Functional Expression of O-linked GlcNAc Transferase

O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. InArabidopsis, the OGT homolog participates in the gibberellin signaling pathway. We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT inEscherichia coli. Kinetic parameters for the purified recombinant enzyme (K m = 1.2 μm for Nup 62; K m = 0.5 μm for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conserved C terminus result in a complete loss of activity. The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates. Removal of the first three tetratricopeptide repeats greatly reduces theO-GlcNAc addition to macromolecular substrates. However, this altered enzyme retains full activity against appropriate synthetic peptides. Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade. Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis. O-GlcNAc transferase (OGT) modifies nuclear pore proteins and transcription factors. InArabidopsis, the OGT homolog participates in the gibberellin signaling pathway. We and others have proposed that mammalian OGT is the terminal step in a glucose-sensitive signal transduction pathway that becomes disregulated in insulin resistance. To facilitate mutational analysis of OGT in the absence of competing endogenous activity, we expressed the 103-kDa human OGT inEscherichia coli. Kinetic parameters for the purified recombinant enzyme (K m = 1.2 μm for Nup 62; K m = 0.5 μm for UDP-GlcNAc) are nearly identical to purified mammalian OGT. Deletions in the highly conserved C terminus result in a complete loss of activity. The N-terminal tetratricopeptide repeat domain is required for optimal recognition of substrates. Removal of the first three tetratricopeptide repeats greatly reduces theO-GlcNAc addition to macromolecular substrates. However, this altered enzyme retains full activity against appropriate synthetic peptides. Autoglycosylation of OGT is augmented when the first six tetratricopeptide repeats are removed showing that these repeats are not required for catalysis. Given its proposed role in modulating insulin action, OGT may modify kinases involved in this signaling cascade. Among the many kinases tested, OGT glycosylates glycogen synthase kinase-3 and casein kinase II, two enzymes critical in the regulation of glycogen synthesis. O-GlcNAc transferase (uridine diphospho-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyl transferase) tetratricopeptide repeat base pair polyacrylamide gel electrophoresis high pressure liquid chromatography glycogen synthase kinase 4-morpholineethanesulfonic acid One ubiquitous form of cytoplasmic and nuclear glycosylation is the addition of a single N-acetylglucosamine inO-glycosidic linkage to serine or threonine residues (1.Haltiwanger R.S. Busby S. Grove K. Li S. Mason D. Medina L. Moloney D. Philipsberg G. Scartozzi R. Biochem. Biophys. Res. Com. 1997; 231: 237-242Crossref PubMed Scopus (95) Google Scholar).O-GlcNAc-modified proteins are typically found in large macromolecular complexes suggesting a role in protein-protein interactions. O-GlcNAc addition is a dynamic process; the half-life of O-GlcNAc residues is significantly shorter than the half-lives of the protein modified (1.Haltiwanger R.S. Busby S. Grove K. Li S. Mason D. Medina L. Moloney D. Philipsberg G. Scartozzi R. Biochem. Biophys. Res. Com. 1997; 231: 237-242Crossref PubMed Scopus (95) Google Scholar, 2.Chou C.F. Smith A.J. Omary M.B. J. Biol. Chem. 1992; 267: 3901-3906Abstract Full Text PDF PubMed Google Scholar, 3.Roquemore E.P. Chevrier M.R. Cotter R.J. Hart G.W. Biochemistry. 1996; 35: 3578-3586Crossref PubMed Scopus (151) Google Scholar). Changes in cell physiology may be accompanied by transient and dramatic alteration inO-GlcNAc levels (4.Kearse K.P. Hart G.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 1701-1705Crossref PubMed Scopus (192) Google Scholar). Such rapid changes have led to proposals that post-translational modification by O-GlcNAc may be analogous to protein phosphorylation. Indeed, in most cases, proteins bearing O-GlcNAc are also found to be phosphorylated (1.Haltiwanger R.S. Busby S. Grove K. Li S. Mason D. Medina L. Moloney D. Philipsberg G. Scartozzi R. Biochem. Biophys. Res. Com. 1997; 231: 237-242Crossref PubMed Scopus (95) Google Scholar, 5.Miller M.W. Caracciolo M.R. Berlin W.K. Hanover J.A. Arch. Biochem. Biophys. 1999; 367: 51-60Crossref PubMed Scopus (71) Google Scholar). The relationship between O-GlcNAc and phosphorylation is obviously complex and may be a key to understanding the function of O-GlcNAc addition. Another clue to understanding the role of O-GlcNAc in cell physiology derives from in vitro studies on the role of the hexosamine biosynthetic pathway in insulin resistance. The hexosamine biosynthetic pathway mediates the metabolic interconversion of intracellular glucose to UDP-GlcNAc, the substrate for OGT. The work of a number of investigators has implicated the hexosamine biosynthetic pathway in insulin resistance (6.Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar, 7.Hebert L.F. Daniels M.C. Zhou J.X. Crook E.D. Turner R.L. Simmons S.T. Neidigh J.L. Zhu J.S. Baron A.D. McClain D.A. J. Clin. Invest. 1996; 98: 930-936Crossref PubMed Scopus (279) Google Scholar, 8.McClain D.A. Crook E.D. Diabetes. 1996; 45: 1003-1009Crossref PubMed Google Scholar, 9.Hawkins M. Barzilai N. Liu R. Hu M.Z. Chen W. Rossetti L. J. Clin. Invest. 1997; 99: 2173-2182Crossref PubMed Scopus (268) Google Scholar). Because OGT uses UDP-GlcNAc as a substrate, it is likely that the OGT participates in some aspect of mammalian insulin resistance (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 11.Hanover J.A. Zhennan L. Lee G. Lubas W.A. Sato S.M. Arch. Biochem. Biophys. 1999; 362: 38-45Crossref PubMed Scopus (114) Google Scholar). A final clue was the identification of a homolog of OGT in the plantArabidopsis. This protein (spindly) corresponds to the protein product of the spy locus responsible for regulating the gibberellin signaling pathway (12.Jacobsen S.E. Binkowski K.A. Olszewski N.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9292-9296Crossref PubMed Scopus (277) Google Scholar). Mutations at this locus yield a phenotype exhibiting constitutive activation of the gibberellin pathway (13.Ogas J. Curr. Biol. 1998; 8: R165-R167Abstract Full Text Full Text PDF PubMed Google Scholar). Although similar mutations have yet to be identified in mammalian OGT, this enzyme is also likely to serve a signaling role. The enzyme responsible for O-linked GlcNAc addition, UDP-N-acetylglucosamine:polypeptide β-N-acetylglucosaminyl transferase (O-GlcNAc transferase, OGT)1 was initially isolated from rat liver as a heterotrimer of 350 kDa, containing two 110-kDa subunits and one 78-kDa subunit (14.Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar). In rabbit red blood cells, however, only the 110-kDa subunit was routinely observed (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). A polyclonal antibody to the 110-kDa rat enzyme recognized both the 110- and 78-kDa subunits by Western blot analysis (15.Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 272: 9308-9315Abstract Full Text Full Text PDF Scopus (607) Google Scholar), suggesting that both proteins had common peptide epitopes. The larger subunit has recently been cloned from humans, nematodes (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar), and rats (15.Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 272: 9308-9315Abstract Full Text Full Text PDF Scopus (607) Google Scholar). Overexpression of the human cDNA, encoding the 103-kDa subunit, in HeLa cells increased O-GlcNAc transferase activity. However, because mammalian cells have endogenous activity, the possibility that the cloned protein was indirectly stimulating endogenous activity could not be rigorously excluded (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, 15.Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 272: 9308-9315Abstract Full Text Full Text PDF Scopus (607) Google Scholar). Here we show that expression of the human OGT cDNA in Escherichia coli, an organism that has no endogenous activity, results in an active enzyme capable of modifying recombinant substrates in vitro. This argues that other subunits are not essential for enzyme activity, although other regulatory subunits may exist in vivo. The cDNAs from humans, rats, and nematodes encode for proteins of molecular mass 103, 116, and 128 kDa, respectively. There are 13 tandem tetratricopeptide repeats (TPRs) in the nematode protein compared with 11 in rat and 9 in human. The TPR is a 34-amino acid repeat containing the 8 following loosely conserved residues: W-L-G-Y-A-F-A-P (16.Lamb J.R. Tugendreich S. Hieter P. Trends. Biochem. Sci. 1995; 20: 257-259Abstract Full Text PDF PubMed Scopus (552) Google Scholar, 17.Das A.K. Cohen P.T.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (710) Google Scholar). A recent crystal structure suggests that each degenerate repeat forms a pair of antiparallel α-helices resulting in a superhelical structure believed to be the site of protein-protein interaction (17.Das A.K. Cohen P.T.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (710) Google Scholar). Here we show that the TPR domain in human OGT is important for glycosylation of proteins such as Nup 62 and casein kinase II as well as peptides such as YSDSPSTST. Yet the first six TPRs are not absolutely required for catalytic activity. The recombinant enzyme is also shown to be capable of autoglycosylation, and removal of the first six TPRs increases this activity. The number of proteins known to be modified by OGT is steadily increasing since it was identified on cytoplasmic and nuclear proteins (18.Holt G.D. Snow C.M. Senior A. Haltiwanger R.S. Gerace L. Hart G.W. J. Cell Biol. 1987; 104: 1157-1164Crossref PubMed Scopus (313) Google Scholar, 19.Hanover J.A. Cohen C.K. Willingham R.S. Park M.K. J. Biol. Chem. 1987; 262: 9887-9894Abstract Full Text PDF PubMed Google Scholar). In this study we show that two enzymes that play a key role in glycogen metabolism, glycogen synthase kinase-3 and casein kinase II, can be modified with recombinant OGT in vitro. These data are consistent with a proposed role of OGT in modulating insulin resistance in mammals. Human OGT, clone Lv4F, (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar) was subcloned into pET 32B (Novagen) using the EcoRV and NotI restrictions sites to make clone A. Clone B was isolated by excising the 441-bpBglII-BglII fragment from clone A and religating with T4 DNA ligase. Clone C was isolated by excising the 726-bpEcoRV-NsiI fragment and religating using a linker made from the following primers (p)ACAGGAAGCTCTGATGCA and (p)TCAGAGCTTCCTGT. Clone D was isolated by subcloning theEcoRI-NotI fragment from Lv4F into pET 32C (Novagen). Clone E was isolated by excising the 1819-bpAflII-NotI fragment from Clone A, filling in the 5′-overhangs with Klenow and using T4 DNA ligase for blunt-end ligation. Clone F was isolated by excising the 1212-bpEspI-EspI fragment from Clone A and religating with T4 DNA ligase. Clone G was isolated by excising the 341-bpXhoI-XhoI fragment from clone A and religating with T4 DNA ligase. All clones were sequenced through the cloning sites to confirm the coding sequence. Cultures of BL21 (DE3) cells containing the pET 32 human OGT clone expression vector were grown overnight at room temperature at 220 rpm in LB media (Digene) supplemented with carbenicillin (50 μg/ml). Cells were centrifuged at 3000 rpm for 5 min in a Beckman GS-6R centrifuge. The pellet was freeze-thawed and resuspended in 1/20 of the original volume in 1 mg/ml lysozyme, 20 mm Tris-HCl, pH 7.4, 2 mm EDTA, 1 mm phenylmethylsulfonyl fluoride, 1 μg/ml chymostatin, 1 μg/ml leupeptin, 1 μg/ml pepstatin, 0.1% Triton X-100. The lysozyme digestion was performed at 30 °C for 15 min. The lysate was sonicated on ice (2 × 10 s, setting 3, Misonix Ultrasonic Processor) until the DNA was The at for 10 min was in at proteins in the were purified on (Novagen) using the The rapid (Novagen) was to the yield of The (Novagen) was to the yield of were performed using OGT, clones in mm Tris-HCl, pH 7.4, 1 and mm as The macromolecular substrates were Nup 62 as W.A. Smith M. C.M. Hanover J.A. Biochemistry. 1995; Scopus Google Scholar) or casein kinase II The was at °C at 220 rpm for min. were by an volume of and for min. was performed using or were with 0.1% in and acid for min and overnight with with was with for 20 by using data were using the and OGT enzyme activity was also using or with Nup as (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar, W.A. Smith M. C.M. Hanover J.A. Biochemistry. 1995; Scopus Google Scholar). The peptide glycosylation was performed as (14.Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar) with The for a OGT peptide or 1 of mm Tris-HCl, pH 7.4, 1 mm and mm in a final volume of The was at °C at 220 rpm for 30 min and by the addition of of mm acid to the The pellet was with of and the was in mm The were with 8 of acid and with 2 of 0.5 m of into the peptides was using The of OGT to the in each was by Western blot analysis using the (Novagen). glycogen synthase kinase human casein kinase II containing both kDa, and kDa, rat casein kinase with a of the C terminus at protein kinase A catalytic protein kinase kinase and rat protein kinase II kDa, containing the first were as purified and recombinant proteins from We isolated human OGT cDNA using the (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar). Because has no transferase activity, this it an for expression and of the we the OGT cDNA into the pET 32 vector for expression in coli. OGT activity was isolated from the as shown in that OGT glycosylation is The enzyme not modify proteins 1 A, in Nup 62 is highly modified A, Because O-GlcNAc glycosylation can be observed in with OGT cDNA, it that this enzyme is for activity, and other mammalian subunits are not of OGT into the pET 32 vector a protein containing an N-terminal A protein can be highly purified in a single step using as shown in 1 with the recombinant OGT to Nup 62 in a enzyme and and data not The is by as had been for the mammalian enzyme (14.Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar). The purified recombinant enzyme a activity and parameters similar to the enzyme isolated from rabbit blood (10.Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar) and rat liver (14.Haltiwanger R.S. Blomberg M.A. Hart G.W. J. Biol. Chem. 1992; 267: 9005-9013Abstract Full Text PDF PubMed Google Scholar) These data that the addition of the N-terminal not significantly enzyme activity. In addition to Nup 62 we to OGT could modify other peptide substrates and A that OGT glycosylation is The enzyme can modify a peptide had been shown to be a substrate for the rat not modify similar peptide was shown not serve as an R.S. G.D. Hart G.W. J. Biol. Chem. Full Text PDF PubMed Google Scholar). Given this we of the kinases known to be involved in insulin signaling be substrates for OGT. B recombinant OGT can modify two mammalian casein kinase II and not protein kinase A, casein kinase protein kinase kinase or protein kinase casein kinase II and glycogen synthase and its activity. with the active recombinant of OGT were made as shown in A to the of OGT that are responsible for catalysis. Human OGT has only 9 tetratricopeptide repeats compared with has and has N-terminal the first repeats the first repeats or the TPR domain and three clones and G were as shown in 5 A. The recombinant mutations were purified using chromatography and with to confirm that the single were for each clone 5 of these was for the to modify Nup casein kinase II, and the peptide of the first tetratricopeptide repeats in clone B results in a loss of activity for Nup removal of the first tetratricopeptide repeats in clone C results in an complete loss of activity Nup 62 as a clones and had no activity suggesting that this of the protein is essential for catalysis. This is consistent with the high of between human and rat cDNA in the There is at the protein in the of rat and human OGT. kinase II, was a substrate than Nup to be modified only by the enzyme clone A. In the peptide was modified by clone B the first three tetratricopeptide suggesting the first three tetratricopeptide repeats were its these data that the tetratricopeptide repeats to the substrate of OGT. Although of the tetratricopeptide repeats are not essential for catalytic activity, to be for optimal glycosylation of O-GlcNAc transferase activity of kinase activity was as The activity was for each in a OGT activity was as The activity was for each In the glycosylation made by the forms of the enzyme 5 we that clone C We of clones A, and C in the absence of an substrate Although levels of A, and C were clones A and B had significantly levels of than clone C We also to terminal O-GlcNAc residues on the purified recombinant enzymes to OGT had modified in coli. clones A and B were highly modified in coli, this could were substrates for in the in vitro The however, that clone C also had the of coli. This that clones A and B are substrates for than clone these data that removal of the first TPRs with the recognition of Nup 62 as a substrate and is addition can as a regulatory modification involved in signal transduction In the OGT homolog is involved in of the gibberellin signaling pathway (12.Jacobsen S.E. Binkowski K.A. Olszewski N.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9292-9296Crossref PubMed Scopus (277) Google Scholar). a large number of nuclear and cytoplasmic proteins have been identified as substrates for OGT. of these proteins are involved in signal II and the transcription nuclear and the transcription modified are the such as large and the has been shown that glycosylation of in its regulatory domain its DNA activity P. J. R. R. 1996; Google Scholar), glycosylation of an peptide its to the S. J.R. Biol. 1997; 17: PubMed Scopus Google Scholar). In this we have shown that recombinant human OGT expressed in is active and capable of modifying recombinant proteins in vitro. This make it to the role that glycosylation in signal The to modify recombinant proteins make it to study the on protein-protein and in vitro. data that OGT glycosylation is We were to glycosylation of proteins isolated with recombinant OGT in the from coli. However, of OGT was Because active OGT can be isolated from coli, has no activity, is not likely to be essential for enzyme activity. However, may play an important role in the regulation of enzyme activity or substrate in vivo. We are a mutational analysis of OGT to residues required for catalytic activity and the substrate Nup 62 is as a substrate, it is recognized and modified by recombinant OGT with nearly identical to the The data we have that the TPR domain is required for the interaction with protein substrates such as Nup Although TPR a the in a domain are and may be involved in protein-protein interactions. In the of a protein responsible for glucose in only of the 10 TPRs are essential and of TPR have been shown to be responsible for of of D. K. 1995; PubMed Scopus Google Scholar). TPRs with the TPRs are involved in of TPRs to be required for of in the of the protein it that TPRs with the signal D. S. EMBO J. 1995; PubMed Scopus Google Scholar). on these we to tetratricopeptide repeats important for substrate data that optimal of Nup 62 and casein kinase II to the TPR Although the first tetratricopeptide repeats are not absolutely for glycosylation of Nup to to substrate In the first three tetratricopeptide repeats are not required for glycosylation of the peptide and in may the of the peptide to the catalytic removal of the first six tetratricopeptide repeats OGT glycosylation of yet this enzyme is active and suggesting that the first six tetratricopeptide repeats are not essential for catalysis. these data that the TPR domain is important in substrate and it be to OGT substrate by with TPR substrate This could a of glycosylation of substrates. OGT was shown to modify two purified recombinant kinases and casein kinase II, two enzymes critical in the regulation of glycogen synthesis. kinases in the such as protein kinase A, casein kinase protein kinase and protein kinase II were not found to be substrates for OGT in vitro. OGT glycosylation of kinases such as and casein kinase II suggests a for between glycosylation and the phosphorylation cascade. of these kinases are involved in glycogen the step in glycogen synthesis. kinase II was isolated as and phosphorylation of glycogen synthase by phosphorylation by casein kinase has been shown to both glycogen synthase and insulin Proc. Natl. Acad. Sci. U. S. A. 1997; PubMed Scopus Google Scholar). OGT may also be implicated in insulin resistance the hexosamine biosynthetic pathway. has been shown that can to increases in UDP-GlcNAc levels A. Daniels M.C. McClain D. W.K. 1998; Full Text PDF PubMed Scopus Google Scholar, J. Liu R. M. Barzilai N. Rossetti L. 1998; PubMed Scopus Google Scholar) and results in increased levels proteins A. Daniels M.C. McClain D. W.K. 1998; Full Text PDF PubMed Scopus Google Scholar, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). This is consistent with the K m of OGT in the and to dynamic changes in intracellular the K m for the enzyme UDP-GlcNAc is similar to that found for UDP-GlcNAc and in to the Biochem. 1987; PubMed Scopus Google Scholar). by to changes in cytoplasmic UDP-GlcNAc OGT glycosylation of cytoplasmic and nuclear proteins may as an intracellular glucose of transcription or proteins in to changes in intracellular glucose a for insulin resistance. final of this a the expression and of OGT in In with these data suggesting that the C terminus is likely to a catalytic domain L.K. Hart G.W. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). synthetic peptides as that OGT has three for the K m of Although of synthetic peptide substrates may some at the catalytic site of OGT, it is that synthetic peptides with K m are in the as protein substrates Nup 62 (K m = 1.2 is that larger protein substrates may with the TPR domain of OGT and glycosylation sites in to the catalytic site be This is similar to the for protein phosphorylation by kinases in the site is important for not phosphorylation In we to as an expression original that proteins expressed in the are by the endogenous OGT S.M. Berlin W.K. C.M. Hanover J.A. 1995; PubMed Scopus Google Scholar). of OGT in the the to the activity of of the recombinant Because an endogenous activity, the expression we have facilitate mutational analysis not in a with high levels of endogenous activity. in also for of large of recombinant required for the structure of OGT as well as We are these