THE JOURNAL OF BIOLOGICAL CHEMISTRY Q 1993 by The American Society for Biochemistry and MolecUtar Biolw, Ine.

Vol. 268, No. 20, Issue of July 16, pp. 14687-14693,1993 Printed in U.S.A.

Characterization of Rabbit Skeletal Muscle Glycogenin TYROSINE 194 IS ESSENTIAL FOR FUNCTION*

(Received for publication, January 19,1993, and in revised form, March 23,1993)

Youjia Cao, Alan M. ~ a h r e n h o l z ~ # , Anna A. DePaoli-Roach, and Peter J. Roachtl From the Department of Biochemistry and Molecular Biology, Indiana University School of Medicine, Indianapolis, Indiana 46202-5122 and $Division of Immunology, Beckman Research Institute of the City of Hope, Dwrrte, California 91010-0269

The biogenesis of glycogen involves a specific initi- ation event mediated by the initiator protein, glyco- genin, which undergoes self-glucosylation to generate an oligosaccharide primer from which the glycogen molecule grows. Rabbit muscle glycogenin was ex- pressed at high levels in Escherichia coli and purified close to homogeneity in a procedure that involved bind- ing to a UDP-agarose affinity column. The resulting protein had subunit molecular weight of 38,000 as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Analysis of peptide fragments by mass spectroscopy indicated that the re- combinant glycogenin was already glucosylated at Tyr-194 and contained from 1 to 8 glucose residues attached. The enzyme was active as a glucosyl trans- ferase and could incorporate a further -5 mol of glu- cose/mol. The apparent K,,, for the glucosyl donor UDP- glucose was 4.5 PM, and the pH optimum was pH 8. Of a number of nucleotides and related compounds sur- veyed, UDP and UTP were the most effective inhibi- tors. There was also a correlation between inhibition and the presence of a pyrophosphate group. Of several oligosaccharides of glucose, only maltose caused sig- nificant inhibition. The glucosylation reaction was first order with respect to glycogenin suggesting that it was intramolecular. The efficacy of the purified glycogenin as a substrate for the elongation reaction catalyzed by glycogen synthase was significantly en- hanced if glycogenin was first allowed to undergo self- glucosylation. The length of the priming oligosaccha- ride is thus important for glycogen synthase action. A mutant of glycogenin, in which Tyr-194 was changed to Phe, behaved identically to the wild-type through purification and in particular bound to the UDP-aga- rose affinity matrix. Despite these indications of the protein’s overall structural integrity, it was unable to self-glucosylate. This result indicates that Tyr-194 is neceesary for glycogenin function and is consistent with Tyr-194 being the sole site of glucosylation.

Glycogen exists in many cell types as a major storage form of glucose. Krisman and Barrengo (1) had first postulated that the de mu0 synthesis of glycogen required an initiating protein factor that was later termed glycogenin (2). Glyco-

* This work was supported in part by National Institutes of Health Grant DK27221. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Lafayette, IN 47907-1153. I Present address: Dept. of Biochemistry, Purdue University, West

TTo whom correspondence should be addressed. “el.: 317-274- 7151; Fax: 317-272-4686.

genin was found not simply to act as a protein backbone but also to be itself an enzyme that catalyzed its own glucosylation (3,4). A model for glycogen biogenesis thus evolved in which glycogenin would grow a priming oligosacch~ide that could then serve as the substrate for elongation by glycogen syn- thase (5,6). Intervention also by the branching enzyme would lead to mature glycogen molecules. Pitcher et ~ l . (7) identified glycogenin as a well known -40-kDa contaminant of rabbit muscle glycogen synthase preparations and proposed that glycogenin existed in a 1:1 stoichiometric complex with the glycogen synthase subunit (8). The complete amino acid se- quence of the rabbit muscle protein was determined by Camp- bell and Cohen (9) and this matched almost exactly the sequence predicted from a cDNA isolated by Viskupic et al. (10). Glucosylation was reported to occur on Tyr residues (11, 12), and Smythe et ~ l . (13) identified Tyr-194 as a site of glucose attachment via a glucose-1-0-tyrosyl linkage. It is not known, however, if Tyr-194 is the only site of glucosylation and whether Tyr-194 is essential for glycogenin function.

A key question in the study of glycogen initiation has been the mechanism by which the glucose is initially attached to Tyr-194. Is there a separate glucosylating enzyme or does glycogenin itself mediate this reaction? If there is a separate enzyme, one would predict that u n m ~ f i e d glycogenin would not be competent for self-glucosylation. Glycogenin isolated from tissues already contains covalently attached glucose, and so it has not been easy to address this issue (14-16). When we succeeded in isolating a cDNA encoding glycogenin, we had hoped that the question could be resolved by expressing m a m m a l i ~ glycogenin in E s c ~ r i c h ~ coli. The production of inactive glycogenin, that required a separate activation step, would have allowed us to seek a distinct initiating enzyme. However, the recombinant glycogenin was already capable of catalyzing self-glucosylation (10). What we did not know was whether this recombinant protein was already glucosylated or not.

The present study describes the production of significant amounts of recombinant rabbit muscle glycogenin in E. coli, and its subsequent purification and biochemical characteriza- tion. We show that the recombinant protein already contains glucose attached to Tyr-194. Site-directed mutagenesis also demonstrated the importance of Tyr-194 for glycogenin func- tion and indicates that this is most likely the sole site of glucosylation in the protein,

EXPERIMENTAL PROCEDURES

Expression and P u r i ~ a ~ i o n of Recombinant Glycogenin The expression essentially followed the methods reported previ-

ously by Viskupic et al. (10). The buffers, all at pH 7.5, were as follows. Homogenizing buffer contained 50 mM HEPES, 1 mM EDTA, 5 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 0.1 mM N”-p-tosyl-L-lysine chloromethyl ketone, and 2 mM benzamidine,

14687

14688 Rabbit Muscle Glycogenin Buffer A contained 20 mM HEPES and 2.5 mM dithiothreitol. Buffer B1 was composed of buffer A plus 5 mM MnC12; buffer B2 additionally contained 1 mM UDP and 0.5 M NaCl. All steps were performed at 4 'C unless noted otherwise.

s tep 1: Cell Induction and Lysis-Cells (strain BL21/DE3) carrying the glycogenin expression vector were cultured in 800 ml of MgZB medium (17) for 8 h and induced with 20 p~ isopropyl-8-D-thiogalac- topyranoside for 3 h. Cells were collected by centrifugation at 5200 x g for 15 min and lysed in homogenizing buffer with glass beads using a Beadbeater (Biospec Products). The crude extract was separated from the glass beads and centrifuged at 10,000 X g for 25 min. The supernatant was collected.

Step 2: Ion Exchange Chromatography on Q-Sephurose Fast Flow- Cell extract was loaded onto a Q-Sepharose column (3 x 18 cm) equilibrated with buffer A and the column was washed with 5 times the bed volume of the same buffer. The column was eluted with a linear gradient of 0-1 M NaCl in buffer A.

Step 3: Ammonium Sulfate Precipitation-The fractions containing glycogenin activity from Step 2 were pooled and precipitated by adding saturated ammonium sulfate to give 30% saturation. The solution was then centrifuged at 9000 X g for 15 min. The precipitate was resuspended in buffer A plus 0.1 M NaC1.

Step 4: Gel Filtration on Sephacryl S-200"Samples (0.5 ml) of resuspended precitate were applied to an S-200 column (1.5 X 93 cm) in buffer A plus 0.1 M NaCl and eluted with same buffer. Active fractions were pooled, concentrated to 0.5-1 ml by Centricon-10 (Amicon), and dialyzed against buffer B1 overnight.

Step 5: Affinib Chromatography on UDP-Hexanohmine-Agarose- Since glycogenin requires Mn2+ for maximal activity, the partially purified glycogenin (0.5 ml at a time) was bound to a UDP-hexano- lamine-agarose column (1 X 10 cm) in the presence of Mn2+ in buffer B1. After washing, the column was eluted with a linear gradient of buffer B1 to buffer B2 containing UDP and salt. The fractions containing glycogenin were dialyzed against buffer A and concen- trated to 0.5-1.0 ml with a Centricon-10.

Step 6: MonoQ Ion-exchange Chromatography-The sample was applied to a Mono Q column (0.5 x 5 cm) and eluted with salt gradient, 0-1 M NaCl, in buffer A. Fractions were collected, dialyzed against buffer A, and stored at -70 "C.

Site-directed Mutagenesis of Glycogenin An SphI-BamHI fragment from the plasmid PET-GN (10) con-

taining the entire glycogenin-coding region was inserted into the BamHI and SphI cloning sites of double stranded M13mp19 DNA. The codon TAC corresponding to Tyr-194 was altered to a phenyl- alanine codon TTC by site-directed mutagenesis (Amersham kit). The mutagenizing oligonucleotide was antisence 5"AGGTAGGA- GgTATAGAAA-3'. The mutation and the sequence of the SphI- BamHl fragment were confirmed by DNA sequencing (18). The mutant DNA was excised from M13 and religated into the SphI- BamHI sites of the PET-& plasmid. The new plasmid, named PET- GNF194, was then transformed into Escherichia coli and expressed as described above for the wild-type glycogenin.

Gel Electrophoresis and Western Blot Analysis Polyacrylamide gel electrophoresis in the presence of SDS followed

the method of Laemmli (19). Protein samples, which were treated with 0.25 volume of sample buffer (60 mM Tris, 10% glycerol, 0.25% bromphenol blue, 2% SDS, and 0.7 M 8-mecaptoethanol) and boiled for 5 min, were subjected to SDS-PAGE' with 4% acrylamide in the stacking gel and 12% in the separating gel. For Western transfer, gels were equilibrated with transfer buffer (containing 190 mM glycine, 20 mM Tris, 0.13 mM SDS (0.37% w/v), and 20% methanol, pH 8.3) and sandwiched between a nitrocellulose membrane and Whatman No. 3MM filter paper soaked with transfer buffer. This sandwich was then placed in a Semi-phor apparatus (Hoefer Scientific Inc.) for 40 min at a constant current of 45 mA (no more than 0.8 mA/cm2 of gel). After transfer, the nitrocellulose membrane was blocked with 5% powdered milk in PBST (20 mM sodium phosphate, pH 7.4, 115 mM sodium chloride, 0.1% Tween 20) for 1 h and incubated with a 1:500 dilution of antiserum for 2 h at room temperature. The mem- brane was then incubated with '%I-protein A (0.28 pCi/ml in 5% dried milk in PBST) to detect the antibody by autoradiography. The

The abbreviations used are: PAGE, polyacrylamide gel electro- phoresis; HPLC, high performance liquid chromatography; FAB, fast atom bombardment; MS, mass spectrometry.

antibody was raised in guinea pigs against a synthetic peptide 3'3DYMGADSFDNIKKKLDTYLQ332 corresponding to the extreme COOH terminus of glycogenin. The synthetic peptide was coupled to keyhole limpet hemocyanin (20) and emulsified with Freund's com- plete adjuvant for inoculation. After 4 weeks, a booster injection was made and the guinea pigs were exsanguinated at the 6th week, the blood collected, and serum prepared.

Glucosyhtion Assays The reaction was generally carried out in 50 mM HEPES buffer

(pH 7.5) in the presence of 5 mM MnSO,, 5 mM dithiothreitol, and 19 p~ UDP-[U-"C]glucose (263 Ci/mol) at 30 "C for 5 min. The total volume of the reaction mixture was 10 pl. In some cases, glycogen synthase, glucose-6-P, and UDP-glucose were added to final concen- trations of 0.016 mg/ml, 5 mM, and 0.5 mM, respectively, for the elongation reaction. Two methods were used to visualize and detect glucose incorporation. (a) The reaction was stopped by adding one- fourth the reaction volume of sample buffer containing SDS and boiled for 5 min. The samples were subjected to SDS-PAGE as described above. The gel was stained with Coomassie Blue R-250, destained with methanol and acetic acid, and dried. The radioactivity of the labeled glycogenin was quantitated with a &Scanner (AMBIS). ( b ) An aliquot of the reaction mixture was spotted on a square of P81 chromatography paper which was then washed with 5% phosphoric acid three times and then washed once with ethanol. The paper was dried under an infrared light, placed in 5% (w/v) 2,5-diphenyloxazole in toluene, and counted in a liquid scintillation counter.

Synthetic Peptide Corresponding to the Glucosyhtion Site A 30-residue peptide (residues 178-207) surrounding Tyr-194, the

glucosylation site, was synthesized and purified by HPLC on a C8 column. Peptide was eluted by a 10-98% gradient of CH,CN in 0.1% trifluoroacetic acid and fractions collected and lyophilized. For the P81 paper assay, described above, 50 PM peptide was incubated with 0.2 pg of purified wild-type or F194 mutant glycogenin at 30 "C for 10 min under the conditions of the normal glycogenin assay. The reactions were stopped by spotting the reaction mixture onto P81 paper and placing in 5% phosphoric acid solution as described above. The peptide, with predicted PI of 10.3, should bind the paper whether or not it is glucosylated.

Mass Spectrometry

Purified recombinant glycogenin was digested with endoproteinase Lys-C (Boehringer Mannheim) in 50 pl of 20 mM Tris-HC1 buffer (pH 8.5) for 18 h at 35 "C. Following proteolysis, peptide mixtures were chromatographed using a Vydac C4 reversed-phase column and a linear gradient formed between buffer containing 0.1% trifluoroa- cetic acid and buffer containing 0.1% trifluoroacetic acid, 90% CH3CN. Fractions were collected and partially dried by Speed-Vac, and aliquots were then applied to a platinum probe with thioglycerol as matrix. FAB mass spectra were obtained using a Finnigan TSQ 700 mass spectrometer equipped with a cesium ion gun operated at 8 keV.

RESULTS

Purification and Characterization of Recombinant Glyco- genin from E. coli-Glycogenin was expressed in E. coli essen- tially as described previously to give an active protein (10). Approximately 50% of the expressed glycogenin was present in the soluble fraction and this protein could be purified close to homogeneity, as described under "Experimental Proce- dures" (Table I and Fig. 1). A key step in the purification procedure was a UDP-hexanolamine- agarose affinity column from which the glycogenin was eluted with buffer containing UDP. Based on the self-glucosylation assay, the recombinant glycogenin was purified -8-fold with -15% yield. By this method, -20 mg of pure protein per liter of E. coli culture could be produced. The purified wild-type protein had appar- ent M, of -38,000 as determined by SDS-PAGE (Fig. 1) and a PI of 4.8 (data not shown).

Recombinant Glycogenin Is Already Glucosyluted-The fact that the recombinant glycogenin is active for self-glucosyla- tion implies that either E. coli contains an activity able to

Rabbit Muscle Glycogenin 14689

TABLE I Purification of recombinunt glycogenin

SkDS Total activity Total protein Specific activity Yield Purification pmolfrnin mg pmolfminfpg ?6 -fold

1. Crude extract 843 940 0.90 100.0 1.0 2. Q-Sepharose 608 414 1.47 72.0 1.6 3. Ammonium sulfate precipitation 430 135 3.19 51.0 3.6 4. Sephacryl S-200 316 64 4.96 37.5 5.5 5. UDP-agarose 158 25 6.26 18.7 7.0 6. Mono Q 122 16 7.53 14.4 8.4

A B

1 2 3 4 5 6 7 1 2

170 212

116

76 94

67

53

43

36

30

FIG. 1. Purification of recombinant rabbit muscle glyco- genin. Fractions from different stages of the purification (see Table I) were analyzed by SDS-PAGE and the gel stained with Coomassie Blue. A, lane I , supernatant of lysed cells; lane 2, pooled fractions after Q-Sepharose; lane 3, resuspended ammonium sulfate precipitate; lane 4, pooled fractions after Sephacryl S-200 gel filtration column; lane 5, pooled fractions after UDP-agarose affinity column; lane 6, peak fraction from Mono Q column. B, purified wild-type glycogenin; lane 2, purified F194 mutant glycogenin.

introduce the first glucose residue into glycogenin or else glycogenin itself can catalyze this reaction, as has been dis- cussed previously (10). I t was therefore of considerable im- portance to know whether the glycogenin purified from E. coli extracts was already modified. The absence of glucosylation would clearly implicate glycogenin as being responsible for the initial step. Glycogenin was cleaved with Lys-C protease and the resulting peptides separated by reversed phase HPLC. From FAB-MS analysis, a peptide corresponding to residues 181-201 of glycogenin was identified in the profile as a rela- tively broad peak (not shown). This peptide contains the residue, Tyr-194, that Smythe et al. (13) had shown to be glucosylated. The averaged mass spectrum (Fig. 2) indicated that in addition to the parent peptide (2474.9 atomic mass units calculated; 2473.3 observed) a series of ions of higher mass were also detected. These represented successive in- creases of 162 mass units, the mass that corresponds to the addition of a glucose residue. The data indicated the presence of up to 8 glucose residues attached to the 181-201 peptide. In Fig. 2, the signal corresponding to +8 glucoses is very weak but in some analyses was more prominent. The signals cor- responding to the parent peptide and peptide plus 4 glucose residues were the strongest. Because of the inherent lability of carbohydrates in FAB-MS, it is difficult distinguish the heterogeneity generated by the analysis from any heteroge- neity of the starting material. Nonetheless, we can conclude that the glycogenin expressed in E. coli was already substan- tially glucosylated. This conclusion is supported by the results of microsequencing the peptide which gave the expected se- quence but with very little signal in the cycle corresponding

to Tyr-194, consistent with this residue being largely in a modified form (Table 11).

Enzymatic Properties of Recombinant Glycogenin-Several kinetic properties of the recombinant protein were examined. When incubated with UDP-[U-14C]glucose, radioactivity was incorporated into glycogenin, as detected by SDS-PAGE and radiography (Fig. 3). The time course of the reaction indicated an initial rapid and approximately linear segment with sub- sequent leveling off (Fig. 3). As the reaction progressed, there was a slight reduction in electrophoretic mobility and a broad- ening of the band corresponding to the 14C-labeled material. From trials at different UDP-glucose and glycogenin concen- trations, the maximum average level of additional glucosyla- tion was up to -5 glucose/glycogenin. This finding would support the idea that the heterogeneity detected by FAB-MS did reflect, a t least in part, a mixture of lengths of oligosac- charide attached to the purified glycogenin before analysis. If all the glycogenin had been maximally glucosylated, it would have been unable to undergo further self-glucosylation. Ki- netic analysis is complicated both by this heterogeneity of the starting material as well as the the fact that the reaction involves a series of successive glucose additions. The initial linear phase of self-incorporation (Fig. 3) was thus used operationally as the measure of glucosylation activity in this and other kinetic analyses. In this way, hyperbolic kinetics with respect to substrate were observed, with an apparent K,,, of 4.5 ~ L M for UDP-glucose (data not shown). A pH optimum of pH 8 was observed (data not shown). The maximal rate of self-glucosylation was 15.4 nmol/min/mg. The reaction rate was first, not second, order with respect to glycogenin concen- tration, consistent with the reaction being intramolecular (Fig. 4).

Glycogenin as a Substrate for Glycogen Synthase-In pre- vious work with unpurified recombinant glycogenin, we had observed that glycogenin could be converted to species with high MI on SDS-PAGE simply by incubation with purified rabbit muscle glycogen synthase in the presence of UDP- glucose, but without any requirement that Mn2+ be present (10). Thus, the recombinant protein appeared fully competent as a glycogen synthase substrate; this result is repeated in Fig. 5 for comparison. Purified recombinant glycogenin, how- ever, had lost this property (Fig. 5). If the purified glycogenin was preincubated with Mn2+ and UDP-glucose, to allow self- glucosylation, a slight reduction in electrophoretic mobility was seen and this material was now able to serve as a substrate for glycogen synthase, as judged by immunoblotting analysis (Fig. 5). Increasing time of preincubation correlated with greater ability of the glycogenin to be a substrate for glycogen synthase (Fig. 6). After 20 min, under the conditions of Fig. 6, all of the labeled glycogenin could be converted to higher MI species upon incubation with glycogen synthase. It is also interesting that relatively discrete forms of glycogenin were

14690 Rabbit Muscle Glycogenin

100

0 W

Z 80 a n

m 60

2

z 3

< W

2 4 0

W J

a 2 0

I 2473.3 +4 I 3123.0

+1 2635.9

+2 2796 .8

+5 3285.3

+6 3448.3 +7

I 3 6 0 8 . 2

2000 2500 3000 3500 4000

MASS / CHARGE FIG. 2. Analysis of glycogenin glucosylation by mass spectrometry. The Lys-C peptide was analyzed by FAB-MS (see “Experimental

Procedures”). The positive ion spectrum is shown in the mass range 2000-4000. The calibrated masses are shown alongside the signals and the integers refer to the number of additional glucose units relative to the unmodified peptide (mass 2473.3).

TABLE I1 Amino acid sequencing of residues 181 -201

of glycogenin Lys-C peptide Cycle Residue Amount Cycle Residue Amount

pmol P m l 1 His 50

20 Phe 5 9 Ser 14 19 Ala 6 8 Leu 35 18 Pro 6 7 Asn 31 17 Leu 8 6 TY r 32 16 TYr 11 5 Ile 32 15 Ser 6 4 Phe 37 14 Tyr 1 3 Pro 26 13 Ile 14 2 Leu 52 12 Ser 8

10 Ser 14 21 LY 8 3 11 Ile 26 22

detected after the addition of glycogen synthase. These had apparent M, of -80,000, -150,000, and one species in excess

Effectors of Glycogenin-The ability of Ca2+ and M F to substitute for Mn2+ in the self-glucosylation assay was tested (Table 111). At 10 mM, neither ion sustained more than 10% of the control activity. The presence of EDTA caused almost total inhibition. Phosphate was weakly inhibitory, resulting in 40% inhibition at 50 mM. Polyphosphates (pyrophosphate, tripolyphosphate, and tetrapolyphosphate) were somewhat more effective although the cyclic trimetaphosphate was not. Several nucleotides and related compounds were screened at high (6-10 mM) concentration. Adenine nucleotides were weakly inhibitory. ADP-glucose caused only 14% inhibition at 6 mM. UTP and UDP were the most effective inhibitors and other experiments (not shown) indicated half-maximal inhibiton by UDP at 100 p~ under the standard reaction

’ At longer times, high M, material is produced that can accumulate a t the top of the separating gel and in the stacking gel, and whose transfer to the nitrocellulose is less reproducible. This material is better visualized by using “C labeling of the glycogenin so that the gel can be analyzed directly by fluorography without transfer (see Ref. 10).

of 200,000.2

conditions. Glucose-6-P had no detectable effect on activity. Glucose and several glucose containing oligosaccharides

were also tested as effectors of glycogenin self-glucosylation (Fig. 7). Of these, maltose inhibited significantly and malto- triose acted very weakly. Glucose, maltotetraose, and malto- pentaose had little if any effect. In the self-glucosylation reaction, it appeared as though inhibition of the initial rapid phase was less effective compared with inhibition at later time points. Thus, half-maximal inhibition by maltose occurred at -4 mM for the early reaction (2-min time point) uersus -1 mM using a later time point (10 min).

Tyr-194 Is Essential for Function-Smythe et al. (13) had reported a single site of glucosylation of glycogenin, namely, at Tyr-194. However, the original report that the modified amino acid was tyrosine had suggested that 2 Tyr residues were involved (11). It was therefore of interest to determine whether Tyr-194 was essential for the self-glucosylation re- action and whether it was the only site of glucosylation. Site- directed mutagenesis was used to convert Tyr-194 to phenyl- alanine to generate the F194 mutant. This protein was ex- pressed at high levels in E. coli and was purified according to the scheme developed for the wild-type recombinant protein. The purification was monitored by SDS-PAGE since the F194 mutant was inactive as judged by self-glucosylation assays. It should be noted that the F194 mutant bound to the UDP- agarose affinity column and was eluted with UDP, suggestive that the nucleoside diphosphate sugar binding site was intact. SDS-PAGE indicated a sharp band corresponding to a poly- peptide of apparent M, -37,000 just a little lower than that of the wild-type (Fig. 1). The stained polypeptide band cor- responding to the wild-type protein was noticeably broader than that of the F194 mutant. The F194 mutant was inactive for self-glucosylation in numerous different analyses (for ex- ample, see Fig. 8). Although the self-glucosylation of glyco- genin appeared to be intramolecular, we considered the pos- sibility that an exogenous acceptor might still be a substrate, even though entropically disadvantaged. Also, modification of an exogenous substrate might be easier to detect in the absence of the intramolecular reaction. Therefore a synthetic

Rabbit Muscle Glycogenin 14691

FIG. 3. Time course of glycogenin self-glucosylation. Purified glyco- genin (0.54 p ~ ) was incubated with UDP-[“C]glucose a t 30 “C as described under “Experimental Procedures.” At the indicated times, aliquots of the re- action mixture were removed and spot- ted on P81 chromatography paper for quantitation by scintillation counting. The inset shows parallel analyses in which samples were subjected to SDS- PAGE followed by autoradiography.

- 20 1 L I I

O ! I I I I I I 0.0 0.2 0.4 0.6 0.8 1.0 1.2

Glycogenin (pg) FIG. 4. Dependence of glycogen self-glucosylation on pro-

tein concentration. The initial rate of glucose incorporation was measured a t the indicated amounts of glycogenin. The reaction vol- ume was 10 p1 and was performed in the presence of 1 mg of bovine serum albumin/ml.

30-residue peptide containing the sequence surrounding Tyr- 194 was tested as a glucose acceptor with wild-type and the F194 mutant glycogenin (Fig. 8). No modification of the peptide was detected using F194. When added to wild-type glycogenin, if anything a slight activation of self-glucosylation was observed. With similar intent, F194 was also incubated with wild-type glycogenin. No major effect on glucosylation was detected. The paper binding assay should detect I 4 C

incorporation into either the peptide or glycogenin protein. In other experiments, the peptide was analyzed by HPLC and found not to be labeled after incubation with wild-type or mutant glycogenin (not shown). By the various tests available to us, there was no evidence that the F194 mutant glycogenin had any ability to transfer glucose to itself or the synthetic ~ e p t i d e . ~

DISCUSSION Expression of significant quantities of active glycogenin in

E. coli has permitted purification of the protein used in the

I 0.3 2 5 10 20 30 40 60 75

1 I I I

Of course, if glycogenin is not responsible for attachment of the first glucose residue, then the appropriate exogenous substrate would be peptide with the initial glucose residue attached.

0 50 100 1 50 200

time (min)

1 2 3 4 5 6 7 8

Mr ( k W

200 - 97 - 68 - 43-

29 - .

FIG. 5. Comparison of purified and unpurified glycogenin as substrates for glycogen synthase. Samples were analyzed by Western blot, the generation of higher M, immunoreactive species indicating the covalent modification of the glycogenin. In lanes 1-4, the samples were crude extracts of E. coli expressing glycogenin. In lanes 5-8, purified recombinant glycogenin was used. The initial treatments were as follows: lanes 1 and 5, glycogenin sample alone; lanes 2 and 6, glycogenin sample reacted with glycogen synthase; lanes 3 and 7, glycogenin sample incubated with UDP-glucose and Mn2+; lanes 4 and 8, glycogenin sample incubated with UDP-glucose and MnZ+ followed by reaction with glycogen synthase. Further details are given under “Results.”

present study. Previous characterizations of glycogenin had either used the limited amounts of protein purified directly from tissue (5, 14) or else protein dissociated from glycogen synthase by treatment with LiBr (3). Several properties of the recombinant glycogenin were similar to those described previously. Included among these would be the micromolar K , for UDP-glucose and the preference for Mn2+ as the divalent cation. The self-glucosylation reaction was intramo- lecular, in agreement with the report of Pitcher et al. (3). The N-acetylation of the native protein (9) does not appear to be necessary for the transglucosylase activity of glycogenin or its ability to serve as a glycogen synthase substrate.

One of the important outcomes of this study is the finding that Tyr-194 was essential for activity and that there were no other sites of glucosylation in glycogenin. This conclusion is based on the lack of activity of the F194 mutant protein in self-glucosylation assays. Site-directed mutagenesis that re- sults in an inactive protein is always subject to the criticism that the mutation has had a general, rather than a specific, detrimental effect on the protein’s functional integrity. In the

14692 Rabbit Muscle Glycogenin

Mr ( k W

or1 200 - 94- 67 - 43.

36 *

30 .

0.5 2 5 10 20

Time (min)

FIG. 6. Effect of glycogenin priming on competence as a glycogen synthase substrate. Glycogenin was allowed to self- glucosylate with 19 p~ UDP-glucose for the indicated times, when aliquots (10 pl) were removed and incubated with 0.2 pg of glycogen synthase, 5 mM glucose-6-P, and 0.5 mM UDP-glucose for 20 min. The samples were finally analyzed by Western blotting.

TABLE I11 Effectors of glycogenin self-glucosylatwn activity

Compound Concentration Activity mM ?6 of control

MnS04 5 100.0 MgSO4 10 5.6 CaCI2 10 EDTA 10

9.2 2.2

UDP-glucuronate 10 45.9 UMP 10 23.6 UDP 10 UTP

3.6 10 5.4

ADP-glucose 6 86.3 AMP 10 91.7 ADP 10 ATP

64.1 10 54.3

Dibutyryl CAMP 10 79.0 cGMP 10 89.7 Glucose-6-P 10 102.0 p-Nitrophenylmannose 10 NAD

107.0 10 76.4

NADP 10 72.0

Phosphate 10 71.8 Pyrophosphate 10 10.2 Tripolyphosphate 10 11.6 Trimetaphosphate 10 88.9 Tetrapolyphosphate 10 8.6

present case, properties of the protein involved in its purifi- cation have remained intact since the mutant went through the isolation procedure like the wild-type. In particular, the ability of the F194 mutant to bind to a UDP-agarose column is a good indicator of the integrity of the active site. We therefore conclude that the lack of activity was most likely due to the specific requirement for Tyr-1942

Survey of some potential effectors indicated that com- pounds containing the uridine moiety were the best inhibitors, UDP for example causing half-maximal inhibition at 100 p ~ .

In addition, a second mutant with Tyr-194 converted to a Thr residue was also inactive for self-glucosylation; thus inactivity is not linked specifically to the presence of the Phe residue nor was a hydroxyl group alone sufficient to support activity.

0.0- 0' 0 10 20 30 40 0 2 5 8 1 0

Time (min) Imanosel (mM)

2

FIG. 7. Effect of oligosaccharides on glycogenin self-gluco- sylation. A, glucose incorporation into glycogenin (0.54 pM) was measured in the presence of several potential inhibitors. The com- pounds (10 mM) were: glucose (open diamonds), maltose (open tri- angles), maltotriose (inverted triangles), maltotetraose (filled squares), and maltopentaose (filled diamonds). The control reaction (open squares) is also shown. B, glucose incorporation at 2 min (open circles) or 10 min (filled circles) is shown as a function of maltose concentration.

1 .o-

3, 0.8- r 0 .-

I I I

d

u- !? a W a + v

> z

L W

+ d

LL

a

?

FIG. 8. Comparison of wild-type and F194 mutant. Glucose incorporation was measured by the P81 paper assay, which would detect both self-glucosylation as well as peptide glucosylation. The wild-type, Y194, or the F194 mutant proteins were present as indi- cated. The presence of 0.05 mM of the synthetic peptide is denoted by PEP (see text). Both Y194 and F194 are 0.2 pg in each assay.

Advantage was taken of this interaction by using the UDP- agarose affinity column in the purification procedure. In contrast, adenine containing compounds were much less ef- fective. For example, ADP-glucose caused only 14% inhibition at 6 mM whereas the K,,, for UDP-glucose was 4.5 pM. Phos- phate was weakly inhibitory but polyphosphates, including pyrophosphate, tripolyphosphate, and tetrapolyphosphate, were significantly more effective. Glucose had no detectable effect on activity. Although this analysis of effectors is far from a complete kinetic study, the results are consistent with the nucleoside diphosphate sugar binding being specified strongly by the uridine and, to a lesser degree, by the pyro- phosphate moieties. Of several oligosaccharides tested, by far the greatest inhibition was seen with maltose, in agreement with the report of Whelan et al. (21). One would expect that the active site must bind UDP-glucose substrate as well as the nonreducing end of the growing oligosaccharide. The reaction product, after formation of the al,4-glycosidic link- age, should thus contain a maltose dimer unit bound to the enzyme and one could hypothesize that maltose would interact in this position. Why maltotetraose or maltopentaose was not inhibitory is unclear, but their lack of effect is consistent also

Rabbit Muscle ~ l y c o g ~ n ~ n 14693

with the observation that maltose was a less effective inhibitor at the earlier stages of the reaction when, on average, shorter oligosaccharide chains would be attached to the glycogenin. That the longer oligosaccharides interact less effectively at the active site is also in agreement with the observation that the self-glucosylation reaction does not continue to form a chain of unlimited length. Presumably, the kinetics of elon- gation decrease so much as a function of chain length that effective growth much beyond eight glucose residues is highly unfavorable.

The FAB-MS analysis of the 181-201 peptide of glycogenin provided direct evidence that the protein isolated from E. coli was already substantially glucosylated and contained as many as 8 glucose residues attached. Had the result been that the protein was unmodified, then we would have been in a position to resolve the question as to whether glycogenin itself or an E. coli activity introduced the first glucose; the observed outcome, although, does not formally settle the issue. The recombinant glycogenin, as purified, was not maximally glu- cosylated, however. First, it could incorporate an additional -5 mol of glucose/mol upon incubation with UDP-glucose, implying that the oligosaccharide was well short of its maxi- mal length, probably 8 glucoses per molecule. Second, the purified protein was ineffective as a primer for glycogen synthase unless it was first allowed to self-glucosylate. In contrast, glycogenin present in a fresh E. coli extract was effective for elongation by glycogen synthase and was presum- ably more highly glucosylated. The most likely explanation for this difference is that some glucose residues were removed during the purification procedure. We have excluded the possibility that glycogenin itself has any form of glycosidase activity? The lability of carbohydrates in FAB-MS makes it difficult to assess accurately the starting glucosylation state of the sample. Since there is no obvious reason why the peptide with four glucoses attached would be especially stable, the prominence of the corresponding ion in the FAB-MS shown in Fig. 2 does suggest that this species was relatively abundant. However, more highly glucosylated species must also have been present and it is likely the material was heterogeneous with respect to glucosylation state.

One of the most i m p o ~ n t conclusions of this study is that the length of the oligosaccharide chain is a critical determi- nant of whether glycogenin can be effectively elongated by

Y. Cao and P. J. Roach, unpublished data.

glycogen synthase. Below some minimal length, the glycogen synthase appears to be ineffective in lengthening the oligo- saccharide. I t is difficult, as discussed above, to define the exact number of glucose units required from our studies, and there may or may not be a discrete switch from incompetence to competence as a glycogen synthase substrate with increas- ing chain length. Still, we have clearly demonstrated that glycogenin can be converted from a poor primer of glycogen synthesis to an effective one via the ~lf-glucosylation reac- tion. This observation is of potential importance for the function of glycogenin in vivo since it implies that control of the glucosylation state of glycogenin could dictate its effec- tiveness for the production of glycogen.

A c k ~ ~ ~ d g ~ e ~ t s - W e acknowledge t h e B i ~ c h e m i s t ~ Biotechnol- ogy Facility of Indiana University which was responsible for synthesis of peptides and oligonucleotides used in this study. We are most grateful to Dr. Stan Hefta, City of Hope, for helpful discussions and Dr. John E. Shively, City of Hope, for providing facilities for FAB- MS.

REFERENCES 1. Krisman, C. R., and Barrengo, R. (1975) Eur. J. Biochem. 62,117-123 2. Kennedy L. D., Kirkman, B. R., Lomako, J., Rodriguez, I. R., and Whelan,

W. J. (1985) in Membrane and Muscle (Berman, M. C., Graves, W., and

3. Pitcher, J., Smythe, C., and Cohen, P. (1988) Eur. J. Biochem. 176,391- Opie, L. A., e&) pp. 65-84, ICSU Press/IRL Press, Oxford

395 4. Smythe, C., and Cohen, P. (1991) Eur~ J. Biochem. 200,625-631 5. Lomako, J., Lomako, W. M., and Whelan, W. d . (1988) FASEB J. 2,3097-

6. Rodriguez, I. R., and Fliesler, S. (1988) Arch. Biochem. Biophys. 260,628-

7. Pltcher, J., Smythe, C., Campbell, D. G., and Cohen, P. (1987) Eur. J.

8. Smythe, C., Watt, P., and Cohen, P. (1990) Eur. J. Biochem 189,199-204

10. Viskupic, E., Cao, Y., Zhang, W., Cheng, C., DePaoli-Roach, A. A., and 9. Campbell, D. G., and Cohen, P. (1989) Eur. J. Biochem. 186 , 119-125

11. Rodriguez, 1. R., and Whelan, W. J. (1985) Bwchem. Biophys. Res. Commun.

12. Lomako, J., Lomako, W. M., and Whelan, W. J. (1992) Carbohydr. Res.

13. Sm he, C., Caudwell, F. B., Ferguson, M., and Cohen, P. (1988) EMBOJ.

14. Smythe, C., Villar-Palasi, C., and Cohen, P. (1989) Eur. J. Bwchem. 183 ,

15. Lomako, J., Lomako, W. M., and Whelan, W. J. (1990) FEBS Lett. 268 ,

16. Lomako, J., Lomako, W. M., and Whelan, W. J. (1990) Biochem. Int. 21,

3103

637

Biochem. 169,497-502

Roach, P. J. (1992) J. Bwl. Chem. 267,25759-25763

132,829-836 2m8Y-l-.- 205-209

8-12

251-2c;n 17.

18.

19. 20. 21.

Studier, F. M., Rosenherg, A. H., Dunn, J. J., and Dubendorff, J. W. (1990)

Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. NatL Acad. Sc i

"_ ""

Methods Enzyrrwl. 186,60-89

U. S. A. 74,5463-5467 Laemmfi, U. K. (1970) Nature 227,680-685 Tamura T., Bauer H., Birr, C., and Pipkom, R. (1983) Cell 34,587-596 Lomakd, J., Lomaio, W. M., and Whelan, W. J. (1990) FEBS Lett. 264,

13-16