Recombinant O-GlcNAc transferase isoforms: identification of O-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates (original) (raw)

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Laboratory of Cell Biology and Biochemistry, NIDDK, National Institutes of Health, Building 8, Room 402, 9000 Rockville Pike, Bethesda, MD 20897-0851, USA

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Laboratory of Cell Biology and Biochemistry, NIDDK, National Institutes of Health, Building 8, Room 402, 9000 Rockville Pike, Bethesda, MD 20897-0851, USA

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Laboratory of Cell Biology and Biochemistry, NIDDK, National Institutes of Health, Building 8, Room 402, 9000 Rockville Pike, Bethesda, MD 20897-0851, USA

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Received:

07 November 2005

Revision received:

12 January 2006

Accepted:

13 January 2006

Published:

23 January 2006

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Brooke D. Lazarus, Dona C. Love, John A. Hanover, Recombinant _O_-GlcNAc transferase isoforms: identification of _O_-GlcNAcase, yes tyrosine kinase, and tau as isoform-specific substrates, Glycobiology, Volume 16, Issue 5, May 2006, Pages 415–421, https://doi.org/10.1093/glycob/cwj078
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Abstract

_O_-linked _N_-acetylglucosaminyltransferase (OGT) catalyzes the transfer of _O_-linked GlcNAc to serine or threonine residues of a variety of substrate proteins, including nuclear pore proteins, transcription factors, and proteins implicated in diabetes and neurodegenerative disorders. We have identified two nucleocytoplasmic isoforms of OGT (ncOGT and sOGT) and one isoform that localizes to the mitochondria (mOGT). These three isoforms contain identical catalytic regions but differ in the number of tetratricopeptide repeat motifs found at the _N_-terminus of each enzyme. We expressed each of these OGT isoforms in a soluble form in Escherichia coli and have used them to identify novel targets including the Src-family tyrosine kinase yes and _O_-GlcNAc-ase. We demonstrate that some substrate proteins, such as Nup62 and casein kinase II, are glycosylated by both ncOGT and mOGT, while others such as _O_-GlcNAcase and tau are specifically modified by ncOGT. The yes kinase was specifically modified by mOGT. The short isoform of OGT (sOGT) did not glycosylate any of the substrates tested, although it retains a potentially active catalytic domain. Our findings demonstrate the potential utility of recombinant OGT in identifying new targets and illustrate the necessity to examine all active isoforms of the enzyme. The identification of a tyrosine kinase and _O_-GlcNAcase as OGT targets suggests the potential for OGT participation in numerous signal transduction cascades.

BCA, bicinchoninic acid, CKII, casein kinase II, mOGT, mitochondrial _O_-linked _N_-acetylglucosaminyltransferase, ncOGT, nucleocytoplasmic _O_-linked _N_-acetylglucosaminyltransferase, NiNTA, nickel-chelating resin, OGA, _O_-linked _N_-acetylglucosaminidase, _O_-GlcNAcase, _O_-linked _N_-acetylglucosaminidase, OGT, _O_-linked _N_-acetylglucosaminyltransferase, PCR, polymerase chain reaction, sOGT, short _O_‐linked _N_-acetylglucosaminyltransferase, TPR, tetratricopeptide repeat, UDP, uridine di-phosphate

Introduction

_O_-linked _N_-acetylglucosaminyltransferase (OGT) is a soluble glycosyltransferase that catalyzes the addition of a single β-_N_-acetylglucosamine (β-GlcNAc) residue, in an _O_-glycosidic linkage, to serine or threonine residues of target proteins (Holt et al., 1987; Kreppel et al., 1997; Lubas et al., 1997). This dynamic form of posttranslational modification is analogous to phosphorylation (Haltiwanger et al., 1997; Miller et al., 1999), suggesting a role in regulation. Indeed, in many cases, proteins bearing _O_-GlcNAc can also be phosphorylated, and this will often occur at the same or adjacent serine or threonine residues (Comer and Hart, 2001; Kamemura et al., 2002). The _O_-GlcNAc modification occurs on a wide variety of proteins such as nuclear pore proteins (Davis and Blobel, 1987; Hanover et al., 1987; Holt et al., 1987), RNA polymerase II (Kelly et al., 1993; Cervoni et al., 1997) and associated transcription factors (Jackson and Tjian, 1988; Yang et al., 2001; Iyer et al., 2003), cytoskeletal proteins (Arnold et al., 1996; Ding and Vandre, 1996; Takahashi et al., 1999), proteosomes (Han and Kudlow, 1997; Zhang et al., 2003), synapsins (Luthi et al., 1991; Cole and Hart, 1999), oncogenic proteins (Chou et al., 1995; Medina et al., 1998), and tumor suppressor proteins (Shaw et al., 1996). This suggests a physiological role for _O_-GlcNAc modification in many important cellular processes such as transport, transcription, cell shape, cell signaling, and apoptosis. However, the mechanisms for regulation and substrate selection still remain largely unknown.

The gene for OGT resides on the X chromosome and is essential for mammalian development (Shafi et al., 2000). It covers >45 Kb of genomic DNA and contains 23 coding exons. The OGT gene is capable of producing three separate transcripts, and each encodes a different OGT isoform. The longest isoform is encoded by all 23 exons and produces a protein of ∼116 kDa. It has been localized to both the nucleus and cytoplasm and has thus been designated as ncOGT (Hanover et al., 2003). The second longest isoform is encoded by exons 5–23 and produces a protein of ∼103 kDa. This isoform contains a mitochondrial targeting sequence at its _N_-terminus and has been localized to mitochondria (Love et al., 2003). It has thus been termed mOGT. The shortest isoform of OGT is encoded by exons 10–23. It produces a protein of ∼78 kDa and has been termed sOGT for its short length (Shafi et al., 2000; Nolte and Muller, 2002; Hanover et al., 2003). Similar to ncOGT, sOGT is ubiquitously expressed within the cell and is localized to both the nucleus and the cytoplasm (data not shown). To date, OGT has been cloned from humans, mouse, rat, Caenorhabditis elegans, Drosophila melanogastor, and Arabidopsis thaliana, encoding all three isoforms (Nolte and Muller, 2002; Hanover et al., 2003). Drosophila melanogastor and C. elegans contain one gene that is predicted to encode just the ncOGT isoform (Hanover et al., 2005), whereas A. thaliana contains two separate genes that encode the proteins Spindly and Secret Agent, both of which have been shown to have OGT activity (Hartweck et al., 2002; Chen et al., 2005).

All OGT isoforms are comprised of three distinct regions, an _N_-terminal domain containing a varying number of tetratricopeptide repeat (TPR) motifs, a linker region, and a C-terminal catalytic domain (Lubas et al., 1997; Kreppel et al., 1997) (Figure 1). All three isoforms have identical catalytic domains but differ in their _N_-termini. Each of the _N_-terminal domains of ncOGT and mOGT has a unique 5′ sequence before their TPR motifs and in mOGT; this region encodes a mitochondrial targeting sequence. All three isoforms contain TPR motifs at their _N_-terminus, with each containing a different number of repeats. The TPR motif consists of 34 amino acids, of which 8 are highly conserved, and is known to mediate protein–protein interactions (Goebl and Yanagida, 1991; Liu et al., 1999; Iyer and Hart, 2003). Multiple TPRs have been shown to create a superhelix, and a minimum of 2.5 TPRs are required for function (Hanover et al., 2003). The three isoforms of OGT (ncOGT, mOGT, and sOGT) have 12.5, 9.5, and 2.5 TPR motifs, respectively (Hanover et al., 2003) (Figure 1). The superhelical structure of the TPR domain contains an amphipathic groove capable of accommodating an alpha helix from the substrate protein (Blatch and Lassle, 1999) and is thought to be involved in determining substrate specificity (Hanover et al., 2003; Iyer et al., 2003). Recently, the TPR domain of ncOGT was crystallized, and the inner surface of the superhelix was shown to contain a ladder of Asn residues (Jinek et al., 2004). This Asn ladder has also been identified in importin-α, a protein known to bind multiple substrates with no known sequence motif.

Fig. 1.

Schematic structure of OGT isoforms. Three isoforms of human OGT are produced from the OGT gene. ncOGT is the longest isoform and contains a unique _N_-terminal sequence, followed by 12 TPR motifs, a linker region, and the catalytic domain. mOGT contains a different _N_-terminal sequence, which also encodes a mitochondrial targeting sequence. The _N_-terminal sequence is then followed by 9 TPR motifs, a linker region, and the catalytic domain. sOGT is the shortest isoform consisting of only 2 TPR motifs, a linker region, and the catalytic domain. The catalytic region in all three isoforms is identical and contains two domains, the CD I domain and the CD II domain.

There are a growing number of proteins reported to contain the _O_-GlcNAc modification, and many approaches are being developed to identify new substrates (Khidekel et al., 2003; Vocadlo et al., 2003; Cieniewski-Bernard et al., 2004), including high-throughput proteomics (Whelan and Hart, 2003). Our approach has been to express recombinant forms of the enzyme in vitro and directly test the ability of each isoform to glycosylate a given target protein. This has the advantage of being highly specific, as it allows us to determine the substrate specificity for each isoform. This is the first report to show in vitro activity of both ncOGT and sOGT and the first time that a difference in substrate specificity has been observed. Furthermore, the results presented in this article provide further evidence that OGT interacts with substrate proteins through its TPR domain, as the three isoforms differ only in this _N_-terminal region. Previous reports have not discussed the possibility that different isoforms may have different functions. However, the results presented here clearly indicate that each isoform may have a separate role in cellular functions, thus allowing the cell a greater degree of control over the regulation of proteins by posttranslational modification.

Results

Expression of recombinant human OGT isoforms in Escherichia coli

The three human isoforms of OGT (ncOGT, mOGT, and sOGT) were produced in an Escherichia coli expression system, which has previously been shown to lack any endogenous OGT activity (Lubas et al., 1995). Figure 2A shows the production of protein in E. coli lysate for each isoform. The pET43.1 vector produces a fusion protein containing an _N_-terminal NusA-tag (54 kDa) as well as an S-tag and a His-tag. Therefore, (1) ncOGT (116 kDa, lane 1) produces a protein that runs at ∼170 kDa, (2) mOGT (103 kDa, lane 2) produces a protein that runs at ∼157 kDa, and (3) sOGT (78 kDa, lane 3) produces a protein that runs at ∼132 kDa. Figure 2B shows the approximate amount of enzyme produced from a 20 mL culture of BL21(DE3) cells, whereas Figure 2C shows each E. coli lysate probed with an anti-His-tag antibody.

Fig. 2.

Production and purification of OGT isoforms. All OGT isoforms were produced in E. coli, which is known to lack the gene for OGT. (A) Lysates containing soluble OGT underwent electrophoresis and were then transferred to nitrocellulose, followed by staining with Ponceau S. Lane 1, ncOGT (#); Lane 2, mOGT (*); Lane 3, sOGT (∼). (B) Western blot analysis of OGT lysates, probing with an anti-His-tag antibody directed against recombinant OGT. Lane 1, ncOGT(#); Lane 2, mOGT(*); Lane 3, sOGT(∼). (C) Approximate amount of soluble OGT protein produced in each lysate. (D) OGT glycosylation assay; isoforms were incubated with Nup62 (1 µg) and C14-labeled UDP-GlcNAc (12 µM) for 30 min. Glycosylation was visualized by fluorography. Lane 1, ncOGT; Lane 2, mOGT; Lane 3, sOGT. (E) Lysate containing soluble sOGT was incubated with C14-labeled UDP-GlcNAc and assay buffer for 30 min. Reaction was stopped and was either electrophoresed (Lane 1) or was incubated with NiNTA beads to separate sOGT from remaining lysate. Lane 2, purified sOGT; Lane 3, E. coli lysate.

To show that the different OGT isoforms are active in vitro, the E. coli supernatants were incubated with uridine di-phosphate (UDP)-[14C]-GlcNAc and Nup62 as described in the experimental procedures. The addition of an _O_-GlcNAc moiety onto Nup62 was visualized in lanes containing ncOGT and mOGT supernatants (Figure 2D, lanes 1 and 2, respectively), showing that functional enzyme was produced in vitro. The approximate Km and Vmax values of ncOGT and mOGT were also determined for Nup62. The Km with respect to Nup62 for ncOGT was lower than that observed for mOGT (25 and 227 nM, respectively). mOGT exhibited a two-fold increase in the Vmax (0.2 and 0.1 pmol/min, respectively). Assays performed at saturation would not be significantly influenced by these minor kinetic differences. No glycosylation of Nup62 was observed with the sOGT supernatant, however a band of ∼130 kDa was seen (Figure 2D, lane 3). This band was thought to be either an autoglycosylation product or a protein present in the E. coli lysate. Purification of sOGT from the lysate by nickel-chelating resin (NiNTA) agarose showed that the protein visualized in Figure 2D (lane 3) was not sOGT autoglycosylation but an unknown E. coli protein (Figure 2E). These data indicate that functional sOGT can be produced in vitro, however it has no activity toward the prototypic OGT substrate, Nup62.

OGT isoforms glycosylate: a unique subset of target proteins

Having shown that the three OGT isoforms were active in vitro, many other substrates were tested. Each of the isoforms were analyzed for their ability to glycosylate casein kinase II (CKII), tau, yes kinase, α-synuclein, and β-synuclein.

The results for ncOGT showed that, in addition to Nup62, ncOGT was also capable of glycosylating CKII and tau (Figure 3A, lanes 1, 2, and 4, respectively). No bands were observed in the lanes corresponding to yes kinase, α-synuclein, or β-synuclein (Figure 3A, lanes 3, 4, and 5 respectively), showing that these proteins do not serve as substrates for ncOGT. The results for mOGT showed that, in addition to Nup62, it was also capable of glycosylating CKII and yes kinase (Figure 3B, lanes 1 and 3, respectively). No bands were observed in lanes corresponding to tau, α-synuclein, and β-synuclein (Figure 3B, lanes 3, 5, and 6, respectively). Lastly, the results for sOGT showed that none of the analyzed substrates were glycosylated by this isoform (data not shown). In each lane, a band of ∼130 kDa was observed, corresponding to the unknown E. coli protein observed previously (Figure 2D); however, none of the other proteins analyzed served as a substrate for sOGT.

Fig. 3.

Substrate specificity of OGT isoforms. Each OGT isoform was analyzed against six proteins to determine whether they were a substrate for glycosylation (see experimental procedures). (A) ncOGT was used in an OGT glycosylation assay with the following proteins for substrates. (1) Nup62, (2) CKII, (3) tau, (4) yes, (5) α-synuclein, and (6) β-synuclein. (B) mOGT was used in an OGT glycosylation assay with the following proteins for substrates. (1) Nup62; (2) CKII; (3) tau; (4) Yes; (5) α-synuclein; (6) β-synuclein. Stoichiometry for positive substrates was determined from phosphoimage quantitation (see experimental procedures). ncOGT glycosylated Nup62, CKII, and tau at ∼1%, 0.013%, and 0.003%, respectively. mOGT-glycosylated Nup62, CKII, and yes at ∼3%, 0.04%, and 0.01%, respectively. These numbers are within the expected range, given that Nup62 is known to be glycosylated at 15 independent sites.

CKII and yes are both kinases involved in many important cellular processes, and the _O_-GlcNAc modification is thought to play an important role in regulating the activity of these enzymes. We therefore wanted to investigate other kinases to determine whether they are substrates for any of the OGT isoforms and are thus regulated by this form of posttranslational modification. Many kinases were analyzed, including the serine or threonine kinases mitogen-activated protein kinase (MAPK), MAPK1, and protein kinase A (PKA). Yes kinase is a tyrosine kinase belonging to the Src family of kinases, thus we also analyzed Src, as well as the Src-family kinases Lck, FynT, LynA, and Fgr. None of these enzymes were shown to be substrates for any of the OGT isoforms and thus do not appear to be _O_-GlcNAc modified (data not shown).

ncOGT glycosylates O-GlcNAcase

_O_-GlcNAcase (OGA) is the enzyme required for removal of _O_-linked GlcNAc residues and is thought to act in conjunction with OGT to regulate the function of substrate proteins. It was of interest to determine whether the OGA enzyme could also serve as a substrate for OGT. Figure 4 shows that OGA can serve as substrate for ncOGT (lane 2) but not mOGT or sOGT (lane 4 and data not shown).

Fig. 4.

Substrate specificity of OGT isoforms against OGA. Each OGT isoform was analyzed for glycosylation of OGA to determine whether it was a substrate (see experimental procedures). (1) ncOGT + Nup62, (2) ncOGT + OGA, (3) mOGT + Nup62, (4) mOGT + OGA, (5) sOGT + OGA. ncOGT glycosylated OGA with a stoichiometry of ∼0.06%, which is within the expected range.

Discussion

Over the past two decades, it has become increasingly obvious that the addition of _O_-GlcNAc to proteins is an important regulatory process in the cell. Some of the consequences of posttranslational modification by OGT include the reversible inhibition of Sp1 degradation by glycosylation of the proteosome (Zhang et al., 2003) as well as up-regulation of calmodulin gene transcription by the glycosylation of Sp1 in response to insulin stimulus (Majumdar et al., 2003). The OGT enzyme can also prevent transcription of RNA polymerase II through association with mSin3A and histone deacetylases (Yang et al., 2002), whereas glycosylation of the transcription factor pancreas duodenum homeobox-1 (PDX-1) increases its DNA binding ability and subsequently effects levels of insulin secretion (Gao et al., 2003). There are many other examples of OGT having a role in crucial signaling pathways, including inactivation of endothelial nitric oxide synthase in diabetics (Musicki et al., 2005), glycosylation of oncogene products, and transcription repressor proteins (for review see Chou and Hart, 2001). It is clear from these and many other published data that understanding the mechanisms of substrate selection and specificity is key to elucidating the many functions of OGT. In this article, we have shown that all three isoforms of OGT are active in vitro, and we have observed yes kinase and _O_-GlcNAcase as novel substrates for mOGT and ncOGT, respectively. We also show that the three isoforms of OGT have different specificities for substrate proteins, indicating that the regulation of proteins by glycosylation is more complex than previously imagined and that each OGT isoform may have a specific, clearly defined role in the cell.

The OGT gene is known to produce three distinct splice variants, referred to in this article as ncOGT (116 kDa), mOGT (103 kDa), and sOGT (74.5 kDa) (Kreppel et al., 1997; Lubas et al., 1997; Hanover et al., 2003). Two of these variants, ncOGT and mOGT, have previously been shown to be distinct isoforms that (1) have different subcellular localizations (ncOGT and mOGT reside in the nucleus/cytoplasm and mitochondria, respectively) and (2) are regulated by separate promoters (Love et al., 2003). It was therefore predicted that they would be functionally distinct, with each having a different set of target proteins and intracellular functions (Love et al., 2003). The third isoform of OGT, sOGT, was predicted from gene analysis and could be detected by immunoblot from cell extracts (Hanover et al., 2003). Production of sOGT is thought to be regulated by the same promoter as mOGT and utilizes an internal initiation methionine that results in a truncated form of the enzyme containing only 2.5 TPR motifs (Hanover et al., 2003). The localization of sOGT is similar to ncOGT (unpublished data) and, due to regulation by a separate promoter, was also predicted to be functionally distinct.

Tissue expression data for each isoform are available on the UCSC human gene sorter website (http://genome.ucsc.edu/cgi-bin/hgNear), and analysis shows that there is some distinction between the isoforms in their pattern of tissue expression. All three isoforms are highly expressed in many different blood cell types, including T cells, B cells, NK cells, and dendritic cells. They are also present in the fetal brain. ncOGT can be detected in areas of the adult brain, including the prefrontal cortex and hypothalamus, and it is also specific to the pancreas and uterus. mOGT appears to have the most limited tissue expression and appears only in the fetal lung, in addition to fetal brain and the blood cells mentioned above. sOGT has the widest array of tissue expression and, in addition to tissues mentioned above, can also be found in thymus, tonsil, whole blood, salivary gland, placenta, and ovary. It also shares some overlap with ncOGT in that it can also be found in pancreatic islet cells. This unique tissue expression supports a role for the specialized functions of each OGT isoform.

Determining the exact function of each OGT isoform has proven difficult in the past. A growing number of substrates continue to be reported, but how these substrates are recognized by OGT and determining the isoform that glycosylates them have been complicated. Only mOGT has been produced in vitro in a pure, active form (Lubas and Hanover, 2000). This is the first report of either ncOGT or sOGT being produced in vitro in an active form, and this has allowed us to look at a range of proteins to determine (1) whether they are substrates for _O_-GlcNAc addition and (2) which of the three isoforms they are substrates for. For the proteins tested in this article, we showed that certain substrates, such as Nup62 and CKII, could be glycosylated by both ncOGT and mOGT, whereas others are specific for an individual isoform, that is, tau is specific for ncOGT and the tyrosine kinase yes is specific for mOGT. Interestingly, there is some evidence that OGT may be phosphorylated by a tyrosine kinase, and, to date, this kinase has not been identified (Kreppel et al., 1997). We also show that two proteins previously thought to be _O_-glycosylated α-synuclein (Shimura et al., 2001) and β-synuclein (Cole and Hart, 2001) are not substrates for any of the isoforms in our assay and may not contain the _O_-GlcNAc modification. Many other kinases were also not _O_-glycosylated by any of the OGT isoforms in this assay; however, these negative findings do not exclude the possibility that these proteins may be modified to an extent that is below our level of detection. There is also a possibility that modification by OGT could be modulated by other processes in the cell, such as additional posttranslational modification of either the substrate protein or OGT itself. In addition, other interacting proteins may need to be recruited to a complex for glycosylation to occur. However, results presented here suggest that OGT is not directly glycosylating these proteins in vitro, in the prototypical manner that is illustrated by Nup62.

The results presented here show that the three isoforms of OGT are distinct in their ability to glycosylate a subset of target proteins within the cell and that this occurs because of the number of TPR repeats in the _N_-terminal region of the enzyme. Production of active OGT isoforms in vitro allows any number of substrates to be tested and for the specificity of each substrate to be determined. Previous in vitro studies did not recognize multiple OGT isoforms and have only utilized mOGT as a source of enzyme. Thus, many substrates for ncOGT (and possibly sOGT) may have been overlooked. It would therefore be of interest to use these different OGT isoforms in a proteomics screen to determine the substrates for each isoform. Although many substrates for OGT have been identified, the function of these modifications has been elusive, and understanding the basis and role of substrate selectivity may eventually help to elucidate the roles of each isoform in many cellular processes.

Materials and methods

Subcloning OGT isoforms into the pET43.1 expression vector

Human ncOGT was amplified by polymerase chain reaction (PCR) from the ncOGT Gateway vector (Invitrogen, Carlsbad, CA), whereas human mOGT and sOGT were amplified by PCR from an existing mOGT construct (Lubas and Hanover, 2000). Each PCR product was then subcloned into the pET43.1(Ek/LIC) vector, following the manufacturer’s instructions (Novagen, San Diego, CA) and confirmed by direct sequencing.

Production of recombinant OGT isoforms using an E. coli protein expression system

The pET43.1(Ek/LIC) expression vectors containing ncOGT, mOGT, and sOGT were transformed into competent BL21(DE3) cells (Novagen). As previously described (Lubas and Hanover, 2000), cells were grown overnight at room temperature with vigorous shaking, in LB media (KD Medical, Columbia, MD) supplemented with 50 µg/mL of ampicillin. Cells were then centrifuged at 3000 rpm (2060 × g) for 10 min in a Beckman GS-6R centrifuge, and the pellet was resuspended in 1/20 of the original volume of lysis buffer containing 20 mM Tris–HCl, pH 7.5, 2 mM EDTA, 1 mg/mL of lysozyme, 0.1% Triton X, and complete mini EDTA-free protease inhibitor cocktail tablet (Roche, Basel, Switzerland). The lysozyme digestion was performed at room temperature for 5 min. The lysate was then subjected to freeze/thaw and sonication on ice (3 × 10 sec) until DNA was sheared. The supernatant obtained after centrifugation at 14,000 × g for 10 min was frozen in aliquots at –70°C.

Total protein levels in the supernatant were determined using the bicinchoninic acid (BCA) protein assay protocol as described by the manufacturer (Pierce Biotechnology, Rockford, IL). Expressed His-tag fusion proteins in the supernatant were detected by immunoblotting with an anti-His-tag antibody (AbCam, Cambridge, UK). OGT concentrations were initially determined by Ponceau S staining of western blots. Image J software was used to determine the level of OGT protein compared with total protein levels determined by BCA. More recent experiments used fast green staining of nitrocellulose, followed by quantitation by infrared imaging using an Odyssey western blot scanner.

O-GlcNAc transferase assay

As described previously (Lubas and Hanover, 2000), bacterial extracts containing human recombinant ncOGT, mOGT, and sOGT were added to a 40 µL reaction mixture containing 50 mM Tris–HCl, pH 7.5, 1 mM dithiothreitol, 12.5 mM MgCl2, 0.2 nCi UDP-[14C]-GlcNAc (American Radiolabeled Chemicals, St. Louis, MD), and 1 µg of recombinant, purified Nup62, prepared as described previously (Lubas et al., 1995). Other substrates utilized in these reactions included CKII, tau, yes kinase, α-synuclein, and β-synuclein. Reactions were incubated for 30 min at 37°C with shaking and were stopped by the addition of 4× SDS–PAGE sample buffer (Invitrogen) and by boiling for 3 min. SDS–PAGE was performed using precast 4–12% NuPage gels (Invitrogen), followed by staining with Simply Blue Safestain (Invitrogen) for 60 min and by destaining with distilled water for 60 min. Glycosylation of Nup62 with [14C]-GlcNAc was visualized after treatment with En3Hance (Perkin Elmer, Wellesley, MA) for 60 min by exposure to a phosphoimage screen, which was then developed on the Fujifilm BAS-1500 phosphoimager. Densitometry of the phosphoimage data was performed with Image Gauge 3.0 software. Stoichiometry of glycosylated substrate proteins was determined using phosphoimage quantitation against a C14-BSA internal control.

Human CKII (α2β2, a = 44 kDa, b = 26 kDa), human tau (60 kDa), human yes kinase (yes, 64 kDa), and human α-synuclein (14.46 kDa) were purchased as purified recombinant proteins from Calbiochem (La Jolla, CA). Purified human recombinant β-synuclein (14.3 kDa) was a kind gift from Dr Nelson Cole.

Glycosylation and purification of sOGT

E. coli lysate containing soluble sOGT was incubated with C14-labeled UDP-GlcNAc and assay buffer, as above, for 30 min. The reaction was then treated in one of two ways. It was either electrophoresed or was incubated with NiNTA agarose beads (Invitrogen). Reactions were bound to the NiNTA beads for 30 min at 4°C, lightly spun, and the supernatant removed for analysis. The sOGT-bound NiNTA beads were washed following the manufacturer’s instructions, and then sOGT was eluted with 250 mM imidazole and collected for analysis.

Conflict of interest statement

None declared.

Acknowledgments

This research was supported (in part) by the Intramural Research Program of the NIH, NIDDK.

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