able to slightly increase its phosphorylation at Ser87 by the CK1 kinase, but inhibit its phosphorylation at S129 by the GRK5, CK1 and PLK3 kinases (Figure 3C). As a result, such cross-talk may also affect α-synuclein aggregation. Another well-demonstrated example is the synthesis of O-GlcNAcylated CK2 which is involved in cell growth and proliferation through its kinase activity (Lebrin et al. 2001). In this case (Figure 3C), an O-GlcNAcylated protein was prepared using the same expressed protein ligation strategy, except that the synthetic peptide was prepared with metabolically stable thio-linked GlcNAc (S-GlcNAc) (Tarrant et al. 2012). Using this protein, it was found that O-GlcNAcylation of Ser347 inhibits its phosphorylation at Thr344 and modulates CK2 kinase substrate selectivity. This methodology is very powerful although the synthesis is challenging and in certain cases protein folding issues may occur. Generation of specific O-GlcNAc antibodies with small OGT substrate peptides A well-studied substrate peptide provides important sequence-specific information which can be used as an antigen to generate modification-site-specific antibodies. Such specific antibodies would be powerful tools for the further validation of O-GlcNAcylation on a specific protein at the more complex endogenous levels. Take the Tau protein for example, as an early attempt with a synthetic glycopeptide (CSPVV-(O-GlcNAc)-SGDTS) as an antigen. Its use led to the generation of a polyclonal antibody (termed 3925) that recognizes Tau with the O-GlcNAc modification at Ser400 in vitro, and both the peptide sequence and the GlcNAc moiety were found to contribute to the antibody specificity (Yuzwa et al. 2011). However, this antibody was also found to recognize a range of other O-GlcNAc proteins, which makes it unsuitable for use in in vivo. Since then, a more specific monoclonal antibody against the Tau protein O-GlcNAcylation at Ser400 (termed OTau (S400)) was generated using the O-GlcNAc peptide (VYKSPVV-(O-GlcNAc))-SGDTSPRH based on a comprehensive study of O-GlcNAcylation by peptide scanning (Smet-Nocca et al. 2011; Cameron et al. 2013). This antibody was successfully applied to study the O-GlcNAcylation of the full tau protein at Ser400 in vitro and in vivo, which may further facilitate the elucidation of the function of Tau in AD (Cameron et al. 2013). There are other examples of site-specific O-GlcNAc antibodies generated by using glycopeptides, such as the monoclonal antibody against Thr58 O-GlcNAc in c-Myc (Kamemura et al. 2002), a polyclonal antibody against Ser1011 O-GlcNAc in IRS-1 (Klein et al. 2009) and a polyclonal antibody against Ser395 O-GlcNAc in TAB1 (Pathak et al. 2012). These works indicate that the sequence information generated in the peptide-based studies of O-GlcNAcylation facilitates the detection of protein O-GlcNAcylation with site-specific antibodies. Specific OGT inhibitors/bisubstrate inhibitors from OGT substrates Specific and potent inhibitors are valuable tools for deeper investigation of the biological functions of OGT. The development of OGT inhibitors is still in its early stages and the number of selective OGT inhibitors with suitable pharmaceutical properties is still quite limited. Current OGT inhibitors through rational designs are mostly small analogs of UDP-GlcNAc and UDP targeting the sugar donor binding site of OGT, such as C-UDP (IC50 = 9 μM) (Dorfmueller et al. 2011), Alloxan (IC50 = 18 μM) (Konrad et al. 2002; Dorfmueller et al. 2011), UDP-GlcNAc glycosyl C-phosphate (UDP-C-GlcNAc, IC50 = 41 μM) (Chang et al. 2006; Hajduch et al. 2008) and a thioglycosyl analog of UDP-GlcNAc (UDP-S-GlcNAc, IC50 = 93 μM) (Dorfmueller et al. 2011). Remarkably, UDP, the byproduct from O-GlcNAcylation, showed the strongest inhibitory effect of them all with an IC50 value of 1.8 μM (Dorfmueller et al. 2011). Although low micromolar inhibitors have been generated by mimicking the sugar donor, they are not cell permeable or show unfavorable side effect by affecting other enzymes that utilizing UDP or UDP-GlcNAc (Dorfmueller et al. 2011). A noteworthy rational approach described is to produce sugar donor mimic UDP-5SGlcNAc in cells by using the metabolic nature of GlcNAc salvage and HBP pathways with a precursor Ac-5SGlcNAc (Gloster et al. 2011). UDP-5SGlcNAc (Ki = 8 μM) produced by this approach has been shown to be able to disrupt OGT activity in cases (Andres et al. 2017), but it might have side effects on cell surface glycosylation and other metabolic pathways. A high throughput screening assay was also used for the discovery of OGT inhibitors, such as a substrate analog displacement assay in which a fluorescent UDP-GlcNAc analog was used as a probe to determine the binding of small molecules by fluorescence polarization (Gross et al. 2005). In this work, a cell permeable OGT inhibitor (OSMI-1, IC50 = 2.7 μM) was identified with a good but not perfect selectivity (Ortiz-Meoz et al. 2015). Recently, a natural product OGT inhibitor L01 was discovered with an IC50 value of 2.7 μM through a structure-based virtual screening and this compound showed lower toxicity and a higher selectivity in vitro and in vivo (Liu et al. 2017). One study reported on developing OGT inhibitors by mimicking the sugar acceptor peptide (Borodkin et al. 2014). The use of the enzyme–substrate interacting motif as a template to develop specific peptide inhibitor has been well-demonstrated in the field of kinases (Nishikawa et al. 2000; van Wandelen et al. 2013), although high affinity may not be easily achieved with a relatively flexible peptide backbone only. In our recent work, we have shown that an OGT substrate peptide in which the serine (Ser420) at the O-GlcNAcylation site was replaced with an alanine, which cannot be O-GlcNAcylated, exhibited ~50% OGT activity inhibition at 500 μM (Shi et al. 2016). A closely related peptide substrate was shown to bind the OGT catalytic site with several hydrogen bonds and Van der Waals interactions (Pathak et al. 2015), so it was hypothesized that the alanine mutant peptide still binds the active site of OGT. These findings provide the possibility that peptide acceptors can be starting points of OGT inhibitor development by taking advantage of the specific contacts with OGT. Conclusion and perspectives In the last decade, the use of peptide substrates has remarkably facilitated the study of O-GlcNAcylation and its processing enzymes at the active sites. OGT was shown to be selective with respect to the amino acids around the modification site, but the current OGT substrate motif or sequon derived from a limited number of peptide substrates is not complete and not yet able to accurately predict substrates. It has to be noted that there might be other synergistical modes of selectivity for OGT with respect to protein substrates, involving TRP repeat binding and holoenzymes, but this is not the case for short peptide substrates. With further profiling of OGT peptide substrates, it should be possible to generate a more comprehensive motif that could be an important predictive tool with applications, e.g., in cancer. Further research by profiling cancer cell lines and patient tissues would lead to the identification of biomarkers with diagnostic and predictive value. Although a significant number of peptides have already been identified as OGT substrates, whether its parental proteins are indeed physiological targets of OGT in cells is mostly unknown. Site-specific O-GlcNAc antibodies would be useful to discover the biological function of a specific O-GlcNAcylation event and its relevance for disease, especially for cancer, diabetes and neurodegenerative disease. Specific and cell permeable OGT inhibitors are still urgently required, not only for research tools to study OGT but also for pharmacological applications. Funding This work was supported by a scholarship (No. 201306180061) to J.S. from the China Scholarship Council. Conflict of interest statement None declared. Abbreviations AD Alzheimer’s disease GlcNAc N-acetyl-d-glucosamine HAT histone acetyltransferase HBP hexosamine biosynthetic pathway OGA O-GlcNAcase OGT O-GlcNAc transferase PTM post-translational modification TPR tetratricopeptide repeats UDP-GlcNAc Uridine 5′-diphospho-N-acetylglucosamine HAT histone acetyltransferase. References Ande SR, Moulik S, Mishra S. 2009. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch. PLoS One . 4: e4586. Google Scholar CrossRef Search ADS PubMed  Anderson JC, Willey CD, Mehta A, Welaya K, Chen D, Duarte CW, Ghatalia P, Arafat W, Madan A, Sudarshan S et al.  . 2015. High throughput kinomic profiling of human clear cell renal cell carcinoma identifies kinase activity dependent molecular subtypes. PLoS One . 10: e0139267. Google Scholar CrossRef Search ADS PubMed  Andres LM, Blong IW, Evans AC, Rumachik NG, Yamaguchi T, Pham ND, Thompson P, Kohler JJ, Bertozzi CR. 2017. Chemical modulation of protein O-GlcNAcylation via OGT inhibition promotes human neural cell differentiation. ACS Chem Biol . 12: 2030– 2039. Google Scholar CrossRef Search ADS PubMed  Banerjee PS, Ma J, Hart GW. 2015. Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria. Proc Natl Acad Sci USA . 112: 6050– 6055. Google Scholar CrossRef Search ADS PubMed  Bond MR, Hanover JA. 2013. O-GlcNAc cycling: a link between metabolism and chronic disease. Annu Rev Nutr . 33: 205– 229. Google Scholar CrossRef Search ADS PubMed  Borodkin VS, Schimpl M, Gundogdu M, Rafie K, Dorfmueller HC, Robinson DA, van Aalten DM. 2014. Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors. Biochem J . 457: 497– 502. Google Scholar CrossRef Search ADS PubMed  Caldwell SA, Jackson SR, Shahriari KS, Lynch TP, Sethi G, Walker S, Vosseller K, Reginato MJ. 2010. Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1. Oncogene . 29: 2831– 2842. Google Scholar CrossRef Search ADS PubMed  Cameron A, Giacomozzi B, Joyce J, Gray A, Graham D, Ousson S, Neny M, Beher D, Carlson G, O’Moore J et al.  . 2013. Generation and characterization of a rabbit monoclonal antibody site-specific for tau O-GlcNAcylated at serine 400. FEBS Lett . 587: 3722– 3728. Google Scholar CrossRef Search ADS PubMed  Capotosti F, Guernier S, Lammers F, Waridel P, Cai Y, Jin J, Conaway JW, Conaway RC, Herr W. 2011. O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1. Cell . 144: 376– 388. Google Scholar CrossRef Search ADS PubMed  Cetinbas N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. 2006. Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants. Biochemistry . 45: 3835– 3844. Google Scholar CrossRef Search ADS PubMed  Chang R, Vo TT, Finney NS. 2006. Synthesis of the C1-phosphonate analog of UDP-GlcNAc. Carbohydr Res . 341: 1998– 2004. Google Scholar CrossRef Search ADS PubMed  Dawson PE, Muir TW, Clark-Lewis I, Kent SB. 1994. Synthesis of proteins by native chemical ligation. Science . 266: 776– 779. Google Scholar CrossRef Search ADS PubMed  De Leon CA, Levine PM, Craven TW, Pratt MR. 2017. The sulfur-linked analogue of O-GlcNAc (S-GlcNAc) is an enzymatically stable and reasonable structural surrogate for O-GlcNAc at the peptide and protein levels. Biochemistry . 56: 3507– 3517. Google Scholar CrossRef Search ADS PubMed  Deng RP, He X, Guo SJ, Liu WF, Tao Y, Tao SC. 2014. Global identification of O-GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome. Proteomics . 14: 1020– 1030. Google Scholar CrossRef Search ADS PubMed  Deng Y, Alicea-Velazquez NL, Bannwarth L, Lehtonen SI, Boggon TJ, Cheng HC, Hytonen VP, Turk BE. 2014. Global analysis of human nonreceptor tyrosine kinase specificity using high-density peptide microarrays. J Proteome Res . 13: 4339– 4346. Google Scholar CrossRef Search ADS PubMed  Dias WB, Cheung WD, Wang Z, Hart GW. 2009. Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification. J Biol Chem . 284: 21327– 21337. Google Scholar CrossRef Search ADS PubMed  Dong DL, Hart GW. 1994. Purification and characterization of an O-GlcNAc selective N-acetyl-beta-d-glucosaminidase from rat spleen cytosol. J Biol Chem . 269: 19321– 19330. Google Scholar PubMed  Dorfmueller HC, Borodkin VS, Blair DE, Pathak S, Navratilova I, van Aalten DM. 2011. Substrate and product analogues as human O-GlcNAc transferase inhibitors. Amino Acids . 40: 781– 792. Google Scholar CrossRef Search ADS PubMed  Durning SP, Flanagan-Steet H, Prasad N, Wells L. 2016. O-Linked beta-N-acetylglucosamine (O-GlcNAc) acts as a glucose sensor to epigenetically regulate the insulin gene in pancreatic beta cells. J Biol Chem . 291: 2107– 2118. Google Scholar CrossRef Search ADS PubMed  Elsen NL, Patel SB, Ford RE, Hall DL, Hess F, Kandula H, Kornienko M, Reid J, Selnick H, Shipman JM et al.  . 2017. Insights into activity and inhibition from the crystal structure of human O-GlcNAcase. Nat Chem Biol . 13: 613– 615. Google Scholar CrossRef Search ADS PubMed  Folkvord S, Flatmark K, Dueland S, de Wijn R, Groholt KK, Hole KH, Nesland JM, Ruijtenbeek R, Boender PJ, Johansen M et al.  . 2010. Prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling. Int J Radiat Oncol Biol Phys . 78: 555– 562. Google Scholar CrossRef Search ADS PubMed  Fong JJ, Nguyen BL, Bridger R, Medrano EE, Wells L, Pan S, Sifers RN. 2012. Beta-N-acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3. J Biol Chem . 287: 12195– 12203. Google Scholar CrossRef Search ADS PubMed  Gallegos KM, Conrady DG, Karve SS, Gunasekera TS, Herr AB, Weiss AA. 2012. Shiga toxin binding to glycolipids and glycans. PLoS One . 7: e30368. Google Scholar CrossRef Search ADS PubMed  Gambaryan AS, Piskarev VE, Yamskov IA, Sakharov AM, Tuzikov AB, Bovin NV, Nifant’ev NE, Matrosovich MN. 1995. Human influenza virus recognition of sialyloligosaccharides. FEBS Lett . 366: 57– 60. Google Scholar CrossRef Search ADS PubMed  Gloster TM, Zandberg WF, Heinonen JE, Shen DL, Deng L, Vocadlo DJ. 2011. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat Chem Biol . 7: 174– 181. Google Scholar CrossRef Search ADS PubMed  Gross BJ, Kraybill BC, Walker S. 2005. Discovery of O-GlcNAc transferase inhibitors. J Am Chem Soc . 127: 14588– 14589. Google Scholar CrossRef Search ADS PubMed  Guinez C, Mir AM, Dehennaut V, Cacan R, Harduin-Lepers A, Michalski JC, Lefebvre T. 2008. Protein ubiquitination is modulated by O-GlcNAc glycosylation. FASEB J . 22: 2901– 2911. Google Scholar CrossRef Search ADS PubMed  Hahne H, Sobotzki N, Nyberg T, Helm D, Borodkin VS, van Aalten DM, Agnew B, Kuster B. 2013. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. J Proteome Res . 12: 927– 936. Google Scholar CrossRef Search ADS PubMed  Hajduch J, Nam G, Kim EJ, Frohlich R, Hanover JA, Kirk KL. 2008. A convenient synthesis of the C-1-phosphonate analogue of UDP-GlcNAc and its evaluation as an inhibitor of O-linked GlcNAc transferase (OGT). Carbohydr Res . 343: 189– 195. Google Scholar CrossRef Search ADS PubMed  Hanover JA, Yu S, Lubas WB, Shin SH, Ragano-Caracciola M, Kochran J, Love DC. 2003. Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene. Arch Biochem Biophys . 409: 287– 297. Google Scholar CrossRef Search ADS PubMed  Hawkins M, Barzilai N, Liu R, Hu M, Chen W, Rossetti L. 1997. Role of the glucosamine pathway in fat-induced insulin resistance. J Clin Invest . 99: 2173– 2182. Google Scholar CrossRef Search ADS PubMed  Hilhorst R, Houkes L, van den Berg A, Ruijtenbeek R. 2009. Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A. Anal Biochem . 387: 150– 161. Google Scholar CrossRef Search ADS PubMed  Himmler A, Drechsel D, Kirschner MW, Martin DW Jr. 1989. Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. Mol Cell Biol . 9: 1381– 1388. Google Scholar CrossRef Search ADS PubMed  Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. 2015. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res . 43: D512– D520. Google Scholar CrossRef Search ADS PubMed  Hovestad-Bijl L, van Ameijde J, Pijnenburg D, Hilhorst R, Liskamp R, Ruijtenbeek R. 2016. Peptide microarrays for real-time kinetic profiling of tyrosine phosphatase activity of recombinant phosphatases and phosphatases in lysates of cells or tissue Samples. Methods Mol Biol . 1447: 67– 78. Google Scholar CrossRef Search ADS PubMed  Iyer SP, Akimoto Y, Hart GW. 2003. Identification and cloning of a novel family of coiled-coil domain proteins that interact with O-GlcNAc transferase. J Biol Chem . 278: 5399– 5409. Google Scholar CrossRef Search ADS PubMed  Iyer SP, Hart GW. 2003. Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity. J Biol Chem . 278: 24608– 24616. Google Scholar CrossRef Search ADS PubMed  Janetzko J, Trauger SA, Lazarus MB, Walker S. 2016. How the glycosyltransferase OGT catalyzes amide bond cleavage. Nat Chem Biol . 12: 899– 901. Google Scholar CrossRef Search ADS PubMed  Jarboe JS, Jaboin JJ, Anderson JC, Nowsheen S, Stanley JA, Naji F, Ruijtenbeek R, Tu T, Hallahan DE, Yang ES et al.  . 2012. Kinomic profiling approach identifies Trk as a novel radiation modulator. Radiother Oncol . 103: 380– 387. Google Scholar CrossRef Search ADS PubMed  Jinek M, Rehwinkel J, Lazarus BD, Izaurralde E, Hanover JA, Conti E. 2004. The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha. Nat Struct Mol Biol . 11: 1001– 1007. Google Scholar CrossRef Search ADS PubMed  Jochmann R, Holz P, Sticht H, Sturzl M. 2014. Validation of the reliability of computational O-GlcNAc prediction. Biochim Biophys Acta . 1844: 416– 421. Google Scholar CrossRef Search ADS PubMed  Kamemura K, Hayes BK, Comer FI, Hart GW. 2002. Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens. J Biol Chem . 277: 19229– 19235. Google Scholar CrossRef Search ADS PubMed  Kim EJ, Kang DO, Love DC, Hanover JA. 2006. Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate. Carbohydr Res . 341: 971– 982. Google Scholar CrossRef Search ADS PubMed  Klein AL, Berkaw MN, Buse MG, Ball LE. 2009. O-linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs. Mol Cell Proteomics . 8: 2733– 2745. Google Scholar CrossRef Search ADS PubMed  Konrad RJ, Zhang F, Hale JE, Knierman MD, Becker GW, Kudlow JE. 2002. Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase. Biochem Biophys Res Commun . 293: 207– 212. Google Scholar CrossRef Search ADS PubMed  Kreppel LK, Hart GW. 1999. Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats. J Biol Chem . 274: 32015– 32022. Google Scholar CrossRef Search ADS PubMed  Krzeslak A, Forma E, Bernaciak M, Romanowicz H, Brys M. 2012. Gene expression of O-GlcNAc cycling enzymes in human breast cancers. Clin Exp Med . 12: 61– 65. Google Scholar CrossRef Search ADS PubMed  Lai S, Winkler DF, Zhang H, Pelech S. 2016. Determination of the substrate specificity of protein kinases with peptide micro- and macroarrays. Methods Mol Biol . 1360: 183– 202. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Jiang J, Gloster TM, Zandberg WF, Whitworth GE, Vocadlo DJ, Walker S. 2012. Structural snapshots of the reaction coordinate for O-GlcNAc transferase. Nat Chem Biol . 8: 966– 968. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Jiang J, Kapuria V, Bhuiyan T, Janetzko J, Zandberg WF, Vocadlo DJ, Herr W, Walker S. 2013. HCF-1 is cleaved in the active site of O-GlcNAc transferase. Science . 342: 1235– 1239. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Nam Y, Jiang J, Sliz P, Walker S. 2011. Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature . 469: 564– 567. Google Scholar CrossRef Search ADS PubMed  Lebrin F, Chambaz EM, Bianchini L. 2001. A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit alpha. Oncogene . 20: 2010– 2022. Google Scholar CrossRef Search ADS PubMed  Lemeer S, Jopling C, Naji F, Ruijtenbeek R, Slijper M, Heck AJ, den Hertog J. 2007. Protein–tyrosine kinase activity profiling in knock down zebrafish embryos. PLoS One . 2: e581. Google Scholar CrossRef Search ADS PubMed  Leney AC, El Atmioui D, Wu W, Ovaa H, Heck AJR. 2017. Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation. Proc Natl Acad Sci USA . 114: E7255– E7261. Google Scholar CrossRef Search ADS PubMed  Li B, Li H, Lu L, Jiang J. 2017. Structures of human O-GlcNAcase and its complexes reveal a new substrate recognition mode. Nat Struct Mol Biol . 24: 362– 369. Google Scholar CrossRef Search ADS PubMed  Li J, Huang CL, Zhang LW, Lin L, Li ZH, Zhang FW, Wang P. 2010. Isoforms of human O-GlcNAcase show distinct catalytic efficiencies. Biochemistry (Mosc) . 75: 938– 943. Google Scholar CrossRef Search ADS PubMed  Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong CX. 2004. O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci USA . 101: 10804– 10809. Google Scholar CrossRef Search ADS PubMed  Liu J, Marchase RB, Chatham JC. 2007. Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels. J Mol Cell Cardiol . 42: 177– 185. Google Scholar CrossRef Search ADS PubMed  Liu X, Li L, Wang Y, Yan H, Ma X, Wang PG, Zhang L. 2014. A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase. FASEB J . 28: 3362– 3372. Google Scholar CrossRef Search ADS PubMed  Liu Y, Ren Y, Cao Y, Huang H, Wu Q, Li W, Wu S, Zhang J. 2017. Discovery of a low toxicity O-GlcNAc Transferase (OGT) inhibitor by structure-based virtual screening of natural products. Sci Rep . 7: 12334. Google Scholar CrossRef Search ADS PubMed  Love DC, Krause MW, Hanover JA. 2010. O-GlcNAc cycling: emerging roles in development and epigenetics. Semin Cell Dev Biol . 21: 646– 654. Google Scholar CrossRef Search ADS PubMed  Lubas WA, Frank DW, Krause M, Hanover JA. 1997. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J Biol Chem . 272: 9316– 9324. Google Scholar CrossRef Search ADS PubMed  Lubas WA, Hanover JA. 2000. Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. J Biol Chem . 275: 10983– 10988. Google Scholar CrossRef Search ADS PubMed  Lynch TP, Ferrer CM, Jackson SR, Shahriari KS, Vosseller K, Reginato MJ. 2012. Critical role of O-linked beta-N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis, and metastasis. J Biol Chem . 287: 11070– 11081. Google Scholar CrossRef Search ADS PubMed  Macauley MS, Vocadlo DJ. 2009. Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr Res . 344: 1079– 1084. Google Scholar CrossRef Search ADS PubMed  Macauley MS, Whitworth GE, Debowski AW, Chin D, Vocadlo DJ. 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem . 280: 25313– 25322. Google Scholar CrossRef Search ADS PubMed  Marotta NP, Lin YH, Lewis YE, Ambroso MR, Zaro BW, Roth MT, Arnold DB, Langen R, Pratt MR. 2015. O-GlcNAc modification blocks the aggregation and toxicity of the protein alpha-synuclein associated with Parkinson’s disease. Nat Chem . 7: 913– 920. Google Scholar CrossRef Search ADS PubMed  Miller CJ, Turk BE. 2016. Rapid identification of protein kinase phosphorylation site motifs using combinatorial peptide libraries. Methods Mol Biol . 1360: 203– 216. Google Scholar CrossRef Search ADS PubMed  Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL, Mucke L. 2015. Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci . 18: 1183– 1189. Google Scholar CrossRef Search ADS PubMed  Nishikawa K, Sawasdikosol S, Fruman DA, Lai J, Songyang Z, Burakoff SJ, Yaffe MB, Cantley LC. 2000. A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase. Mol Cell . 6: 969– 974. Google Scholar CrossRef Search ADS PubMed  Onodera Y, Nam JM, Bissell MJ. 2014. Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways. J Clin Invest . 124: 367– 384. Google Scholar CrossRef Search ADS PubMed  Ortiz-Meoz RF, Jiang J, Lazarus MB, Orman M, Janetzko J, Fan C, Duveau DY, Tan ZW, Thomas CJ, Walker S. 2015. A small molecule that inhibits OGT activity in cells. ACS Chem Biol . 10: 1392– 1397. Google Scholar CrossRef Search ADS PubMed  Ortiz-Meoz RF, Merbl Y, Kirschner MW, Walker S. 2014. Microarray discovery of new OGT substrates: the medulloblastoma oncogene OTX2 is O-GlcNAcylated. J Am Chem Soc . 136: 4845– 4848. Google Scholar CrossRef Search ADS PubMed  Pathak S, Alonso J, Schimpl M, Rafie K, Blair DE, Borodkin VS, Albarbarawi O, van Aalten DMF. 2015. The active site of O-GlcNAc transferase imposes constraints on substrate sequence. Nat Struct Mol Biol . 22: 744– 750. Google Scholar CrossRef Search ADS PubMed  Pathak S, Borodkin VS, Albarbarawi O, Campbell DG, Ibrahim A, van Aalten DM. 2012. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J . 31: 1394– 1404. Google Scholar CrossRef Search ADS PubMed  Rafie K, Raimi O, Ferenbach AT, Borodkin VS, Kapuria V, van Aalten DMF. 2017. Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats. Open Biol . 7: 170078. Google Scholar CrossRef Search ADS PubMed  Rao FV, Dorfmueller HC, Villa F, Allwood M, Eggleston IM, van Aalten DM. 2006. Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis. EMBO J . 25: 1569– 1578. Google Scholar CrossRef Search ADS PubMed  Roth C, Chan S, Offen WA, Hemsworth GR, Willems LI, King DT, Varghese V, Britton R, Vocadlo DJ, Davies GJ. 2017. Structural and functional insight into human O-GlcNAcase. Nat Chem Biol . 13: 610– 612. Google Scholar CrossRef Search ADS PubMed  Schimpl M, Borodkin VS, Gray LJ, van Aalten DM. 2012. Synergy of peptide and sugar in O-GlcNAcase substrate recognition. Chem Biol . 19: 173– 178. Google Scholar CrossRef Search ADS PubMed  Schimpl M, Zheng X, Borodkin VS, Blair DE, Ferenbach AT, Schuttelkopf AW, Navratilova I, Aristotelous T, Albarbarawi O, Robinson DA et al.  . 2012. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis. Nat Chem Biol . 8: 969– 974. Google Scholar CrossRef Search ADS PubMed  Selvan N, Williamson R, Mariappa D, Campbell DG, Gourlay R, Ferenbach AT, Aristotelous T, Hopkins-Navratilova I, Trost M, van Aalten DMF. 2017. A mutant O-GlcNAcase enriches Drosophila developmental regulators. Nat Chem Biol . 13: 882– 887. Google Scholar CrossRef Search ADS PubMed  Shafi R, Iyer SP, Ellies LG, O’Donnell N, Marek KW, Chui D, Hart GW, Marth JD. 2000. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc Natl Acad Sci USA . 97: 5735– 5739. Google Scholar CrossRef Search ADS PubMed  Sharif S, Shi J, Bourakba M, Ruijtenbeek R, Pieters RJ. 2017. Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray. Anal Biochem . 532: 12– 18. Google Scholar CrossRef Search ADS PubMed  Shen DL, Gloster TM, Yuzwa SA, Vocadlo DJ. 2012. 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. J Biol Chem . 287: 15395– 15408. Google Scholar CrossRef Search ADS PubMed  Shen DL, Liu TW, Zandberg W, Clark T, Eskandari R, Alteen MG, Tan HY, Zhu Y, Cecioni S, Vocadlo D. 2017. Catalytic promiscuity of O-GlcNAc transferase enables unexpected metabolic engineering of cytoplasmic proteins with 2-azido-2-deoxy-glucose. ACS Chem Biol . 12: 206– 213. Google Scholar CrossRef Search ADS PubMed  Shi J, Sharif S, Ruijtenbeek R, Pieters RJ. 2016. Activity based high-throughput screening for novel O-GlcNAc transferase substrates using a dynamic peptide microarray. PLoS One . 11: e0151085. Google Scholar CrossRef Search ADS PubMed  Shi J, Tomasic T, Sharif S, Brouwer AJ, Anderluh M, Ruijtenbeek R, Pieters RJ. 2017. Peptide microarray analysis of the cross-talk between O-GlcNAcylation and tyrosine phosphorylation. FEBS Lett . 591: 1872– 1883. Google Scholar CrossRef Search ADS PubMed  Singleton KD, Wischmeyer PE. 2008. Glutamine induces heat shock protein expression via O-glycosylation and phosphorylation of HSF-1 and Sp1. JPEN J Parenter Enteral Nutr . 32: 371– 376. Google Scholar CrossRef Search ADS PubMed  Smet-Nocca C, Broncel M, Wieruszeski JM, Tokarski C, Hanoulle X, Leroy A, Landrieu I, Rolando C, Lippens G, Hackenberger CP. 2011. Identification of O-GlcNAc sites within peptides of the Tau protein and their impact on phosphorylation. Mol BioSyst . 7: 1420– 1429. Google Scholar CrossRef Search ADS PubMed  Stanford SM, Krishnamurthy D, Kulkarni RA, Karver CE, Bruenger E, Walker LM, Ma CT, Chung TD, Sergienko E, Bottini N et al.  . 2014. pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays. Methods . 65: 165– 174. Google Scholar CrossRef Search ADS PubMed  Swamy M, Pathak S, Grzes KM, Damerow S, Sinclair LV, van Aalten DM, Cantrell DA. 2016. Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy. Nat Immunol . 17: 712– 720. Google Scholar CrossRef Search ADS PubMed  Tarrant MK, Rho HS, Xie Z, Jiang YL, Gross C, Culhane JC, Yan G, Qian J, Ichikawa Y, Matsuoka T et al.  . 2012. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis. Nat Chem Biol . 8: 262– 269. Google Scholar CrossRef Search ADS PubMed  Torreno-Pina JA, Castro BM, Manzo C, Buschow SI, Cambi A, Garcia-Parajo MF. 2014. Enhanced receptor–clathrin interactions induced by N-glycan-mediated membrane micropatterning. Proc Natl Acad Sci USA . 111: 11037– 11042. Google Scholar CrossRef Search ADS PubMed  Trinidad JC, Barkan DT, Gulledge BF, Thalhammer A, Sali A, Schoepfer R, Burlingame AL. 2012. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics . 11: 215– 229. Google Scholar CrossRef Search ADS PubMed  Trinidad JC, Schoepfer R, Burlingame AL, Medzihradszky KF. 2013. N- and O-glycosylation in the murine synaptosome. Mol Cell Proteomics . 12: 3474– 3488. Google Scholar CrossRef Search ADS PubMed  van Wandelen LT, van Ameijde J, Ismail-Ali AF, van Ufford HC, Vijftigschild LA, Beekman JM, Martin NI, Ruijtenbeek R, Liskamp RM. 2013. Cell-penetrating bisubstrate-based protein kinase C inhibitors. ACS Chem Biol . 8: 1479– 1487. Google Scholar CrossRef Search ADS PubMed  Vosseller K, Trinidad JC, Chalkley RJ, Specht CG, Thalhammer A, Lynn AJ, Snedecor JO, Guan S, Medzihradszky KF, Maltby DA et al.  . 2006. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics . 5: 923– 934. Google Scholar CrossRef Search ADS PubMed  Wang Z, Gucek M, Hart GW. 2008. Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc. Proc Natl Acad Sci USA . 105: 13793– 13798. Google Scholar CrossRef Search ADS PubMed  Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW. 2010. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci Signal . 3: ra2. Google Scholar PubMed  Withers SG, Davies GJ. 2012. The case of the missing base. Nat Chem Biol . 8: 952– 953. Google Scholar CrossRef Search ADS PubMed  Wu HY, Lu CT, Kao HJ, Chen YJ, Chen YJ, Lee TY. 2014. Characterization and identification of protein O-GlcNAcylation sites with substrate specificity. BMC Bioinf . 15: S1. Google Scholar CrossRef Search ADS   Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS, Cho JW. 2006. Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability. Nat Cell Biol . 8: 1074– 1083. Google Scholar CrossRef Search ADS PubMed  Yuzwa SA, Shan X, Macauley MS, Clark T, Skorobogatko Y, Vosseller K, Vocadlo DJ. 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol . 8: 393– 399. Google Scholar CrossRef Search ADS PubMed  Yuzwa SA, Yadav AK, Skorobogatko Y, Clark T, Vosseller K, Vocadlo DJ. 2011. Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody. Amino Acids . 40: 857– 868. Google Scholar CrossRef Search ADS PubMed  Zachara NE, Vosseller K, Hart GW. 2011. Detection and analysis of proteins modified by O-linked N-acetylglucosamine. Curr Protoc Protein Sci . Chapter 12: Unit12 18. Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow JE. 2003. O-GlcNAc modification is an endogenous inhibitor of the proteasome. Cell . 115: 715– 725. Google Scholar CrossRef Search ADS PubMed  Zhang S, Roche K, Nasheuer HP, Lowndes NF. 2011. Modification of histones by sugar beta-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated. J Biol Chem . 286: 37483– 37495. Google Scholar CrossRef Search ADS PubMed  Zhong J, Martinez M, Sengupta S, Lee A, Wu X, Chaerkady R, Chatterjee A, O’Meally RN, Cole RN, Pandey A et al.  . 2015. Quantitative phosphoproteomics reveals crosstalk between phosphorylation and O-GlcNAc in the DNA damage response pathway. Proteomics . 15: 591– 607. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Glycobiology Oxford University Press

Demystifying O-GlcNAcylation: hints from peptide substrates

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Abstract

Abstract O-GlcNAcylation, analogous to phosphorylation, is an essential post-translational modification of proteins at Ser/Thr residues with a single β-N-acetylglucosamine moiety. This dynamic protein modification regulates many fundamental cellular processes and its deregulation has been linked to chronic diseases such as cancer, diabetes and neurodegenerative disorders. Reversible attachment and removal of O-GlcNAc is governed only by O-GlcNAc transferase and O-GlcNAcase, respectively. Peptide substrates, derived from natural O-GlcNAcylation targets, function in the catalytic cores of these two enzymes by maintaining interactions between enzyme and substrate, which makes them ideal models for the study of O-GlcNAcylation and deglycosylation. These peptides provide valuable tools for a deeper understanding of O-GlcNAc processing enzymes. By taking advantage of peptide chemistry, recent progress in the study of activity and regulatory mechanisms of these two enzymes has advanced our understanding of their fundamental specificities as well as their potential as therapeutic targets. Hence, this review summarizes the recent achievements on this modification studied at the peptide level, focusing on enzyme activity, enzyme specificity, direct function, site-specific antibodies and peptide substrate-inspired inhibitors. O-GlcNAc transferase, O-GlcNAcase, O-linked N-acetylglucosamine modification, peptide substrates, post-translational modification Introduction O-GlcNAcylation is a ubiquitous post-translational modification (PTM) of serine or threonine residues with a single O-linked β-N-acetylglucosamine (O-GlcNAc) on numerous proteins. In contrast to complex glycans that typically function on the cell surface by mediating numerous ligand–receptor interactions (Gambaryan et al. 1995; Gallegos et al. 2012; Torreno-Pina et al. 2014), O-GlcNAc modifications of proteins ubiquitously occur inside the cells where they regulate many basic cellular processes. Proteomics studies estimated that ~19% of the O-GlcNAcylated proteins, including NF-κB, p53 and STAT, are involved in regulation of gene expression (Hahne et al. 2013). O-GlcNAcylation regulates protein activity and integrates cellular signaling transduction by itself or in combination with intensive cross-talk with other PTMs (Guinez et al. 2008), especially phosphorylation (Wang et al. 2008; Ande et al. 2009; Zhong et al. 2015). Histone proteins are also targets of O-GlcNAcylation, which is suggested to control the cell cycle and cell survival (Zhang et al. 2011; Fong et al. 2012). In addition, O-GlcNAc is also involved in the regulation of proteasome function by modifying the Rpt2 subunit (Zhang et al. 2003). To date, ca. 1492 O-GlcNAcylation sites have been recorded in the PhosphoSitePlus® database (www.phosphosite.org) (Hornbeck et al. 2015) and aberrant regulation of O-GlcNAcylation has been linked to chronic diseases such as type 2 diabetes, cancer and Alzheimer’s disease (AD; Liu et al. 2004; Banerjee et al. 2015). In the case of cancer, modulation of O-GlcNAcylation by inhibitors showed an anticancer effect by inhibiting breast tumor growth and invasion, suggesting that O-GlcNAcylation is a potential therapeutic target for cancer treatment (Caldwell et al. 2010). Like protein phosphorylation, the O-GlcNAc modification of proteins is also reversible, but it is under the control of only a pair of antagonistic enzymes. O-GlcNAc transferase (OGT) catalyzes the addition of GlcNAc onto the protein using uridine 5′-diphosphate-N-acetylglucosamine (UDP-GlcNAc) as the sugar donor, while O-GlcNAcase (OGA) removes the O-GlcNAc from the modified protein. Remarkably, OGT activity is largely influenced by the intracellular concentration of UDP-GlcNAc which is the metabolic end product of the hexosamine biosynthetic pathway (HBP; Kreppel and Hart 1999; Swamy et al. 2016). Since the HBP integrates metabolisms of glucose, acids (acetate), amino acids (glutamine) and nucleotides (UTP) to generate UDP-GlcNAc, O-GlcNAcylation is considered as a nutrient sensitive PTM (Hawkins et al. 1997; Liu et al. 2007; Singleton and Wischmeyer 2008; Onodera et al. 2014; Durning et al. 2016). The gene encoding OGT is localized on the X-chromosome in human and mouse and is considered to be essential in the mammalian development (Shafi et al. 2000). Although only one gene is responsible for OGT, it can generate three separate transcripts that encode three different isoforms of the enzyme. All OGT isoforms consist of a tetratricopeptide repeats (TPR) domain, a linker region and a C-terminal catalytic domain. In humans, all three OGT isoforms have an identical catalytic domain but differ in the number of TPR repeats, each of which includes ca. 34 amino acids (Lubas et al. 1997; Hanover et al. 2003). The longest one contains 13.5 TPR repeats (Jinek et al. 2004), is localized in both the nucleus and the cytoplasm and has been termed as nc-OGT. The second longest one has 9.5 TPR repeats, contains a mitochondrial targeting sequence and has been termed m-OGT. The shortest one has only 2.5 TPR repeats, is expressed in both the nucleus and the cytoplasm and is termed s-OGT, referring to the short nature of the TPR tail. Crystal structures of the OGT catalytic pocket with bound substrates have revealed that an ordered bi-bi mechanism was involved in the glycosylation reaction where the sugar donor UDP-GlcNAc binds first and facilitates the binding of the acceptor peptide (Lazarus et al. 2011). UDP-GlcNAc is bound in the OGT catalytic site by evolutionarily conserved residues important for catalytic activity. The peptide is bound in the OGT catalytic site by its amide backbone interaction with OGT side chains and the UDP moiety with an extended conformation. In the transfer of GlcNAc, the α-phosphate of UDP-GlcNAc is possibly used as a catalytic base, together with catalytical residues of OGT such as Lys842 (Schimpl, Zheng et al. 2012), although the identity of the base has not been firmly established (Withers and Davies 2012). Surprisingly, OGT was also shown to have a proteolytic activity in the maturation process of the cell cycle host cell factor 1 (HCF1) and the cleavage of HCF1 occurs within the OGT catalytic domain in a UDP-GlcNAc dependent manner (Capotosti et al. 2011; Lazarus et al. 2013). The underlying mechanism for this unexpected activity of OGT seems to involve glycosylation of the HCF1 glutamate side chain and conversion of this glycosylation product to an internal pyroglutamate which undergoes backbone cleavage (Janetzko et al. 2016). Studies of TPR repeat structures and interactors have demonstrated that TPR repeats form important protein–protein interactions which might contribute to the substrate specificity of OGT (Iyer and Hart 2003; Iyer et al. 2003; Jinek et al. 2004; Lazarus et al. 2013). Recent studies further suggested that OGT recognizes the protein substrate TAB1 through extensive interactions with the TPR domain, suggesting an essential role of TPR in OGT recognition of physiological substrates (Rafie et al. 2017). In addition, it was proposed that transient interactions of OGT and its partner interactors may form holoenzymes which might be substrate-specific (Deng et al. 2014). While aspects of TPR recognition and holoenzymes formation may play significant roles, all OGT substrates will additionally have to obey the specificity constraints imposed upon them especially from the −3 to +3 positions around the modification sites by the characteristics of the OGT active site, which can be efficiently studied with short peptide OGT substrates (Lazarus et al. 2012, 2013; Schimpl, Zheng et al. 2012). The human OGA gene is localized on chromosome 10q24 and it has been implicated in AD (Kim et al. 2006). It has been reported that humans have two OGA splicing transcripts which produce the long OGA (OGA-L or OGA-FL) and the small nuclear variant of OGA (OGA-S or OGA-NV), respectively (Li et al. 2010). OGA-L is a 103 kDa protein that typically has an N-terminal catalytic domain, a stalk domain and a C-terminal histone acetyltransferase (HAT) like domain. OGA-S (76 kDa) does not have the HAT like domain and it shows a 400-fold lower hydrolase efficiency than OGA-L, although they have similar catalytic properties (Kim et al. 2006; Macauley and Vocadlo 2009; Li et al. 2010). Despite ample progress in the investigation of human OGA, its mechanisms and inhibitors have been mostly derived from studying the bacterial homologs of OGA. In the hydrolysis reaction by bacterial CpOGA, Asp401 (Asp175 in human OGA) forms major hydrogen bonds with the sugar portion of the substrate, while Asp298 (Asp285 in human OGA) protonates the glycosidic bond (Rao et al. 2006; Selvan et al. 2017). As a boost for research on the human OGA, it should be noted that crystal structures of human OGA became available recently (Elsen et al. 2017; Li et al. 2017; Roth et al. 2017). In agreement with bacterial OGA studies, it was found that human OGA mainly uses Asp285 to anchor the GlcNAc moiety of the substrate through hydrogen bonds and uses Asp174 as a catalytic base and Asp175 as a catalytic acid (Cetinbas et al. 2006; Li et al. 2017). Recent research work on demystifying O-GlcNAc processing enzymes, using short peptide substrates, has seen progress and further advanced our understanding of this essential PTM. This motivated us to summarize the usefulness of peptide substrates to gain insights into the properties of OGT and OGA. This review covers five aspects of the use of peptides: (1) it describes peptide substrate-based methods for the study of O-GlcNAcylation; (2) it summarizes the OGT and OGA substrate preferences; (3) it discusses the incorporation of O-GlcNAc into peptides as well as proteins by chemical synthesis; (4) it summarizes the generation of site-specific O-GlcNAc antibodies using glycopeptides and (5) it discusses the early development of OGT inhibitors utilizing the peptide substrate information. Identifying activities of O-GlcNAc processing enzymes using peptide-based approaches OGT activity profiling using peptide substrates The relevance of the use of short peptides, as substrates, to profile activity and acquire substrate specificity for kinases and phosphatases has been shown on numerous occasions for kinases and phosphatases, which has lead to great successes in the field of phosphorylation (Jarboe et al. 2012; Deng Y et al. 2014; Stanford et al. 2014; Hovestad-Bijl et al. 2016; Miller and Turk 2016). By taking advantage of their flexibility and availability, peptide substrates of OGT have become efficient tools to predict OGT activity (Lubas and Hanover 2000; Shen et al. 2017) as well as a means to study aspects of binding and catalysis in crystal structures of complexes (Lazarus et al. 2011; Schimpl, Borodkin et al. 2012). One commonly used peptide substrate of OGT is the CKII_340_352 peptide derived from the α-subunit of casein kinase II which is O-GlcNAcylated at residue Ser347 in vitro and in vivo (Kreppel and Hart 1999; Zachara et al. 2011; Tarrant et al. 2012). Several excellent OGT peptide substrates have also been discovered through different means including RBL2_410_422 (Pathak et al. 2015; Shi et al. 2016), TAB1_389_401 (Pathak et al. 2012; Schimpl, Borodkin et al. 2012) and ZO-3_357_371 (Table I) (Shi et al. 2017). All these peptide substrates have individual protein origins, and therefore provide entries into the study of the biology of the parent proteins. Table I. A list of peptides mentioned in this review Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Table I. A list of peptides mentioned in this review Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  Primary sequence  Protein  Start and end  Modification site  PGGSTPVSSANMM  CKII  340–352  Ser347  PVSVPYSSAQSTS  TAB1  389–401  Ser395  KENSPCVTPVSTA  RBL2  410–422  Ser420  RESSYDIYRVPSSQS  ZO-3  357–371  Ser369  QVAHSGAKAS  hOGA  401–410  Ser405  VYKSPVVSGDTSPRH  Tau  393–407  Ser400  KTAPVQLWVDSTPPPGTRVRA  p53  139–159  Ser149  In recent years, protein and peptides arrays of different formats have been applied to identify O-GlcNAcylation and the study of OGT activities (Deng RP et al. 2014; Ortiz-Meoz et al. 2014; Pathak et al. 2015; Shi et al. 2016, 2017). A human protein microarray containing 8000 unique proteins has been successfully used to study OGT activity and hundreds of novel proteins have been identified as OGT substrates with diverse functions, including kinases, transcription factors and apoptosis-related proteins (Ortiz-Meoz et al. 2014). However, the protein array cannot be used to generate straightforward sequence information. While it has its own valuable uses, the use of small peptides focuses more readily on the specificity of the enzyme. The use of a peptide microarray to study OGT offers advantages in generating inherent sequence-specificity information. As such, the method is complementary to the protein array where additional protein–protein interactions may additionally determine the specificity. The first example of a peptide microarray study described a library of 720 synthesized biotinylated peptides with sequences derived from the human proteome, using a scintillation proximity assay (Pathak et al. 2015). In this assay (Figure 1A), the OGT reaction was carried out in 384-well polypropylene plates, with one peptide in each well, in the presence of radio labeled UDP-[3H]GlcNAc. Then, the O-GlcNAcylated peptides were immobilized on a streptavidin coated scintillant flashplate, which allowed the direct quantification of radio labeled peptide by using scintillation counting. This approach revealed comprehensive sequence information which is discussed in Section 3. Disadvantages include the fact that this approach involved radio labeling which requires time for signal development and is therefore less convenient for real time kinetic analysis. Fig. 1. View largeDownload slide Schematic depiction of high-throughput assays for activity of OGT and OGA against peptide substrates. (A) Scintillation proximity-based OGT assay. (B) Peptide microarray-based real time OGT assay. (C) Glycopeptide microarray-based OGA assay. Fig. 1. View largeDownload slide Schematic depiction of high-throughput assays for activity of OGT and OGA against peptide substrates. (A) Scintillation proximity-based OGT assay. (B) Peptide microarray-based real time OGT assay. (C) Glycopeptide microarray-based OGA assay. A different peptide microarray with kinetics monitoring was described for OGT activity by our group (Figure 1B; Shi et al. 2016). This dynamic peptide microarray technology was initially developed for kinase activity profiling (Lemeer et al. 2007; Anderson et al. 2015). In the kinase field, the use of immobilized peptide sequences derived from knowledge of protein phosphorylation sites has proven to be highly valuable for the study of kinase activity (Hilhorst et al. 2009). In this array, each peptide was prepared with additional Cys-Gly residues at the N-terminus, which allows immobilization of a maleimide functionalized porous membrane surface. However, the nature of this immobilization chemistry also makes the array results less quantifiable when studying peptides with multiple cysteines, due to additional non-terminal attachment modes. The OGT reaction was performed in the presence of a fluorescently labeled detection antibody. The reaction mixture can be pumped up and down periodically through the membrane during the entire reaction time and the fluorescent signal of only the bound fluorescent antibody is acquired at regular intervals. This is possible by temporarily placing the solution with unbound fluorescent antibody below the chip, outside the detector range (Figure 1B). As a result, this enables a kinetic reading of the enzymatic modification of each peptide on the array without removing the unbound antibody by extra washing. O-GlcNAcylation of peptides with this array can be achieved not only by using purified OGT enzyme but also by using a cell lysate. In addition, this array-based identification of OGT substrates from peptides known to contain phosphorylation sites, also made it possible to study cross-talk between these two modifications (Shi et al. 2017), which will be discussed in Section 4. Kinase activity profiling with such arrays has been demonstrated to be of relevance for cancer diagnosis and help in the prediction of efficacy of cancer drugs for a specific patient (Folkvord et al. 2010), so ultimately O-GlcNAcylation on peptide arrays may exhibit a similar medicinal relevance. Although these peptide-based techniques provide a tool for OGT substrate identification, these hits were not validated at the protein level. Detection of OGA activities with peptide substrates In vitro OGA activity is normally detected by using the color producing compound p-nitrophenyl 2-acetamido-2-deoxy-β-d-glucopyranoside (pNPGlcNAc) and the fluorogenic compounds 4-methylumbelliferyl-2-acetamido-2-deoxy-β-d-glucopyranoside (MuGlcNAc) and fluorescein di(N-acetyl-beta-d-glucosamine) (FDGlcNAc) as substrates (Dong and Hart 1994; Macauley et al. 2005; Kim et al. 2006). However, these compounds are not natural substrates of OGA and cannot fully reflect OGA activity with a natural glycopeptidic substrate. Similarly to the OGT array assay, a fluorescent method for the detection of OGT activity was used to study the removal of GlcNAc from a more natural substrate (Figure 1C). This peptide microarray-based approach was recently described to measure OGA activity from both purified OGA as well as OGA present in cell lysates derived from several cancer cell lines (Sharif et al. 2017). A series of glycopeptides as OGA substrates was prepared based on peptides which are known OGT targets and these were immobilized on the array. The array was subjected to purified OGA or cell lysates, followed by detection of the remaining O-GlcNAc on each peptide with a fluorescent antibody. In addition, a more comprehensive version of this glycopeptide microarray with more diversity has to be used to further validate its potential to profile OGA catalytic properties. Essential role of amino acids around the O-GlcNAc site in substrate recognition OGT substrate recognition It was recently reported that OGT has a peptide sequence preference around its modification site on short peptide substrates (Jochmann et al. 2014; Liu et al. 2014; Pathak et al. 2015). A high density peptide microarray is an ideal platform to study such enzymatic specificities (Lai et al. 2016). In an array analysis of 720 Ser/Thr peptides for OGT activity, ~10% of these peptides were identified as OGT substrates (Pathak et al. 2015), suggesting that OGT has the ability to specifically recognize a substrate by its primary sequence among hundreds of peptides (Shi et al. 2016, 2017). With the sequence information of these identified peptides, an unbiased OGT substrate motif was obtained as [TS][PT][VT]S/T[RLV][ASY] (from −3 to +2 with respect to O-GlcNAcylation site) which was also verified in select cases by crystal structures (Pathak et al. 2015). This motif is generally in line with the motif PPV[S/T]SATT (Liu et al. 2014) and PPV[S/T]TA (Wu et al. 2014) obtained by frequency analysis of known OGT substrates. The clear preference of Pro at the −2 site by OGT enables the peptide substrate to display an extended conformation, which facilitates its binding to the OGT active site (Lazarus et al. 2011; Pathak et al. 2015). Interestingly, it was also reported that OGT has a strictly negative preference for Pro at the +1 subsite, which could prevent O-GlcNAcylation at a Ser/Thr/Pro rich region even if other sites match with the substrate motif (Leney et al. 2017). Despite this progress, the current motifs are not able to accurately predict OGT peptide substrates. We have found that only a few of our experimentally identified novel substrates matched with these reported motifs and some matched peptides in our experiments were not experimentally identified as a substrate (Shi et al. 2016, 2017). Nevertheless, for the best discovered hit peptide from our experiments the motif successfully identified the modification site. This site was confirmed through mutation experiments by making a series of peptides (Shi et al. 2017). Regardless of predictable accuracy, all these findings strongly suggest that the primary sequence is a very important factor in determining the substrate specificity of OGT. It should be noted, also, that no obvious consensus motif has been found by proteomic studies of O-GlcNAc proteins from murine synaptosome although moderate preferences for Pro (−2 and −3 positions) and Val (−1 or −3 positions) by OGT have been observed (Trinidad et al. 2012, 2013; Vosseller et al. 2006). There is a possibility that in vivo OGT might achieve substrate specificity following a less stringent motif and that other determinants such as the TPR repeats and holoenzymes are involved as discussed in Section 1. Regardless of this, it should still be possible to generate a more complete and comprehensive OGT substrate motif, since short peptides only interact with OGT at the active site, which would be a very valuable tool for OGT protein substrate prediction and identification. OGA substrate recognition The investigation of human OGA specificity was primarily limited by the lack of available OGA crystal structures with bound substrate. Hence, a bacterial OGA orthologue such as CpOGA was widely used as a model to explore human OGA catalytic properties (Rao et al. 2006; Schimpl, Borodkin et al. 2012). The crystal structure of CpOGA in complex with synthetic glycopeptides derived from p53 (Yang et al. 2006), TAB1 and OGA (Table I) was used to gain insights into the human OGA specificity (Schimpl, Borodkin et al. 2012). In this structure, extensive hydrogen bonds were observed between the GlcNAc moiety and CpOGA (Schimpl, Borodkin et al. 2012), suggesting an important role of GlcNAc for OGA substrate binding and recognition. Interestingly, although no peptide side chain was found to be involved in direct interaction with the enzyme, a combination of intramolecular hydrogen bonds and hydrophobic interactions between the peptide backbone and CpOGA forced the glycopeptide into a conserved “V-shape” conformation that makes it possible to access the CpOGA active site (Schimpl, Borodkin et al. 2012). In addition, kinetic studies showed that human OGA has similar catalytic parameters for five O-GlcNAcylated proteins and a glycopeptide derived from a protein substrate is a better substrate than the protein itself. These data further suggest that GlcNAc is the primary determinant in OGA catalysis but amino acid residues may hinder (Shen et al. 2012). Now that crystal structures of human OGA have recently become available (Elsen et al. 2017; Li et al. 2017; Roth et al. 2017) with, e.g., the complexed O-GlcNAcylated p53_144-154 peptide, again an essential role of GlcNAc for OGA substrates recognition is indicated (Li et al. 2017). However, in this case also the peptide side chains were involved in hydrophobic interactions with OGA. Substitution of substrate peptide residues and OGA residues involved in the interaction, both lead to a reduction of substrate binding (Li et al. 2017). Although O-GlcNAc is the dominant structural feature for OGA substrate recognition, additional specific interactions involving substrate amino acids might exist, which could lead to differential preferences of substrates by OGA. Whether OGA requires a distinct substrate motif could be further studied, e.g., by using an array of synthetic glycopeptides. Based on the above fact that the sugar moiety is the major determinant for OGA activity, a hydrolytically stable sulfur-linked glycopeptide (S-GlcNAc) was synthesized and incorporated into the protein α-synuclein (De Leon et al. 2017) where indeed stability against OGA was observed. Global regulation of O-GlcNAc cycling While several comprehensive reviews have discussed the regulatory mechanisms of O-GlcNAc cycling (Love et al. 2010; Bond and Hanover 2013), recent findings add to this picture. O-GlcNAcylation cycling is critically controlled by many factors (Figure 2). OGT activity is greatly dependent on the intracellular concentration of UDP-GlcNAc, which is a metabolic product of the HBP together with fatty acid metabolism, amino acid metabolism and nucleotide metabolism (Hawkins et al. 1997; Liu et al. 2007; Singleton and Wischmeyer 2008; Onodera et al. 2014). The altered levels of OGT and OGA in cancer cells suggests the involvement of regulation in the enzyme expression levels (Krzeslak et al. 2012; Lynch et al. 2012; Sharif et al. 2017). UDP, a product of the O-GlcNAcylation reaction, might act as a feedback inhibitory regulator of OGT, considering its strong binding to the enzyme of ca. 2 μM (Dorfmueller et al. 2011). The extensive cross-talk of phosphorylation and O-GlcNAcylation represent novel regulatory mechanisms from the substrate side, which is slowly becoming more clear (Dias et al. 2009; Wang et al. 2010; Leney et al. 2017; Shi et al. 2017). Furthermore, the inherent specificity of especially OGT (Shi et al. 2016) and now seemingly also of OGA, enables their ability to selectively act on substrate proteins based on their amino acid sequence (Li et al. 2017). Finally, the role of OGT’s TPR repeats have been implicated in the substrate preference based on protein–protein interactions with the substrate protein (Rafie et al. 2017). Globally, it seems that to maintain a normal O-GlcNAcylation status, the addition of O-GlcNAc involves stringent and multiple levels of regulation, while for the removal of O-GlcNAc this seems to be less so. Nevertheless, additional OGT/OGA sequence specificities and cross-talk mechanisms will likely be uncovered in the future. Fig. 2. View largeDownload slide The global regulation of O-GlcNAc cycling. The cellular O-GlcNAcylation of proteins can be controlled by factors including OGT expression, inherent specificity (TPR and consensus sequence), nutrient availability, UDP-GlcNAc availability, UDP feedback inhibition, cross-talk with phosphorylation, OGA expression and OGA’s robust hydrolysis ability. Fig. 2. View largeDownload slide The global regulation of O-GlcNAc cycling. The cellular O-GlcNAcylation of proteins can be controlled by factors including OGT expression, inherent specificity (TPR and consensus sequence), nutrient availability, UDP-GlcNAc availability, UDP feedback inhibition, cross-talk with phosphorylation, OGA expression and OGA’s robust hydrolysis ability. Direct role of O-GlcNAcylation from synthetic glycopeptide to glycoprotein Elucidating the physiological function of a specific O-GlcNAcylation event on a specific protein is a challenging task because of the complex natural context of proteins within the cell. Removing of a putative modification site by gene mutation has been widely performed to indirectly observe the effect of a specific PTM in both in vitro and in vivo situations, resulting often in unconfirmed hypotheses. Using synthetic peptides is an efficient way to unambiguously introduce specific PTMs in a peptide which can subsequently be incorporated into a protein using advanced chemical ligation protocols. This then enables the direct study of the role of that modification. The advantage of peptide chemistry in studying a specific PTM has been well-demonstrated in the study of the Tau protein, which is linked to assembly and stabilization of microtubules for the proper function of neurons (Himmler et al. 1989; Smet-Nocca et al. 2011). Misfolding and aggregation of Tau has been implicated in AD and other neurodegenerative disorders, which is mainly caused by abnormal PTMs including phosphorylation and O-GlcNAcylation (Yuzwa et al. 2012; Morris et al. 2015). Using a synthetic phosphorylated Tau peptide as an OGT substrate or an O-GlcNAcylated Tau peptide as a kinase substrate, mass spectrometry experiments revealed a reciprocal relationship between phosphorylation (on Serines 396 and 404) and O-GlcNAcylation (on serine 400) in peptide Tau-392_411 (Smet-Nocca et al. 2011). The phosphate group at Ser400 completely prevents O-GlcNAcylation of this peptide by direct occupation of the O-GlcNAcylation site, and the phosphate groups at Ser396 and Ser404 led to a decreased O-GlcNAcylation level at Ser400. In the other direction, O-GlcNAcylation of Ser400 reduced the phosphorylation of Ser404 mediated by the Cdk2 kinase complex and completely suppressed the phosphorylation of Ser396/400/404 mediated by GSK3β (Figure 3A). Fig. 3. View largeDownload slide Study of O-GlcNAcylation and its cross-talk in a direct manner using synthetic peptide and synthetic proteins. Mass spectrometry-based study of cross-talk on Tau_392-411 (A) and peptide microarray-based study of cross-talk on ZO-3_357-371 (B) with synthetic glycopeptide and phosphopeptide. *This kinetic peptide microarray assay is described in Figure 1B. (C) Functional study of O-GlcNAc modification on CK2α and α-synuclein using synthetic glycoprotein. Fig. 3. View largeDownload slide Study of O-GlcNAcylation and its cross-talk in a direct manner using synthetic peptide and synthetic proteins. Mass spectrometry-based study of cross-talk on Tau_392-411 (A) and peptide microarray-based study of cross-talk on ZO-3_357-371 (B) with synthetic glycopeptide and phosphopeptide. *This kinetic peptide microarray assay is described in Figure 1B. (C) Functional study of O-GlcNAc modification on CK2α and α-synuclein using synthetic glycoprotein. Recently, a dynamic peptide microarray approach has been described to study the interplay between O-GlcNAc and tyrosine phosphorylation in a direct and real time manner (Shi et al. 2017). In this work, dual substrates of OGT and the Jak2 tyrosine kinase were identified from an array of peptides derived from known tyrosine kinase protein substrates. With these dual substrates, the effect of prior O-GlcNAcylation by OGT on the subsequent tyrosine phosphorylation by Jak2 was determined, and vice versa. This approach revealed that tyrosine phosphorylation of six peptides prevented their subsequent O-GlcNAcylation, whereas no correlation was found in the other direction. Peptides derived from the ZO-3 protein were synthesized with a phosphate group on Tyr364 and a GlcNAc group on Ser369, respectively. They were immobilized on the microarray to further validate the identified cross-talk kinetically (Figure 3B). Besides using purified enzymes, this cross-talk was also observed using a HeLa cell lysate as the source of both OGT and the kinase. Although useful kinetics information was achieved in this case, future studies should also confirm these observations with in vivo experiments. This type of cross-talk, might prove to be more general with additional studies (Leney et al. 2017). A modified peptide can be employed to synthetically access a modified full-length OGT protein substrate through a semi-synthetic strategy using native chemical ligation (Dawson et al. 1994; Smet-Nocca et al. 2011). This enables validation of its physiological functions at the protein level. Recently, a synthesis of O-GlcNAcylated α-synuclein by ligating synthesized peptide and expressed protein portions, has indicated that O-GlcNAcylation of this protein has an inhibitory effect on its aggregation, suggesting a crucial role of O-GlcNAcylation in the progression of AD and Parkinson’s disease (Marotta et al. 2015). In this study, the O-GlcNAc modified peptide was prepared by an Fmoc-based solid phase peptide synthesis strategy, resulting in a C-terminal thioester and an N-terminal thioproline-protected cysteine. Through an expressed protein ligation strategy (Figure 3C), the N-terminal cysteine of a recombinant protein segment was trans-esterified with the C-terminus of the above peptide and then rapidly rearranged to form a stable peptide bond. Similarly, the ligation of the newly formed construct and a recombinant protein segment with a C-terminal thioesters was carried out. After radical-based desulfurization of the introduced extra cysteine, a full-length α-synuclein containing an O-GlcNAc modification was obtained. The synthetic protein showed secondary structure characteristics that were the same as those derived from the unmodified protein. With this synthetic O-GlcNAc modified α-synuclein, it was demonstrated that a single O-GlcNAcylation at Thr72 has a notable and substoichiometric inhibitory effect on α-synuclein aggregation, suggesting that modulating a specific O-GlcNAcylation is perhaps a viable therapeutic strategy for PD. In addition, O-GlcNAcylation of this protein at Thr72 was shown to be able to slightly increase its phosphorylation at Ser87 by the CK1 kinase, but inhibit its phosphorylation at S129 by the GRK5, CK1 and PLK3 kinases (Figure 3C). As a result, such cross-talk may also affect α-synuclein aggregation. Another well-demonstrated example is the synthesis of O-GlcNAcylated CK2 which is involved in cell growth and proliferation through its kinase activity (Lebrin et al. 2001). In this case (Figure 3C), an O-GlcNAcylated protein was prepared using the same expressed protein ligation strategy, except that the synthetic peptide was prepared with metabolically stable thio-linked GlcNAc (S-GlcNAc) (Tarrant et al. 2012). Using this protein, it was found that O-GlcNAcylation of Ser347 inhibits its phosphorylation at Thr344 and modulates CK2 kinase substrate selectivity. This methodology is very powerful although the synthesis is challenging and in certain cases protein folding issues may occur. Generation of specific O-GlcNAc antibodies with small OGT substrate peptides A well-studied substrate peptide provides important sequence-specific information which can be used as an antigen to generate modification-site-specific antibodies. Such specific antibodies would be powerful tools for the further validation of O-GlcNAcylation on a specific protein at the more complex endogenous levels. Take the Tau protein for example, as an early attempt with a synthetic glycopeptide (CSPVV-(O-GlcNAc)-SGDTS) as an antigen. Its use led to the generation of a polyclonal antibody (termed 3925) that recognizes Tau with the O-GlcNAc modification at Ser400 in vitro, and both the peptide sequence and the GlcNAc moiety were found to contribute to the antibody specificity (Yuzwa et al. 2011). However, this antibody was also found to recognize a range of other O-GlcNAc proteins, which makes it unsuitable for use in in vivo. Since then, a more specific monoclonal antibody against the Tau protein O-GlcNAcylation at Ser400 (termed OTau (S400)) was generated using the O-GlcNAc peptide (VYKSPVV-(O-GlcNAc))-SGDTSPRH based on a comprehensive study of O-GlcNAcylation by peptide scanning (Smet-Nocca et al. 2011; Cameron et al. 2013). This antibody was successfully applied to study the O-GlcNAcylation of the full tau protein at Ser400 in vitro and in vivo, which may further facilitate the elucidation of the function of Tau in AD (Cameron et al. 2013). There are other examples of site-specific O-GlcNAc antibodies generated by using glycopeptides, such as the monoclonal antibody against Thr58 O-GlcNAc in c-Myc (Kamemura et al. 2002), a polyclonal antibody against Ser1011 O-GlcNAc in IRS-1 (Klein et al. 2009) and a polyclonal antibody against Ser395 O-GlcNAc in TAB1 (Pathak et al. 2012). These works indicate that the sequence information generated in the peptide-based studies of O-GlcNAcylation facilitates the detection of protein O-GlcNAcylation with site-specific antibodies. Specific OGT inhibitors/bisubstrate inhibitors from OGT substrates Specific and potent inhibitors are valuable tools for deeper investigation of the biological functions of OGT. The development of OGT inhibitors is still in its early stages and the number of selective OGT inhibitors with suitable pharmaceutical properties is still quite limited. Current OGT inhibitors through rational designs are mostly small analogs of UDP-GlcNAc and UDP targeting the sugar donor binding site of OGT, such as C-UDP (IC50 = 9 μM) (Dorfmueller et al. 2011), Alloxan (IC50 = 18 μM) (Konrad et al. 2002; Dorfmueller et al. 2011), UDP-GlcNAc glycosyl C-phosphate (UDP-C-GlcNAc, IC50 = 41 μM) (Chang et al. 2006; Hajduch et al. 2008) and a thioglycosyl analog of UDP-GlcNAc (UDP-S-GlcNAc, IC50 = 93 μM) (Dorfmueller et al. 2011). Remarkably, UDP, the byproduct from O-GlcNAcylation, showed the strongest inhibitory effect of them all with an IC50 value of 1.8 μM (Dorfmueller et al. 2011). Although low micromolar inhibitors have been generated by mimicking the sugar donor, they are not cell permeable or show unfavorable side effect by affecting other enzymes that utilizing UDP or UDP-GlcNAc (Dorfmueller et al. 2011). A noteworthy rational approach described is to produce sugar donor mimic UDP-5SGlcNAc in cells by using the metabolic nature of GlcNAc salvage and HBP pathways with a precursor Ac-5SGlcNAc (Gloster et al. 2011). UDP-5SGlcNAc (Ki = 8 μM) produced by this approach has been shown to be able to disrupt OGT activity in cases (Andres et al. 2017), but it might have side effects on cell surface glycosylation and other metabolic pathways. A high throughput screening assay was also used for the discovery of OGT inhibitors, such as a substrate analog displacement assay in which a fluorescent UDP-GlcNAc analog was used as a probe to determine the binding of small molecules by fluorescence polarization (Gross et al. 2005). In this work, a cell permeable OGT inhibitor (OSMI-1, IC50 = 2.7 μM) was identified with a good but not perfect selectivity (Ortiz-Meoz et al. 2015). Recently, a natural product OGT inhibitor L01 was discovered with an IC50 value of 2.7 μM through a structure-based virtual screening and this compound showed lower toxicity and a higher selectivity in vitro and in vivo (Liu et al. 2017). One study reported on developing OGT inhibitors by mimicking the sugar acceptor peptide (Borodkin et al. 2014). The use of the enzyme–substrate interacting motif as a template to develop specific peptide inhibitor has been well-demonstrated in the field of kinases (Nishikawa et al. 2000; van Wandelen et al. 2013), although high affinity may not be easily achieved with a relatively flexible peptide backbone only. In our recent work, we have shown that an OGT substrate peptide in which the serine (Ser420) at the O-GlcNAcylation site was replaced with an alanine, which cannot be O-GlcNAcylated, exhibited ~50% OGT activity inhibition at 500 μM (Shi et al. 2016). A closely related peptide substrate was shown to bind the OGT catalytic site with several hydrogen bonds and Van der Waals interactions (Pathak et al. 2015), so it was hypothesized that the alanine mutant peptide still binds the active site of OGT. These findings provide the possibility that peptide acceptors can be starting points of OGT inhibitor development by taking advantage of the specific contacts with OGT. Conclusion and perspectives In the last decade, the use of peptide substrates has remarkably facilitated the study of O-GlcNAcylation and its processing enzymes at the active sites. OGT was shown to be selective with respect to the amino acids around the modification site, but the current OGT substrate motif or sequon derived from a limited number of peptide substrates is not complete and not yet able to accurately predict substrates. It has to be noted that there might be other synergistical modes of selectivity for OGT with respect to protein substrates, involving TRP repeat binding and holoenzymes, but this is not the case for short peptide substrates. With further profiling of OGT peptide substrates, it should be possible to generate a more comprehensive motif that could be an important predictive tool with applications, e.g., in cancer. Further research by profiling cancer cell lines and patient tissues would lead to the identification of biomarkers with diagnostic and predictive value. Although a significant number of peptides have already been identified as OGT substrates, whether its parental proteins are indeed physiological targets of OGT in cells is mostly unknown. Site-specific O-GlcNAc antibodies would be useful to discover the biological function of a specific O-GlcNAcylation event and its relevance for disease, especially for cancer, diabetes and neurodegenerative disease. Specific and cell permeable OGT inhibitors are still urgently required, not only for research tools to study OGT but also for pharmacological applications. Funding This work was supported by a scholarship (No. 201306180061) to J.S. from the China Scholarship Council. Conflict of interest statement None declared. Abbreviations AD Alzheimer’s disease GlcNAc N-acetyl-d-glucosamine HAT histone acetyltransferase HBP hexosamine biosynthetic pathway OGA O-GlcNAcase OGT O-GlcNAc transferase PTM post-translational modification TPR tetratricopeptide repeats UDP-GlcNAc Uridine 5′-diphospho-N-acetylglucosamine HAT histone acetyltransferase. References Ande SR, Moulik S, Mishra S. 2009. Interaction between O-GlcNAc modification and tyrosine phosphorylation of prohibitin: implication for a novel binary switch. PLoS One . 4: e4586. Google Scholar CrossRef Search ADS PubMed  Anderson JC, Willey CD, Mehta A, Welaya K, Chen D, Duarte CW, Ghatalia P, Arafat W, Madan A, Sudarshan S et al.  . 2015. High throughput kinomic profiling of human clear cell renal cell carcinoma identifies kinase activity dependent molecular subtypes. PLoS One . 10: e0139267. Google Scholar CrossRef Search ADS PubMed  Andres LM, Blong IW, Evans AC, Rumachik NG, Yamaguchi T, Pham ND, Thompson P, Kohler JJ, Bertozzi CR. 2017. Chemical modulation of protein O-GlcNAcylation via OGT inhibition promotes human neural cell differentiation. ACS Chem Biol . 12: 2030– 2039. Google Scholar CrossRef Search ADS PubMed  Banerjee PS, Ma J, Hart GW. 2015. Diabetes-associated dysregulation of O-GlcNAcylation in rat cardiac mitochondria. Proc Natl Acad Sci USA . 112: 6050– 6055. Google Scholar CrossRef Search ADS PubMed  Bond MR, Hanover JA. 2013. O-GlcNAc cycling: a link between metabolism and chronic disease. Annu Rev Nutr . 33: 205– 229. Google Scholar CrossRef Search ADS PubMed  Borodkin VS, Schimpl M, Gundogdu M, Rafie K, Dorfmueller HC, Robinson DA, van Aalten DM. 2014. Bisubstrate UDP-peptide conjugates as human O-GlcNAc transferase inhibitors. Biochem J . 457: 497– 502. Google Scholar CrossRef Search ADS PubMed  Caldwell SA, Jackson SR, Shahriari KS, Lynch TP, Sethi G, Walker S, Vosseller K, Reginato MJ. 2010. Nutrient sensor O-GlcNAc transferase regulates breast cancer tumorigenesis through targeting of the oncogenic transcription factor FoxM1. Oncogene . 29: 2831– 2842. Google Scholar CrossRef Search ADS PubMed  Cameron A, Giacomozzi B, Joyce J, Gray A, Graham D, Ousson S, Neny M, Beher D, Carlson G, O’Moore J et al.  . 2013. Generation and characterization of a rabbit monoclonal antibody site-specific for tau O-GlcNAcylated at serine 400. FEBS Lett . 587: 3722– 3728. Google Scholar CrossRef Search ADS PubMed  Capotosti F, Guernier S, Lammers F, Waridel P, Cai Y, Jin J, Conaway JW, Conaway RC, Herr W. 2011. O-GlcNAc transferase catalyzes site-specific proteolysis of HCF-1. Cell . 144: 376– 388. Google Scholar CrossRef Search ADS PubMed  Cetinbas N, Macauley MS, Stubbs KA, Drapala R, Vocadlo DJ. 2006. Identification of Asp174 and Asp175 as the key catalytic residues of human O-GlcNAcase by functional analysis of site-directed mutants. Biochemistry . 45: 3835– 3844. Google Scholar CrossRef Search ADS PubMed  Chang R, Vo TT, Finney NS. 2006. Synthesis of the C1-phosphonate analog of UDP-GlcNAc. Carbohydr Res . 341: 1998– 2004. Google Scholar CrossRef Search ADS PubMed  Dawson PE, Muir TW, Clark-Lewis I, Kent SB. 1994. Synthesis of proteins by native chemical ligation. Science . 266: 776– 779. Google Scholar CrossRef Search ADS PubMed  De Leon CA, Levine PM, Craven TW, Pratt MR. 2017. The sulfur-linked analogue of O-GlcNAc (S-GlcNAc) is an enzymatically stable and reasonable structural surrogate for O-GlcNAc at the peptide and protein levels. Biochemistry . 56: 3507– 3517. Google Scholar CrossRef Search ADS PubMed  Deng RP, He X, Guo SJ, Liu WF, Tao Y, Tao SC. 2014. Global identification of O-GlcNAc transferase (OGT) interactors by a human proteome microarray and the construction of an OGT interactome. Proteomics . 14: 1020– 1030. Google Scholar CrossRef Search ADS PubMed  Deng Y, Alicea-Velazquez NL, Bannwarth L, Lehtonen SI, Boggon TJ, Cheng HC, Hytonen VP, Turk BE. 2014. Global analysis of human nonreceptor tyrosine kinase specificity using high-density peptide microarrays. J Proteome Res . 13: 4339– 4346. Google Scholar CrossRef Search ADS PubMed  Dias WB, Cheung WD, Wang Z, Hart GW. 2009. Regulation of calcium/calmodulin-dependent kinase IV by O-GlcNAc modification. J Biol Chem . 284: 21327– 21337. Google Scholar CrossRef Search ADS PubMed  Dong DL, Hart GW. 1994. Purification and characterization of an O-GlcNAc selective N-acetyl-beta-d-glucosaminidase from rat spleen cytosol. J Biol Chem . 269: 19321– 19330. Google Scholar PubMed  Dorfmueller HC, Borodkin VS, Blair DE, Pathak S, Navratilova I, van Aalten DM. 2011. Substrate and product analogues as human O-GlcNAc transferase inhibitors. Amino Acids . 40: 781– 792. Google Scholar CrossRef Search ADS PubMed  Durning SP, Flanagan-Steet H, Prasad N, Wells L. 2016. O-Linked beta-N-acetylglucosamine (O-GlcNAc) acts as a glucose sensor to epigenetically regulate the insulin gene in pancreatic beta cells. J Biol Chem . 291: 2107– 2118. Google Scholar CrossRef Search ADS PubMed  Elsen NL, Patel SB, Ford RE, Hall DL, Hess F, Kandula H, Kornienko M, Reid J, Selnick H, Shipman JM et al.  . 2017. Insights into activity and inhibition from the crystal structure of human O-GlcNAcase. Nat Chem Biol . 13: 613– 615. Google Scholar CrossRef Search ADS PubMed  Folkvord S, Flatmark K, Dueland S, de Wijn R, Groholt KK, Hole KH, Nesland JM, Ruijtenbeek R, Boender PJ, Johansen M et al.  . 2010. Prediction of response to preoperative chemoradiotherapy in rectal cancer by multiplex kinase activity profiling. Int J Radiat Oncol Biol Phys . 78: 555– 562. Google Scholar CrossRef Search ADS PubMed  Fong JJ, Nguyen BL, Bridger R, Medrano EE, Wells L, Pan S, Sifers RN. 2012. Beta-N-acetylglucosamine (O-GlcNAc) is a novel regulator of mitosis-specific phosphorylations on histone H3. J Biol Chem . 287: 12195– 12203. Google Scholar CrossRef Search ADS PubMed  Gallegos KM, Conrady DG, Karve SS, Gunasekera TS, Herr AB, Weiss AA. 2012. Shiga toxin binding to glycolipids and glycans. PLoS One . 7: e30368. Google Scholar CrossRef Search ADS PubMed  Gambaryan AS, Piskarev VE, Yamskov IA, Sakharov AM, Tuzikov AB, Bovin NV, Nifant’ev NE, Matrosovich MN. 1995. Human influenza virus recognition of sialyloligosaccharides. FEBS Lett . 366: 57– 60. Google Scholar CrossRef Search ADS PubMed  Gloster TM, Zandberg WF, Heinonen JE, Shen DL, Deng L, Vocadlo DJ. 2011. Hijacking a biosynthetic pathway yields a glycosyltransferase inhibitor within cells. Nat Chem Biol . 7: 174– 181. Google Scholar CrossRef Search ADS PubMed  Gross BJ, Kraybill BC, Walker S. 2005. Discovery of O-GlcNAc transferase inhibitors. J Am Chem Soc . 127: 14588– 14589. Google Scholar CrossRef Search ADS PubMed  Guinez C, Mir AM, Dehennaut V, Cacan R, Harduin-Lepers A, Michalski JC, Lefebvre T. 2008. Protein ubiquitination is modulated by O-GlcNAc glycosylation. FASEB J . 22: 2901– 2911. Google Scholar CrossRef Search ADS PubMed  Hahne H, Sobotzki N, Nyberg T, Helm D, Borodkin VS, van Aalten DM, Agnew B, Kuster B. 2013. Proteome wide purification and identification of O-GlcNAc-modified proteins using click chemistry and mass spectrometry. J Proteome Res . 12: 927– 936. Google Scholar CrossRef Search ADS PubMed  Hajduch J, Nam G, Kim EJ, Frohlich R, Hanover JA, Kirk KL. 2008. A convenient synthesis of the C-1-phosphonate analogue of UDP-GlcNAc and its evaluation as an inhibitor of O-linked GlcNAc transferase (OGT). Carbohydr Res . 343: 189– 195. Google Scholar CrossRef Search ADS PubMed  Hanover JA, Yu S, Lubas WB, Shin SH, Ragano-Caracciola M, Kochran J, Love DC. 2003. Mitochondrial and nucleocytoplasmic isoforms of O-linked GlcNAc transferase encoded by a single mammalian gene. Arch Biochem Biophys . 409: 287– 297. Google Scholar CrossRef Search ADS PubMed  Hawkins M, Barzilai N, Liu R, Hu M, Chen W, Rossetti L. 1997. Role of the glucosamine pathway in fat-induced insulin resistance. J Clin Invest . 99: 2173– 2182. Google Scholar CrossRef Search ADS PubMed  Hilhorst R, Houkes L, van den Berg A, Ruijtenbeek R. 2009. Peptide microarrays for detailed, high-throughput substrate identification, kinetic characterization, and inhibition studies on protein kinase A. Anal Biochem . 387: 150– 161. Google Scholar CrossRef Search ADS PubMed  Himmler A, Drechsel D, Kirschner MW, Martin DW Jr. 1989. Tau consists of a set of proteins with repeated C-terminal microtubule-binding domains and variable N-terminal domains. Mol Cell Biol . 9: 1381– 1388. Google Scholar CrossRef Search ADS PubMed  Hornbeck PV, Zhang B, Murray B, Kornhauser JM, Latham V, Skrzypek E. 2015. PhosphoSitePlus, 2014: mutations, PTMs and recalibrations. Nucleic Acids Res . 43: D512– D520. Google Scholar CrossRef Search ADS PubMed  Hovestad-Bijl L, van Ameijde J, Pijnenburg D, Hilhorst R, Liskamp R, Ruijtenbeek R. 2016. Peptide microarrays for real-time kinetic profiling of tyrosine phosphatase activity of recombinant phosphatases and phosphatases in lysates of cells or tissue Samples. Methods Mol Biol . 1447: 67– 78. Google Scholar CrossRef Search ADS PubMed  Iyer SP, Akimoto Y, Hart GW. 2003. Identification and cloning of a novel family of coiled-coil domain proteins that interact with O-GlcNAc transferase. J Biol Chem . 278: 5399– 5409. Google Scholar CrossRef Search ADS PubMed  Iyer SP, Hart GW. 2003. Roles of the tetratricopeptide repeat domain in O-GlcNAc transferase targeting and protein substrate specificity. J Biol Chem . 278: 24608– 24616. Google Scholar CrossRef Search ADS PubMed  Janetzko J, Trauger SA, Lazarus MB, Walker S. 2016. How the glycosyltransferase OGT catalyzes amide bond cleavage. Nat Chem Biol . 12: 899– 901. Google Scholar CrossRef Search ADS PubMed  Jarboe JS, Jaboin JJ, Anderson JC, Nowsheen S, Stanley JA, Naji F, Ruijtenbeek R, Tu T, Hallahan DE, Yang ES et al.  . 2012. Kinomic profiling approach identifies Trk as a novel radiation modulator. Radiother Oncol . 103: 380– 387. Google Scholar CrossRef Search ADS PubMed  Jinek M, Rehwinkel J, Lazarus BD, Izaurralde E, Hanover JA, Conti E. 2004. The superhelical TPR-repeat domain of O-linked GlcNAc transferase exhibits structural similarities to importin alpha. Nat Struct Mol Biol . 11: 1001– 1007. Google Scholar CrossRef Search ADS PubMed  Jochmann R, Holz P, Sticht H, Sturzl M. 2014. Validation of the reliability of computational O-GlcNAc prediction. Biochim Biophys Acta . 1844: 416– 421. Google Scholar CrossRef Search ADS PubMed  Kamemura K, Hayes BK, Comer FI, Hart GW. 2002. Dynamic interplay between O-glycosylation and O-phosphorylation of nucleocytoplasmic proteins: alternative glycosylation/phosphorylation of THR-58, a known mutational hot spot of c-Myc in lymphomas, is regulated by mitogens. J Biol Chem . 277: 19229– 19235. Google Scholar CrossRef Search ADS PubMed  Kim EJ, Kang DO, Love DC, Hanover JA. 2006. Enzymatic characterization of O-GlcNAcase isoforms using a fluorogenic GlcNAc substrate. Carbohydr Res . 341: 971– 982. Google Scholar CrossRef Search ADS PubMed  Klein AL, Berkaw MN, Buse MG, Ball LE. 2009. O-linked N-acetylglucosamine modification of insulin receptor substrate-1 occurs in close proximity to multiple SH2 domain binding motifs. Mol Cell Proteomics . 8: 2733– 2745. Google Scholar CrossRef Search ADS PubMed  Konrad RJ, Zhang F, Hale JE, Knierman MD, Becker GW, Kudlow JE. 2002. Alloxan is an inhibitor of the enzyme O-linked N-acetylglucosamine transferase. Biochem Biophys Res Commun . 293: 207– 212. Google Scholar CrossRef Search ADS PubMed  Kreppel LK, Hart GW. 1999. Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats. J Biol Chem . 274: 32015– 32022. Google Scholar CrossRef Search ADS PubMed  Krzeslak A, Forma E, Bernaciak M, Romanowicz H, Brys M. 2012. Gene expression of O-GlcNAc cycling enzymes in human breast cancers. Clin Exp Med . 12: 61– 65. Google Scholar CrossRef Search ADS PubMed  Lai S, Winkler DF, Zhang H, Pelech S. 2016. Determination of the substrate specificity of protein kinases with peptide micro- and macroarrays. Methods Mol Biol . 1360: 183– 202. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Jiang J, Gloster TM, Zandberg WF, Whitworth GE, Vocadlo DJ, Walker S. 2012. Structural snapshots of the reaction coordinate for O-GlcNAc transferase. Nat Chem Biol . 8: 966– 968. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Jiang J, Kapuria V, Bhuiyan T, Janetzko J, Zandberg WF, Vocadlo DJ, Herr W, Walker S. 2013. HCF-1 is cleaved in the active site of O-GlcNAc transferase. Science . 342: 1235– 1239. Google Scholar CrossRef Search ADS PubMed  Lazarus MB, Nam Y, Jiang J, Sliz P, Walker S. 2011. Structure of human O-GlcNAc transferase and its complex with a peptide substrate. Nature . 469: 564– 567. Google Scholar CrossRef Search ADS PubMed  Lebrin F, Chambaz EM, Bianchini L. 2001. A role for protein kinase CK2 in cell proliferation: evidence using a kinase-inactive mutant of CK2 catalytic subunit alpha. Oncogene . 20: 2010– 2022. Google Scholar CrossRef Search ADS PubMed  Lemeer S, Jopling C, Naji F, Ruijtenbeek R, Slijper M, Heck AJ, den Hertog J. 2007. Protein–tyrosine kinase activity profiling in knock down zebrafish embryos. PLoS One . 2: e581. Google Scholar CrossRef Search ADS PubMed  Leney AC, El Atmioui D, Wu W, Ovaa H, Heck AJR. 2017. Elucidating crosstalk mechanisms between phosphorylation and O-GlcNAcylation. Proc Natl Acad Sci USA . 114: E7255– E7261. Google Scholar CrossRef Search ADS PubMed  Li B, Li H, Lu L, Jiang J. 2017. Structures of human O-GlcNAcase and its complexes reveal a new substrate recognition mode. Nat Struct Mol Biol . 24: 362– 369. Google Scholar CrossRef Search ADS PubMed  Li J, Huang CL, Zhang LW, Lin L, Li ZH, Zhang FW, Wang P. 2010. Isoforms of human O-GlcNAcase show distinct catalytic efficiencies. Biochemistry (Mosc) . 75: 938– 943. Google Scholar CrossRef Search ADS PubMed  Liu F, Iqbal K, Grundke-Iqbal I, Hart GW, Gong CX. 2004. O-GlcNAcylation regulates phosphorylation of tau: a mechanism involved in Alzheimer’s disease. Proc Natl Acad Sci USA . 101: 10804– 10809. Google Scholar CrossRef Search ADS PubMed  Liu J, Marchase RB, Chatham JC. 2007. Glutamine-induced protection of isolated rat heart from ischemia/reperfusion injury is mediated via the hexosamine biosynthesis pathway and increased protein O-GlcNAc levels. J Mol Cell Cardiol . 42: 177– 185. Google Scholar CrossRef Search ADS PubMed  Liu X, Li L, Wang Y, Yan H, Ma X, Wang PG, Zhang L. 2014. A peptide panel investigation reveals the acceptor specificity of O-GlcNAc transferase. FASEB J . 28: 3362– 3372. Google Scholar CrossRef Search ADS PubMed  Liu Y, Ren Y, Cao Y, Huang H, Wu Q, Li W, Wu S, Zhang J. 2017. Discovery of a low toxicity O-GlcNAc Transferase (OGT) inhibitor by structure-based virtual screening of natural products. Sci Rep . 7: 12334. Google Scholar CrossRef Search ADS PubMed  Love DC, Krause MW, Hanover JA. 2010. O-GlcNAc cycling: emerging roles in development and epigenetics. Semin Cell Dev Biol . 21: 646– 654. Google Scholar CrossRef Search ADS PubMed  Lubas WA, Frank DW, Krause M, Hanover JA. 1997. O-Linked GlcNAc transferase is a conserved nucleocytoplasmic protein containing tetratricopeptide repeats. J Biol Chem . 272: 9316– 9324. Google Scholar CrossRef Search ADS PubMed  Lubas WA, Hanover JA. 2000. Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. J Biol Chem . 275: 10983– 10988. Google Scholar CrossRef Search ADS PubMed  Lynch TP, Ferrer CM, Jackson SR, Shahriari KS, Vosseller K, Reginato MJ. 2012. Critical role of O-linked beta-N-acetylglucosamine transferase in prostate cancer invasion, angiogenesis, and metastasis. J Biol Chem . 287: 11070– 11081. Google Scholar CrossRef Search ADS PubMed  Macauley MS, Vocadlo DJ. 2009. Enzymatic characterization and inhibition of the nuclear variant of human O-GlcNAcase. Carbohydr Res . 344: 1079– 1084. Google Scholar CrossRef Search ADS PubMed  Macauley MS, Whitworth GE, Debowski AW, Chin D, Vocadlo DJ. 2005. O-GlcNAcase uses substrate-assisted catalysis: kinetic analysis and development of highly selective mechanism-inspired inhibitors. J Biol Chem . 280: 25313– 25322. Google Scholar CrossRef Search ADS PubMed  Marotta NP, Lin YH, Lewis YE, Ambroso MR, Zaro BW, Roth MT, Arnold DB, Langen R, Pratt MR. 2015. O-GlcNAc modification blocks the aggregation and toxicity of the protein alpha-synuclein associated with Parkinson’s disease. Nat Chem . 7: 913– 920. Google Scholar CrossRef Search ADS PubMed  Miller CJ, Turk BE. 2016. Rapid identification of protein kinase phosphorylation site motifs using combinatorial peptide libraries. Methods Mol Biol . 1360: 203– 216. Google Scholar CrossRef Search ADS PubMed  Morris M, Knudsen GM, Maeda S, Trinidad JC, Ioanoviciu A, Burlingame AL, Mucke L. 2015. Tau post-translational modifications in wild-type and human amyloid precursor protein transgenic mice. Nat Neurosci . 18: 1183– 1189. Google Scholar CrossRef Search ADS PubMed  Nishikawa K, Sawasdikosol S, Fruman DA, Lai J, Songyang Z, Burakoff SJ, Yaffe MB, Cantley LC. 2000. A peptide library approach identifies a specific inhibitor for the ZAP-70 protein tyrosine kinase. Mol Cell . 6: 969– 974. Google Scholar CrossRef Search ADS PubMed  Onodera Y, Nam JM, Bissell MJ. 2014. Increased sugar uptake promotes oncogenesis via EPAC/RAP1 and O-GlcNAc pathways. J Clin Invest . 124: 367– 384. Google Scholar CrossRef Search ADS PubMed  Ortiz-Meoz RF, Jiang J, Lazarus MB, Orman M, Janetzko J, Fan C, Duveau DY, Tan ZW, Thomas CJ, Walker S. 2015. A small molecule that inhibits OGT activity in cells. ACS Chem Biol . 10: 1392– 1397. Google Scholar CrossRef Search ADS PubMed  Ortiz-Meoz RF, Merbl Y, Kirschner MW, Walker S. 2014. Microarray discovery of new OGT substrates: the medulloblastoma oncogene OTX2 is O-GlcNAcylated. J Am Chem Soc . 136: 4845– 4848. Google Scholar CrossRef Search ADS PubMed  Pathak S, Alonso J, Schimpl M, Rafie K, Blair DE, Borodkin VS, Albarbarawi O, van Aalten DMF. 2015. The active site of O-GlcNAc transferase imposes constraints on substrate sequence. Nat Struct Mol Biol . 22: 744– 750. Google Scholar CrossRef Search ADS PubMed  Pathak S, Borodkin VS, Albarbarawi O, Campbell DG, Ibrahim A, van Aalten DM. 2012. O-GlcNAcylation of TAB1 modulates TAK1-mediated cytokine release. EMBO J . 31: 1394– 1404. Google Scholar CrossRef Search ADS PubMed  Rafie K, Raimi O, Ferenbach AT, Borodkin VS, Kapuria V, van Aalten DMF. 2017. Recognition of a glycosylation substrate by the O-GlcNAc transferase TPR repeats. Open Biol . 7: 170078. Google Scholar CrossRef Search ADS PubMed  Rao FV, Dorfmueller HC, Villa F, Allwood M, Eggleston IM, van Aalten DM. 2006. Structural insights into the mechanism and inhibition of eukaryotic O-GlcNAc hydrolysis. EMBO J . 25: 1569– 1578. Google Scholar CrossRef Search ADS PubMed  Roth C, Chan S, Offen WA, Hemsworth GR, Willems LI, King DT, Varghese V, Britton R, Vocadlo DJ, Davies GJ. 2017. Structural and functional insight into human O-GlcNAcase. Nat Chem Biol . 13: 610– 612. Google Scholar CrossRef Search ADS PubMed  Schimpl M, Borodkin VS, Gray LJ, van Aalten DM. 2012. Synergy of peptide and sugar in O-GlcNAcase substrate recognition. Chem Biol . 19: 173– 178. Google Scholar CrossRef Search ADS PubMed  Schimpl M, Zheng X, Borodkin VS, Blair DE, Ferenbach AT, Schuttelkopf AW, Navratilova I, Aristotelous T, Albarbarawi O, Robinson DA et al.  . 2012. O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis. Nat Chem Biol . 8: 969– 974. Google Scholar CrossRef Search ADS PubMed  Selvan N, Williamson R, Mariappa D, Campbell DG, Gourlay R, Ferenbach AT, Aristotelous T, Hopkins-Navratilova I, Trost M, van Aalten DMF. 2017. A mutant O-GlcNAcase enriches Drosophila developmental regulators. Nat Chem Biol . 13: 882– 887. Google Scholar CrossRef Search ADS PubMed  Shafi R, Iyer SP, Ellies LG, O’Donnell N, Marek KW, Chui D, Hart GW, Marth JD. 2000. The O-GlcNAc transferase gene resides on the X chromosome and is essential for embryonic stem cell viability and mouse ontogeny. Proc Natl Acad Sci USA . 97: 5735– 5739. Google Scholar CrossRef Search ADS PubMed  Sharif S, Shi J, Bourakba M, Ruijtenbeek R, Pieters RJ. 2017. Measuring O-GlcNAc cleavage by OGA and cell lysates on a peptide microarray. Anal Biochem . 532: 12– 18. Google Scholar CrossRef Search ADS PubMed  Shen DL, Gloster TM, Yuzwa SA, Vocadlo DJ. 2012. 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. J Biol Chem . 287: 15395– 15408. Google Scholar CrossRef Search ADS PubMed  Shen DL, Liu TW, Zandberg W, Clark T, Eskandari R, Alteen MG, Tan HY, Zhu Y, Cecioni S, Vocadlo D. 2017. Catalytic promiscuity of O-GlcNAc transferase enables unexpected metabolic engineering of cytoplasmic proteins with 2-azido-2-deoxy-glucose. ACS Chem Biol . 12: 206– 213. Google Scholar CrossRef Search ADS PubMed  Shi J, Sharif S, Ruijtenbeek R, Pieters RJ. 2016. Activity based high-throughput screening for novel O-GlcNAc transferase substrates using a dynamic peptide microarray. PLoS One . 11: e0151085. Google Scholar CrossRef Search ADS PubMed  Shi J, Tomasic T, Sharif S, Brouwer AJ, Anderluh M, Ruijtenbeek R, Pieters RJ. 2017. Peptide microarray analysis of the cross-talk between O-GlcNAcylation and tyrosine phosphorylation. FEBS Lett . 591: 1872– 1883. Google Scholar CrossRef Search ADS PubMed  Singleton KD, Wischmeyer PE. 2008. Glutamine induces heat shock protein expression via O-glycosylation and phosphorylation of HSF-1 and Sp1. JPEN J Parenter Enteral Nutr . 32: 371– 376. Google Scholar CrossRef Search ADS PubMed  Smet-Nocca C, Broncel M, Wieruszeski JM, Tokarski C, Hanoulle X, Leroy A, Landrieu I, Rolando C, Lippens G, Hackenberger CP. 2011. Identification of O-GlcNAc sites within peptides of the Tau protein and their impact on phosphorylation. Mol BioSyst . 7: 1420– 1429. Google Scholar CrossRef Search ADS PubMed  Stanford SM, Krishnamurthy D, Kulkarni RA, Karver CE, Bruenger E, Walker LM, Ma CT, Chung TD, Sergienko E, Bottini N et al.  . 2014. pCAP-based peptide substrates: the new tool in the box of tyrosine phosphatase assays. Methods . 65: 165– 174. Google Scholar CrossRef Search ADS PubMed  Swamy M, Pathak S, Grzes KM, Damerow S, Sinclair LV, van Aalten DM, Cantrell DA. 2016. Glucose and glutamine fuel protein O-GlcNAcylation to control T cell self-renewal and malignancy. Nat Immunol . 17: 712– 720. Google Scholar CrossRef Search ADS PubMed  Tarrant MK, Rho HS, Xie Z, Jiang YL, Gross C, Culhane JC, Yan G, Qian J, Ichikawa Y, Matsuoka T et al.  . 2012. Regulation of CK2 by phosphorylation and O-GlcNAcylation revealed by semisynthesis. Nat Chem Biol . 8: 262– 269. Google Scholar CrossRef Search ADS PubMed  Torreno-Pina JA, Castro BM, Manzo C, Buschow SI, Cambi A, Garcia-Parajo MF. 2014. Enhanced receptor–clathrin interactions induced by N-glycan-mediated membrane micropatterning. Proc Natl Acad Sci USA . 111: 11037– 11042. Google Scholar CrossRef Search ADS PubMed  Trinidad JC, Barkan DT, Gulledge BF, Thalhammer A, Sali A, Schoepfer R, Burlingame AL. 2012. Global identification and characterization of both O-GlcNAcylation and phosphorylation at the murine synapse. Mol Cell Proteomics . 11: 215– 229. Google Scholar CrossRef Search ADS PubMed  Trinidad JC, Schoepfer R, Burlingame AL, Medzihradszky KF. 2013. N- and O-glycosylation in the murine synaptosome. Mol Cell Proteomics . 12: 3474– 3488. Google Scholar CrossRef Search ADS PubMed  van Wandelen LT, van Ameijde J, Ismail-Ali AF, van Ufford HC, Vijftigschild LA, Beekman JM, Martin NI, Ruijtenbeek R, Liskamp RM. 2013. Cell-penetrating bisubstrate-based protein kinase C inhibitors. ACS Chem Biol . 8: 1479– 1487. Google Scholar CrossRef Search ADS PubMed  Vosseller K, Trinidad JC, Chalkley RJ, Specht CG, Thalhammer A, Lynn AJ, Snedecor JO, Guan S, Medzihradszky KF, Maltby DA et al.  . 2006. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics . 5: 923– 934. Google Scholar CrossRef Search ADS PubMed  Wang Z, Gucek M, Hart GW. 2008. Cross-talk between GlcNAcylation and phosphorylation: site-specific phosphorylation dynamics in response to globally elevated O-GlcNAc. Proc Natl Acad Sci USA . 105: 13793– 13798. Google Scholar CrossRef Search ADS PubMed  Wang Z, Udeshi ND, Slawson C, Compton PD, Sakabe K, Cheung WD, Shabanowitz J, Hunt DF, Hart GW. 2010. Extensive crosstalk between O-GlcNAcylation and phosphorylation regulates cytokinesis. Sci Signal . 3: ra2. Google Scholar PubMed  Withers SG, Davies GJ. 2012. The case of the missing base. Nat Chem Biol . 8: 952– 953. Google Scholar CrossRef Search ADS PubMed  Wu HY, Lu CT, Kao HJ, Chen YJ, Chen YJ, Lee TY. 2014. Characterization and identification of protein O-GlcNAcylation sites with substrate specificity. BMC Bioinf . 15: S1. Google Scholar CrossRef Search ADS   Yang WH, Kim JE, Nam HW, Ju JW, Kim HS, Kim YS, Cho JW. 2006. Modification of p53 with O-linked N-acetylglucosamine regulates p53 activity and stability. Nat Cell Biol . 8: 1074– 1083. Google Scholar CrossRef Search ADS PubMed  Yuzwa SA, Shan X, Macauley MS, Clark T, Skorobogatko Y, Vosseller K, Vocadlo DJ. 2012. Increasing O-GlcNAc slows neurodegeneration and stabilizes tau against aggregation. Nat Chem Biol . 8: 393– 399. Google Scholar CrossRef Search ADS PubMed  Yuzwa SA, Yadav AK, Skorobogatko Y, Clark T, Vosseller K, Vocadlo DJ. 2011. Mapping O-GlcNAc modification sites on tau and generation of a site-specific O-GlcNAc tau antibody. Amino Acids . 40: 857– 868. Google Scholar CrossRef Search ADS PubMed  Zachara NE, Vosseller K, Hart GW. 2011. Detection and analysis of proteins modified by O-linked N-acetylglucosamine. Curr Protoc Protein Sci . Chapter 12: Unit12 18. Zhang F, Su K, Yang X, Bowe DB, Paterson AJ, Kudlow JE. 2003. O-GlcNAc modification is an endogenous inhibitor of the proteasome. Cell . 115: 715– 725. Google Scholar CrossRef Search ADS PubMed  Zhang S, Roche K, Nasheuer HP, Lowndes NF. 2011. Modification of histones by sugar beta-N-acetylglucosamine (GlcNAc) occurs on multiple residues, including histone H3 serine 10, and is cell cycle-regulated. J Biol Chem . 286: 37483– 37495. Google Scholar CrossRef Search ADS PubMed  Zhong J, Martinez M, Sengupta S, Lee A, Wu X, Chaerkady R, Chatterjee A, O’Meally RN, Cole RN, Pandey A et al.  . 2015. Quantitative phosphoproteomics reveals crosstalk between phosphorylation and O-GlcNAc in the DNA damage response pathway. Proteomics . 15: 591– 607. Google Scholar CrossRef Search ADS PubMed  © The Author(s) 2018. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

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GlycobiologyOxford University Press

Published: Mar 22, 2018

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