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Biochemical and Biophysical Research Communications 288, 1136–1140 (2001)

doi:10.1006/bbrc.2001.5902, available online at http://www.idealibrary.com on

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urification of the O-Glycosylated Protein p135 anddentification as O-GlcNAc Transferase

obert J. Konrad,*,1 Joseph F. Tolar,† John E. Hale,‡ Michael D. Knierman,‡erald W. Becker,‡ and Jeffrey E. Kudlow†

Department of Diagnostic and Experimental Medicine and ‡Lilly Research Laboratories, Eli Lilly and Company,ndianapolis, Indiana 46285; and †Department of Medicine, University of Alabama at Birminghamchool of Medicine, Birmingham, Alabama 35233

eceived October 8, 2001

for protein glycosylation (8, 9). O-glycosylation occurswotvNz(

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We have previously shown that rat pancreatic isletsontain a predominant 135 kDa O-glycosylated proteinp135) that is recognized by immunoprecipitation and

estern blotting with anti-O-GlcNAc antibody. In thisaper, we show that p135 is also detectable in other ratissues including brain, heart, liver, spleen, and lung,ut not kidney. To identify p135, the protein was pu-ified from rat brain using a multistep procedure in-luding selective absorption with anti-O-GlcNAc anti-ody. After electrophoresis, and Coomassie staining,he protein was excised from the gel for tryptic diges-ion. Next, O-methylisourea was used to convert lysineesidues to homoarginine to increase the sequenceoverage, and MALDI-TOF mass spectrometry detec-ion was performed. MALDI-TOF identified p135 as rat-GlcNAc transferase (OGT), an identity confirmed byC/MS of individual peptides. The identification of135 as OGT is consistent with previous reports of theissue distribution of OGT, as well as reports that OGTs itself O-glycosylated. © 2001 Academic Press

Key Words: O-glycosylation; O-GlcNAc transferase;-GlcNAc; p135; auto-O-glycosylation.

We have previously shown that pancreatic islets con-ain a predominant 135 kDa O-glycosylated protein ofnknown identity (p135) (1–3). In the b-cell, as in otherell types, the pathway involving O-glycosylationtarts with the conversion of glucose to glucose-6-hosphate and then to fructose-6-phosphate. The en-yme glutamine: fructose-6-phosphate amidotrans-erase (GFAT) converts fructose-6-phosphate tolucosamine-6-phosphate (4–7), providing the UDP-N-cetylglucosamine (UDP-GlcNAc), which is necessary

1 To whom correspondence should be addressed at Department ofiagnostic and Experimental Medicine, Lilly Corporate Center, Eliilly and Company, Indianapolis, Indiana 46285. Fax: (317) 276-198. E-mail: konrad_robert@lilly.com.

1136006-291X/01 $35.00opyright © 2001 by Academic Pressll rights of reproduction in any form reserved.

hen O-GlcNAc transferase (OGT) catalyzes O-linkagef the monosaccharide N-acetylglucosamine (GlcNAc)o serine and/or threonine residues (10–17). Con-ersely, O-GlcNAc is removed by O-GlcNAc-selective-acetyl-b-D-glucosaminidase (O-GlcNAcase), the en-

yme which removes O-linked sugar from protein18, 19).

We have previously attempted to identify p135 usingsolated islets and clonal b-cell lines as a source ofrotein. Due to the limited amount of tissue that can bebtained from such sources, our prior attempts at p135dentification yielded insufficient amounts of proteinor sequencing. To circumvent these problems, we haveaken a two-step approach. First, we have shown that135 is also present in other rat tissues and have usedat brain as a tissue source in order to have access toreatly increased amounts of protein as a startingoint. Second, we have utilized a recently describedechnique to increase MALDI-TOF sequence coveragey converting lysine residues present in the p135 tryp-ic digest to homoarginines (20). Together, these twoodifications have allowed us to use MALDI-TOF to

dentify O-glycosylated p135 as O-GlcNAc transferaseOGT), a result that we have subsequently confirmedsing LC/MS mass spectrometry. This identification isonsistent with previous reports in the literature show-ng the tissue distribution and O-glycosylation of OGT.

ATERIALS AND METHODS

Islet isolation and analysis of islets and other primary tissues for-glycosylated p135. Islets were isolated aseptically from 3–4ale Sprague–Dawley rats as previously described (1–3). For-glycosylated p135 immunoprecipitation, 1 ml of ice-cold lysisuffer was added to each sample, and O-glycosylated p135 wasmmunoprecipitated with RL2 antibody as previously described (1–). This antibody binds O-glycosylated p135 (1), and immunoprecipi-ation of p135 by RL2 is specifically blocked by GlcNAc (1). Followingmmunoprecipitation, Western blotting was performed with theame antibody as previously described (1–3).

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Vol. 288, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Purification of p135 from rat brain. Three to four rat brainsere washed extensively, chopped, and manually homogenized in 20M pH 7.40 sodium phosphate buffer containing 10 mM MgCl2.rior to processing the tissue, the buffer was supplemented with 1M phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, and 1g/ml aprotinin. The homogenization buffer was kept continuouslytirring on ice. The ratio of tissue volume to buffer during homoge-ization was approximately 1:3. Afterward, rat brain homogenateas placed on ice for 30 min and centrifuged at 14,000g at 4°C forh. After centrifugation, the pellet was discarded, and the cytosolic-

ich supernatant placed on ice for 30 min. Ice-cold lysis buffer (50M Hepes, pH 7.40, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA,mM EGTA, 20 mM NaF, 20 mM Na4P2O7, 1 mM NaVO4, 1 mg/ml

acitracin, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/mleupeptin, 1 mg/ml aprotinin) was added to the supernatant in a ratiof 4:1. The cytosolic rich fraction in lysis buffer was centrifuged againt 14,000g at 4°C for 1 h, after which time the pellet was discardednd the supernatant transferred to a fresh 50-ml conical tube.The sample was precleared with 100 ml of protein A trisacryl beads

Pierce) for 1 h at 4°C. After initial pre-clearing, the sample wasentrifuged for 5 min at 2000g at 4°C. Supernatant was removed andransferred to a new 50 ml conical tube. The supernatant was pre-leared a second time with 200 ml of protein A trisacryl beads (Pierce)readsorbed with 1 mg of rabbit anti-mouse antibody (Sigma) for 2 ht 4°C. Following this second preclearing, O-glycosylated p135 wasmmunoprecipitated with 10 ml of RL2 antibody for 4 h at 4°C.fterward, 20 ml of protein A trisacryl beads preadsorbed with 100g of rabbit anti-mouse antibody were then added, and the incuba-ion was continued overnight at 4°C.

The following day, trisacryl beads were washed three times withash buffer 1 (150 mM NaCl, 10 mM Hepes, pH 7.40, 1% Triton-100, and 0.1% SDS) and twice with wash buffer 2 (10 mM Hepes,H 7.40, 1% Triton X-100, and 0.1% SDS) in order to remove anynbound protein from the beads. After the final washing step, beadsere transferred to a 1.5-ml conical microfuge tube and 25 ml of 23

ample buffer (100 mM Tris, pH 6.80, 4% SDS, 20% glycerol, and 20g/liter bromophenol blue) was added. The tube was vortexed for0 s, boiled for 5 min, vortexed for an additional 30 s, and the sampleun on a 1-mm-thick 7.5% SDS–PAGE gel, which had been prerun at75 V for 10 min in order to eliminate reactive groups in the poly-crylamide. Colored molecular weight markers (Amersham) wereun on a nonadjacent lane of the gel. Following electrophoresis for 1 ht 175 V, the gel was washed twice with distilled water, stained forh with Bio-Rad R-250 Coomassie stain, and detained for 3 h with

everal rinses of Bio-Rad destaining solution. After destaining, theel was again washed twice with distilled water. The Coomassie-tained band corresponding to p135 was cut out of the gel and storedt –20°C for subsequent analysis.

In-gel digestion of p135. A standard in-gel procedure was utilized21) and adapted for use on a Tecan robot. Protein bands visualizedith Coomassie blue were excised, cut int 3–4 pieces and destainedy extensive washing with 50% acetonitrile, 50 mM NH4HCO3. Pro-ein was reduced in the gel with 50 ml of 50 mM DTT in 50mMH4HCO3 for 60 min at 37°C. The DTT solution was removed and

FIG. 1. Tissue expression of p135. p135 from rat tissues wasmmunoprecipitated with RL2 anti-O-GlcNAc antibody, separated,nd transferred to nitrocellulose for subsequent Western blottingith the same antibody. A predominant 135 kDa O-glycosylatedrotein that comigrates with islet p135 was detected in brain, heart,iver, lung, and spleen, but not kidney.

1137

or 15 min at 37°C. The alkylating solution was removed, gel piecesere washed once with 100 mM NH4HCO3 then with acetonitrile,nd dried by placing the gel pieces at 37°C for 10 min. Dried gelieces were swollen in 10 ml of trypsin (33 mg/ml) in 50 mMH4HCO3, pH 8.0, and digested overnight at 37°C. After digestioneptides were extracted and modified with 1 M O-methylisourea,100M Na2CO3 (adjusted to pH 10 with 5 N NaOH) for 1 h at 37°C.eptide extracts were desalted on C-18 Zip tips (Millipore, Bedford,A) by washing with 0.1% trifluoroacetic acid and eluting with 6 ml

f 50% acetonitrile, 0.05% trifluoroacetic acid.

MALDI-TOF mass spectrometry. MALDI-TOF spectra were ob-ained on a Perceptive Biosystems Voyager DE Pro mass spectrom-ter. An aliquot (1 mL) of the desalted tryptic peptide mixture pre-ared above was mixed with matrix (a-cyano 4-hydroxy cinnamiccid) on the sample target and allowed to dry before collecting data.he instrument was calibrated with a mixture of angiotensin I,ubstance P, and ACTH (Sigma). Internal calibration was achievedsing the trypsin autolysis products with protonated masses of 842.5nd 2253.12 (modified with O-methylisourea).

LC/MS/MS analysis of p135 tryptic peptides. The remaining 5 mlf desalted tryptic peptides were reduced in volume to approximatelyml with a stream of helium. Six microliters of DI water were added

o the remaining solution and injected into a capillary HPLC at-ached to a Finnigan LCQ deca ion trap mass spectrometer. ThePLC system consisted of a 300 mm 3 150 mm PepMap C18 column

LC Packings) run at 2–3 ml/min. Peptides were loaded at 5% aceto-itrile 0.1% formic acid and eluted with a linear gradient from 5% to0% acetonitrile, 0.1% formic acid in 30 min. The column effluentas interfaced to the LCQ deca with a custom nanospray source that

ncorporated a sheath gas of nitrogen to assist in the electrospray.he LCQ deca was set to scan from 300 to 2000 amu and take a zoomcan and MS/MS spectra on any ion above 5e5 units in intensity.S/MS was performed with wideband activation, and isolation withamu, and relative collision energy of 45. Dynamic exclusion was

nabled for 2 min at a width of 2 amu.

Database searching. MALDI-TOF mass spectra were searchedgainst a nonredundant protein sequence database available from

FIG. 2. MALDI-TOF spectra of a tryptic digest of p135. Aoomassie-stained gel band containing rat brain p135 was excised

rom the gel, digested with trypsin, and chemically modified asescribed under Materials and Methods prior to analysis by MALDI-OF mass spectrometry. Ions from the spectrum were searchedgainst a nonredundant protein sequence database, and an excellentatch to rat OGT was found (Table 1). The spectrum is labeled with

hose ions whose m/z values correspond to tryptic peptides of OGT.

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Vol. 288, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

he National Center for Biotechnology Information using the pro-ram Knexus (Proteometrix). Peptide masses were adjusted to ac-ount for the modification of lysine residues by O-methylisourea142.06). Methionine oxidation and N-terminal conversion of glu-amine to pyroglutamate were considered as common modificationsuring the search. MS/MS data were searched against the sameonredundant protein sequence database using the program SequestFinnigan). The same peptide modifications considered above werelso allowed in these searches.

FIG. 3. Confirmation of peptide identities by MS/MS. An LC/Methods. Two of the spectra obtained are shown here. (A) MS/MS sp

he b and y ions that match peptide GQLQEAIEHYR, corresponding93.3 ion. The spectrum is labeled with the b and y ions that match

1138

ESULTS

Figure 1 shows results from an experiment in which-glycosylated proteins from various rat tissues were

mmunoprecipitated with RL2 anti-O-GlcNAc antibodynd analyzed by subsequent Western blotting with theame antibody. As the figure shows, rat brain, heart,

MS experiment was carried out as detailed under Materials andrum of the doubly charged 672.3 ion. The spectrum is labeled withresidues 93–103 of OGT. (B) MS/MS spectra of the double-chargedptide EYQAGDFEAAER corresponding to residues 21–32 of OGT.

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Vol. 288, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

iver, lung, and spleen contained a predominant 135Da O-glycosylated protein that comigrated with p135rom rat islets. In contrast, no p135 band was detectedn kidney. In light of these data, we used rat brain as aissue source from which to purify p135 for subsequentdentification.

Following purification of p135, the Coomassie-tained band was excised and processed as describednder Materials and Methods. MALDI-MS analysisf the extracted, modified peptides yielded a spec-rum with a number of intense peaks (Fig. 2). Aearch of the masses of these peaks (all peaks with aignal to noise ratio above 3) against the NCBI non-edundant mammalian protein database usingNEXUS software resulted in a match with highrobability to rat N-acetylglucosamine-peptide N-acetyl-lucosaminyltransferase (O-GlcNAc transferase, OGT).pproximately 30% of the protein sequence was coveredy the 57 peptides from the spectrum that matched.While this match was very convincing, we confirmed

his identification by injecting a portion of the peptideigest onto a capillary C-18 column attached to an ionrap mass spectrometer. Peptides that were elutedith an acetonitrile gradient were subjected to frag-entation in the ion trap and the resultant MS/MS

pectra were searched against the NCBI nonredundantatabase. This analysis resulted in several high quality

Summary of Mass Spectrometr

From–toExperimental

massTheoretical

mass Di

38–644 861.46 861.4790–898 1012.60 1012.6124–732 1025.60 1025.6233–39 1029.46 1029.4692–1000 1047.57 1047.5773–981 1092.56 1092.5864–73 1115.59 1115.6133–742 1145.58 1145.6179–489 1269.72 1269.7393–103 1342.66 1342.6621–32 1384.60 1384.59 231–443 1391.66 1391.6797–311 1654.87 1654.73 212–327 1821.86 1821.9280–396 1870.79 1870.8945–961 1887.91 1887.9974–90 1934.93 1934.9911–430 2159.08 2159.1240–59 2326.21 2326.17 275–294 2326.21 2326.15 223–842 2463.19 2463.15 268–889 2471.11 2471.243–366 2596.11 2596.34

Note. The experimental mass values are read directly from the Malculated for tryptic peptides of OGT and the corresponding sequencasses is shown. Three ions were selected for confirmatory LC/MS/

1139

S/MS spectra that matched peptides in N-acetyl-lucosamine-peptide N-acetylglucosaminyltransferase.wo of these spectra are shown in Fig. 3 and demonstratehe high confidence in assignment of the identity of therotein. All of the peptides identified by MALDI andS/MS analysis are summarized in Table 1.

ISCUSSION

The results above identify rat brain O-glycosylated135 as O-GlcNAc transferase OGT. The fact that rat-GlcNAc transferase has a predicted moleculareight of 119.03 but should run at 135 kDa on anDS–PAGE reducing gel is not unexpected in light ofrevious reports that human OGT, with a predictedW of 103 kDa, runs between 110–116 kDa on an

DS–PAGE gel (5–7). In addition, the fact that therotein was purified based on its recognition by annti-O-GlcNAc antibody indicates that it is posttrans-ationally modified by at least one O-GlcNAc, and this

ay also explain why it should run at a higher molec-lar weight than its predicted value.Our demonstration that OGT is itself a substrate for

uto-O-glycosylation is consistent with previous evi-ence in the literature showing that O-GlcNAc trans-erase itself is O-glycosylated (6). Recently, Hanovernd colleagues demonstrated that when the first six

Identification of p135 as OGT

ence LC/MS/MS Sequence

01 X NELFALR01 IIFSPVAPK02 IVLNGIDLK00 HCMQLW00 ISSPLFNTK02 LGTDLEYLK02 SAHFSTLAIK03 AFLDSLPDVK01 LVSIVAEQLEK00 X GQLQEAIEHYR01 X EYQAGDFEAAE01 X DSGNIPEAIASYR14 GSVAEAEDCYNTAL06 LCPTHADSLNNLANIK10 ISPTFADAYSNMGNTL08 VAASQLTCLGCLELIAK06 QNPLLAEAYSNLGNVY04 AIQINPAFADAHSNLASIHK04 QEPDNTGVLLLLSSIHFQC06 AIELQPHFPDAYCNLANAL04 SQYGLPEDAIVYCNFNQLY09 FPAVGEPNIQQYAQNMGLPQ23 ALEVFPEFAAAHSNLASVLQQQ

DI-TOF spectrum shown in Fig. 2. The theoretical mass values areindicated. The difference between the experimental and theoreticalanalysis as shown below.

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Vol. 288, No. 5, 2001 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

utoglycosylation of the enzyme is augmented (6).hese observations of OGT autoglycosylation are inter-sting in light of prior observations by Hart and co-orkers that OGT is exquisitely regulated the concen-

ration of intracellular UDP-GlcNAc (7).Based on our data, it is likely that rat islet p135 is

lso O-GlcNAc transferase. This conclusion is sup-orted by the tissue distribution of p135, which wasetected in islets, brain, heart, liver, lung, and spleen,ut not the kidney. This pattern is very similar to whatas been previously reported in the literature withegard to OGT. Hart and colleagues have demon-trated that OGT was virtually undetectable in theidney, yet was detectable in all other tissues analyzed9). Likewise, Hanover and colleagues have demon-trated by Northern blot analysis of human tissueshat OGT mRNA is also virtually undetectable in theidney, in contrast to other tissues examined (10).In conclusion, our results demonstrate that rat p135

s an O-glycosylated protein detected in islets, brain,eart, liver, lung, and spleen, but not kidney, and

dentify p135 as OGT. This identification of p135 asGT is consistent with previous literature showingGT’s tissue distribution as well as its O-glycosylation

6, 9, 10).

EFERENCES

1. Konrad R. J., Janowski, K. M., and Kudlow, J. E. (2000) Glucoseand streptozotocin stimulate p135 O-glycosylation in panreaticislets. Biochem. Biophys. Res. Commun. 267, 26–32.

2. Konrad, R. J., Liu, K., and Kudlow, J. E. (2000) A modifiedmethod of islet isolation preserves the ability of b-cells to regu-late O-linked protein glycosylation in response to glucose andstreptozotocin. Arch. Biochem. Biophys. 381, 92–98.

3. Konrad, R. J., Mikolaenko, I., Tolar, J. F., Liu, K., and Kudlow,J. E. (2001) The potential mechanism of the diabetogenic actionof streptozotocin: Inhibition of pancreatic b-cell O-GlcNAc-selective N-acetyl-b-D-glucosaminidase. Biochem. J. 356, 31–41.

4. McKnight, G. U., Mudd, S. L., Mathewes, S. L., Traxinger, R. R.,Marshall, S., Sheppard, P. O., and O’Hara, P. J. (1992) Molecularcloning, CDNA sequence, and bacterial expression of humanglutamine fructose-6-phosphate amidotransferase. J. Biol.Chem. 267, 25208–25212.

5. Sayeski, P. P., Wang, D., Su, K., Han, I.-O., and Kudlow, J. E.(1997) Cloning and partial characterization of the mouseglutamine:fructose-6-phosphate amidotransferase (GFAT) genepromoter. Nucleic Acids Res. 25, 1458–1466.

6. Sayeski, P. P., Paterson, A. J., and Kudlow, J. E. (1994) The

1140

encoding cDNA sequence. Gene 140, 289 –290.7. Kornfeld, R. (1967) Studies on L-glutamine D-fructose 6-phos-

phate amidotransferase: Feedback inhibition by uridine diphos-phate-N-acetylglucosamine. J. Biol. Chem. 242, 3135–3141.

8. Hart, G. W. (1997) Dynamic O-GIcNAcylation of nuclear andcytoskeletal proteins. Annu. Rev. Biochem. 66, 315–335.

9. Kreppel L. K., Blomberg, M. A., and Hart, G. W. (1997) Dynamicglycosylation of nuclear and cytosolic proteins. Cloning and char-acterization of a unique O-GIcNAc transferase with multipletetratricopeptide repeats. J. Biol. Chem. 272, 9308–9315.

0. Lubas, W. A., Frank, D. W., Krause, M., and Hanover, J. A.(1997) O-Linked GIcNAc transferase is a conserved nucleocyto-plasmic protein containing tetratricopeptide repeats. J. Biol.Chem. 272, 9316–9324.

1. Lubas, W. A., and Hanover, J. A. (2000) Functional expression ofO-linked GlcNAc transferase. Domain structure and substratespecificity. J. Biol. Chem. 275, 10983–10988.

2. Shafi, R., Iyer, S. P., Ellies, L. G., O’Donnell, N., Marek, K. W.,Chui, D., Hart, G. W., and Marth, J. D. (2000) The O-GlcNActransferase gene resides on the X chromosome and is essentialfor embryonic stem cell viability and mouse ontogeny. Proc. Natl.Acad. Sci. USA 23; 97, 5735–5739.

3. Kreppel, L. K., and Hart, G. W. (1999) Regulation of a cytosolicand nuclear O-GlcNAc transferase. Role of tetratricopeptide re-peats J. Biol. Chem. 274, 32015–3222.

4. Hanover, J. A., Lai, Z., Le G., Lubas, W. A., and Sato, S. M.(1999) Elevated O-linked N-acetylglucosamine metabolism inpancreatic b-cells. Arch. Biochem Biophys. 367, 51–60.

5. Roos, M. D., Wen, X., Kaihong, S., Clark, J. A., Xiaoyong, Y.,Chin, E., Paterson, A. J., and Kudlow, J. E. (1998) Streptozo-tocin, an analog of N-acetylglucosamine, blocks the removal ofO-GlcNAc from intracellular proteins. Proc. Assoc. Am Phys.110, 1–11.

6. Liu, K., Paterson, A. J., Chin, E., and Kudlow, J. E. (2000)Glucose stimulates protein modification by O-linked GlcNAc inpancreatic b-cells: Linkage of O-linked GlcNAc to b-cell death.Proc. Natl. Acad. Sci. USA 97, 2820–2855.

7. Akimoto, Y., Kreppel, L. K., Hirano, H., and Hart, G. W. (1999)Localization of the O-linked N-acetylglucosamine transferase inrat pancreas. Diabetes 48, 2407–2413.

8. Dong, D. L., and Hart, G. W. (1994) Purification and character-ization of an O-GIcNAc selective N-acetyl-b-D-glucosaminidasefrom rat spleen cytosol. J. Biol. Chem. 269, 19321–19330.

9. Gao, Y., Wells, L., Comer, F. I., Parker, G. J., and Hart, G. W.(2001) Dynamic O-glycosylation of nuclear and cytosolic pro-teins: Cloning and characterization of a neutral, cytosolic b-N-acetylglucosaminidase from human brain J. Biol. Chem. 276,9838–9845.

0. Hale, J. E., Butler, J. P., Knierman, M. D., and Becker, G. W.(2000) Increased sensitivity of tryptic peptide detection byMALDI-TOF mass spectrometry is achieved by conversion oflysine to homoarginine Anal. Biochem. 287, 110–117.