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The intramolecular autoglucosylation of monomeric glycogenin
Biochemical and Biophysical Research Communications 371 (2008) 328–332
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The intramolecular autoglucosylation of monomeric glycogenin Soledad Bazán 1, Federico M. Issoglio 1, María E. Carrizo, Juan A. Curtino * Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 4 April 2008 Available online 23 April 2008
Key words: Glycogenin Proteoglycogen Autoglucosylation Autoxylosylation Reaction mechanism Molecular size
a b s t r a c t The ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60–0.85 lM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step. Ó 2008 Elsevier Inc. All rights reserved.
Proteoglycogen [1], the glycogenin-bound glycogen of eukaryotic organisms, is the final product of the de novo biosynthesis of the polysaccharide. This process begins with the autoglucosylation of the 38 kDa protein glycogenin to produce the primer for the full polymerization and branching by glycogen synthase and branching enzyme [2–6]. Using UDP-glucose as substrate donor and acting as substrate acceptor and reaction product, glycogenin catalyzes two chemically different autoglucosylation reactions, the glucosylation of a tyrosine hydroxyl group and the formation of a-1,4 glucosidic linkages by subsequent glucosylations. Glycogenin also catalyzes the transglucosylation of exogenous acceptors [7–9], the hydrolysis of UDP-glucose [10,11] and it can use UDP-xylose as an alternative sugar donor [12,13]. Recombinant glycogenin expressed in wild-type Escherichia coli is partially autoglucosylated within the bacterial cell from endogenous UDP-glucose [14] to acquire four to seven attached glucose residues [15,16]. Expressed in an E. coli mutant defective in UDPglucose synthesis, the recombinant protein is produced as the non-glucosylated form apo-glycogenin [13,17]. Native glycogenin released from purified muscle proteoglycogen [18] and recombinant glycogenin are truncated at the C-terminal end by protease
Abbreviations: IPTG, isopropylthiogalactopyranoside; TLCK, N-p-tosyl-L-lysine chloromethyl ketone. * Corresponding author. Fax: +54 351 4334074. E-mail address: [email protected] (J.A. Curtino). 1 S.B. and F.M.I. contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.076
activity to be converted into a more stable 31 kDa species without loss of activity. The protease activity responsible for truncation of the purified enzyme preparations escaped conventional protease inhibition mixtures (Bazán and Curtino, unpublished result). The incubation of recombinant glycogenin with UDP-[14C]glucose in the presence of Mn2+ ions results in auto[14C]glucosylation of the protein. Glycogenin containing labeled maltosaccharides of 6–13 glucose units is produced after cessation of the autoglucosylation reaction [16]. A first order reaction kinetics, independent of glycogenin concentration, was described for the autoglucosylation of glycogenin, which supported an intramolecular mechanism of reaction [3,15,19]. Gel filtration studies showed that full length glycogenin as well as the enzyme species truncated at residue 270, existed as 95% dimer with less than 5% tetramer [11]. Based on the kinetic results and the gel filtration data, the authors concluded that autoglucosylation might occur by intradimer intersubunit glucosylation [11,19]. The concentration dependence, second order rate autoglucosylation of glycogenin was also reported [10]. The activity–concentration curve pointed to pass the origin at low protein concentrations, as it would be the case if the dimeric form of the enzyme was the only active autoglucosylating species. Resolving the reaction mechanism by which glycogenin autoglucosylates imply to consider, in addition to the specific autoglucosylation rate at different enzyme concentrations, the oligomerization state of the enzyme, particularly at the low concentrations at which the specific rate is measured. The 3D structure
S. Bazán et al. / Biochemical and Biophysical Research Communications 371 (2008) 328–332
of glycogenin, showing enzyme protomers forming dimers, was resolved using 210–450 lM glycogenin, and studies indicating the enzyme existed in solution mainly as dimer, were carried out by gel filtration of 17–20 lM glycogenin [11]. These protein concentrations are well above the 0.05–2.5 lM range at which the protein concentration dependence of autoglucosylation has been studied [15,19]. The autoglucosylation kinetics of apo-glycogenin has not been studied and that of the already glucosylated form was measured without considering the substrate donor waste produced during autoglucosylation by the unproductive hydrolysis of UDPglucose. In addition to lowering the UDP-glucose concentration, the hydrolytic release of glucose would produce a concomitant increase in the concentration of UDP which might compete with UDP-glucose for binding into the active site. In the present work, we analyze the oligomeric state of non-glucosylated and glucosylated glycogenin at low- and high-protein concentrations, and the enzyme concentration dependence of the specific autoglucosylation rate at 0.05–10 lM of both glycogenin species. The specific autoxylosylation rate of non-glucosylated and glucosylated glycogenin was also analyzed. Materials and methods Materials. Escherichia coli strain ER 2566 was from New England Biolabs. Vector pET15b was from Novagen and the E. coli strain CGSC 4997, a galactose-minus strain lacking UDP-glucose pyrophosphorylase activity, was obtained from the E. coli Genetic Stock Center, Department of Molecular, Cellular, and Developmental Biology, Yale University. The rabbit muscle 38-kDa glycogenin clone used as PCR template was a gift of Dr. P. Roach (Indiana University School of Medicine). UDP-[14C]glucose (320 mCi/mmol) was purchased from Instituto de Investigaciones Bioquímicas Fundación Leloir (Buenos Aires, Argentina) and UDP-[14C]xylose (264 mCi/mmol) was from Perkin-Elmer Life Sciences, Inc. Chelating-Sepharose was from Amersham Biosciences. Except as indicated, all other reagents came from Sigma. Glycogenin expression and purification. In order to obtain the truncated forms of glycogenin at residue 270, primers (forward primer, 50 -GGTGGTCTCGAGACAGATC AGGCCTTTGTG-30 ; reverse primer 50 -GGTGGTGGATCCCTAGGTATCTTGGACAAG-30 ) containing an XhoI site (upstream) and a BamHI site (downstream) were used to polymerase chain reaction amplify the fragment of interest. The resultant cDNA was in frame inserted in the vector pET15b, downstream the His6-tag encoding sequence of the plasmid. For the preparation of the truncated already glucosylated species the plasmid was transformed into the E. coli strain ER2566. The truncated non-glucosylated species was prepared by expression in the CGSC 4997 E. coli cell-line, previously lysogenized for site-specific integration of a T7 RNA polymerase gene with a kDE3 lysogenization kit (Novagen) according to the manufacturer guidelines. For expression of the recombinant proteins, one-liter culture grown to A600 = 0.6–0.8 was induced with 0.3 mM IPTG for 14 h at 20 °C. The cells were collected by centrifugation, resuspended in 20 mM Tris–HCl buffer, pH 8.0, containing 0.5 M NaCl, 0.1% Triton X-100, 20 lM PMSF and 0.14 mM TLCK, sonicated and centrifuged in order to collect the clarified lysate containing the protein of interest. For purification of the His6-glycogenins wt-31 and apo-31, the clarified lysate was passed over a 10 ml Ni2+-column equilibrated with Ni-column buffer (20 mM Tris–HCl, pH 8.0, containing 0.5 M NaCl and 10 mM imidazole). The column was washed to give baseline UV absorbance with the same buffer containing 50 mM imidazole and the protein was eluted rising the imidazole content up to 200 mM. Fractions containing glycogenin were dialyzed against 20 mM Tris–HCl buffer, pH 7.5, containing 0.24 M sucrose, and stored at 20 °C. Molecular mass determination by gel filtration. The molecular mass of glycogenin in solution was determined on a Superdex-75 column (24 ml) equilibrated in 0.1 M phosphate buffer pH 7.4 containing 0.1 M NaCl. The column, with a void volume of 8.0 ml, was standardized using blue dextran, alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and chymotrypsin (25 kDa). Glycogenin was subjected to autoglucosylation by incubation with 80 lM UDP-[14C]glucose for 30 min as indicated below and 0.1 ml of 20 lM or 0.5 lM of the labeled enzyme was loaded into de column. The column was operated at 0.5 ml/min and 0.3-ml-fractions were collected and radioactivity counted in a liquid scintillation counter. Where mentioned, the elution of unlabeled glycogenin from the column was monitored by UV absorbance. Autoglycosylation assays. Glycogenin autoglucosylation was assayed by incubation at 30 °C with 80 lM UDP-[14C]glucose for 3 min or 20 lM UDP-[14C]glucose for 5 min, in the presence of 0.1 M Mes buffer, pH 7.0, containing 5 mM MnSO4 and 1 mg/ml of BSA. Where indicated, 80 lM UDP-[14C]xylose replaced UDP[14C]glucose and the incubation time was 5 min. Aliquots were removed from the reaction mixture and the labeled protein precipitated by addition of trichloroacetic acid, washed and counted as elsewhere described [18].
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HPTLC. The radioactive components of the incubated mixtures were separated on HPTLC plates in the running solvent ethanol/1.0 M ammonium acetate/1.0 M acetic acid (150:8:52, by vol) as elsewhere described [16]. After running the plates were air-dried and subjected to autoradiography. The radioactive signals were detected using a Fujifilm FLA-3000 Imaging System and the images were quantified with the ImageGauge Software. Other methods. Protein concentration was determined by the method of Bradford [21] using BSA as standard.
Results and discussion The aggregation state of glycogenin The determination of the molecular size of the enzyme by gel filtration of low concentration of protein was facilitated by the use of the auto[14C]glucosylated glycogenin. Gel filtration studies carried out by loading 17–20 lM protein solutions into the column showed that the glucosylated and non-glucosylated glycogenin species truncated at residue 270, respectively, existed mainly (95%) as dimer of molecular mass 58 kDa [11]. Coincident with this result, the passage through a Superdex-75 column of a 20-lM solution of the non-glucosylated truncated species apo-31 resulted in elution of a 59 kDa protein, corresponding to the size of the dimer form. However, when loaded at 0.5 lM, elution of the protein was shifted to that of a monomer of molecular mass 34 kDa (Fig. 1A). The gel filtration of the glucosylated truncated species wt-31, loaded at 20 lM and 0.5 lM (Fig. 1B), and of 20 lM unlabeled
Fig. 1. Gel filtration of glycogenin. The 20 lM (h) and 0.5 lM (s) solutions of apo31 (A) and wt-31 (B) were loaded into a Superdex-75 column after auto[14C]glucosylation from UDP-[14C]glucose. The inserts correspond to the eluted fractions extended to the void volume of the column. For details see Materials and methods.
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31 kDa glycogenin (not shown), showed the same elution patterns of the apo-enzyme for the dimer and monomer species. No labeled species of higher size was observed at neither of the concentration analyzed (see inserts in Fig. 1A and B). Kinetics and mechanism of autoglucosylation Taking into account the hydrolytic activity of glycogenin toward UDP-glucose [10,11], it was important to carry out the present kinetic studies assuring saturation with the donor substrate throughout the entire range of enzyme concentration analyzed. This was accomplished by incubation with 80 lM UDP-glucose for three minutes instead of five minutes with 20 lM UDP-glucose which was the experimental condition previously used to analyze the protein concentration dependence of the reaction rate [3,15,19]. The hydrolytic activity toward UDP-glucose was reported to be higher for the non-glucosylated than the partially glucosylated enzyme [11]. Analyzing the apo-31 species, the activity–concentration curves resulting from incubation under both of the abovementioned conditions were quite different (Fig. 3A and B). The analysis by HPTLC of the incubated reaction mixtures, clearly indicated that the hydrolytic release of glucose, expressed as percentage of the total radioactive compounds resolved in the chromatogram, was higher for the assay system containing the lower UDP-glucose concentration (Fig. 2). Exhaustion of the sugar nucleotide was observed at the highest enzyme concentration when the 20 lM UDP-glucose containing incubation system was used. Analyzing the autoglucosylation time-course of apo-glycogenin by mass spectrometry, it was determined that maltosaccharides of
Fig. 2. HPTLC analysis of the reaction products of autoglucosylation. Aliquots from each incubated enzyme concentration of the activity–concentration curves of apo-31 in Fig. 3A and B, corresponding to the autoglucosylations with 80 lM UDP[14C]glucose for 3 min (A), and 20 lM UDP-14C]glucose for 5 min (B) were subjected to HPTLC and the autoradiographic signals of UDP-[14C]glucose (s) and [14C]glucose (h) were quantified as indicated in Materials and methods. The percentage of UDP-glucose and glucose was calculated as the corresponding radioactive product divided by the total radioactive products shown in each line of the autoradiogram (insert). GN, glycogenin.
four to eight glucose units are formed at four minutes reaction [17]. Thus, measuring the autoglucosylation rate of apo-glycogenin by a short incubation with UDP-[14C]glucose allowed us to describe the dependence of the reaction kinetics on the enzyme concentration for incorporation into the tyrosine hydroxyl group of the first glucose units, up to the formation of about three to six or seven a1,4 glycosidic linkages. The apo-glycogenin used was the species truncated at residue 270, apo-31, which showed to be more stable to proteolytic degradation than the particularly susceptible full length apo-38 species. Truncation of recombinant glycogenin at residue 270 resulted in a fully active form, with similar activities to the intact enzyme [11]. The specific autoglucosylation rate of apo-31 was partially dependent on the enzyme concentration (Fig. 3A), showing half reduction at the low concentrations at which apo-31 existed as monomer (Fig. 1A). For a reaction requiring a dimeric form of the enzyme as the unique active species, dissociation at low concentration should result in an activity–concentration curve passing through the origin, the characteristic behavior of a second order
Fig. 3. Specific rate of autoglucosylation against glycogenin concentration. Apo-31 (A,B) and wt-31 (C) were incubated at the indicated concentrations with 80 lM UDP-[14C]glucose for 3 min (A,C) and 20 lM UDP-[14C]glucose for 5 min (B), and the [14C]glucosylated protein was measured as indicated in Materials and methods. The results represent mean values of three independent experiments for each concentration and error bars show the standard deviation.
S. Bazán et al. / Biochemical and Biophysical Research Communications 371 (2008) 328–332
reaction. Contrarily, the specific reaction rate curve of apo-31 leveled at the lower glycogenin concentrations without been affected by further dilution. Thus, the activity–concentration curve revealed first order kinetics occurring at two levels of specific reaction rate, according to the concentrations at which the enzyme existed as monomer or dimer. The first order rate observed at high concentrations is consistent with the proposed intra-dimer intersubunit glucosylation of dimeric glycogenin, while that occurring at low concentrations is consistent with an intramolecular glucosylation of the monomeric enzyme. From half the specific rate difference between the monomeric and dimeric enzyme forms (Fig. 3A), an apparent dissociation constant of 0.85 lM was calculated. As above mentioned, recombinant glycogenin is expressed in wild-type E. coli as a partially glucosylated enzyme containing four to seven glucose units. Accordingly, it was possible to study the rate of autoglucosylation of glycogenin after having acquired an intermediate grown maltosaccharide, by measuring the specific rate of auto[14C]glucosylation of the already glucosylated recombinant form. The wt-31 species showed a pattern in the activity–concentration curve similar to apo-31, with one third reduction of the dimeric specific autoglucosylation rate at the concentrations corresponding to the monomeric form of the enzyme (Fig. 3C). An apparent dissociation constant of 0.6 lM, calculated from the activity–concentration curve, indicated a slightly higher dimerization affinity for glucosylated than non-glucosylated glycogenin. The overall conclusion drawn from the results in Fig. 3A and C is that both, the dimeric and monomeric forms of non-glucosylated and partially glucosylated glycogenin are able to catalyze the autoglucosylation by an intramolecular mechanism of reaction. The comparison of the autoglucosylation rate of monomeric and dimeric glycogenin was extended to the xylosylation reaction. As it was reported before [13], the autoxylosylation of apo-glycogenin and already glucosylated glycogenin results in the incorporation of a unique sugar unit into the hydroxyl group of the tyrosine residue or the C40 -hydroxyl group of glycogenin-linked glucose, respectively. In a difference with the autoglucosylation rate, which represents the average of a variable number of glucose units transferred per glycogenin molecule, the autoxylosylation rate refers to the kinetics for the transfer of one molecular proportion of xylose. Autoxylosylation might thus reveal how the rate of incorporation of a sugar unit into the tyrosine hydroxyl is, related to the incorporation into the glucose hydroxyl. The specific reaction rate for the incorporation of xylose into apo-31 and wt-31 was about 20% lower for monomeric than dimeric glycogenin (Table 1). Independently of the aggregation state, autoxylosylation of the apo-enzyme occurred at half the specific rate of the glucosylated enzyme. In spite of the sugar difference, this result might explain the low rate of diminution of apo-glycogenin relative to the increase in polymerization degree of the glucosylated species, reported during the autoglucosylation time-course analyzed by mass spectrometry [17]. Crystallographic studies of glycogenin showed that the hydroxyl group on the tyrosine residue implicated in the binding of glucose, is at least 15 Å from the C1” position of the glucose residue on the UDP-glucose molecule bound in the same glycogenin monomer, a distance which would not permit an intramonomer reaction
Table 1 Specific autoxylosylation rate of apo-31 and wt-31 Glycogenin (lM)
3 0.3
Specific rate (pmol/min/pmol) 102 apo-31
wt-31
3.19 ± 0.10 2.51 ± 0.02
6.18 ± 0.01 4.83 ± 0.07
Results are means ± SD of three independent experiments.
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for initiation of the autoglucosylation [11,20]. However, a conformational change permitting the necessary motion to reduce the 15 Å distance appear quite plausible considering the dynamics of a protein in solution. This would bring the tyrosine residue into direct contact with UDP-glucose, allowing formation of the covalent intermediate between the active site nucleophile and the donor for the intramonomer glucosylation. In conclusion, in the present work, we described by the first time the existence of monomer glycogenin in solution at concentration below 0.60–0.85 lM, the protein concentration independent autoglucosylation rate of the enzyme at the dimer and monomer states, and the lower reaction rate for autoglycosylation of the tyrosine than glucose hydroxyl group. The results provide the most direct evidence to date that besides dimeric glycogenin, the monomeric form of the enzyme is able to accomplish the incorporation of the first and subsequent glucose units by efficient intramolecular autoglucosylation. Referring to which intramolecular reaction mechanism, intramonomer or intradimer glucosylation, would actually occur in vivo, it is conceivable that just after synthesized, a molecule of glycogenin is able to initiate its autoglucosylation at least for acquiring the first(s) sugar unit(s), without the need to reach the concentration required for dimerization. Whenever dimerization occurs, its autoglucosylation rate will be two fold higher than the glucosylation rate of monomer glycogenin. Thus, the monomer and dimer conversion determining the different rate of autoglucosylation would allow glycogenin to modulate the de novo biosynthesis of proteoglycogen at the initial step of the glucose polymerization. Acknowledgments We thank Drs. J. M. Romero and G. Montich for carefully and critically reading the manuscript. This work was supported by grants from CONICET, FONCyT and SECyT-UNC. S.B. was a Fellow of CONICET. M.E.C. and J.A.C. are Career Investigators of CONICET. References [1] M.A. Aon, J.A. Curtino, Evidence for the glycoprotein nature of retina glycogen, Eur. J. Biochem. 140 (1984) 557–566. [2] J. Pitcher, C. Smythe, D.G. Campbell, P. Cohen, Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin, Eur. J. Biochem. 169 (1987) 497–502. [3] J. Pitcher, C. Smythe, P. Cohen, Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle, Eur. J. Biochem. 176 (1988) 391–395. [4] J. Lomako, W.M. Lomako, W.J. Whelan, A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis, FASEB J. 2 (1988) 3097–3103. [5] C. Smythe, P. Cohen, The discovery of glycogenin and the priming mechanism for glycogen biogenesis, Eur. J. Biochem. 200 (1991) 625–631. [6] C. Cheng, J. Mu, I. Farkas, D. Huang, M.G. Goebel, P.J. Roach, Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae, Mol. Cell. Biol. 15 (1995) 6632–6640. [7] J. Lomako, W.M. Lomako, W.J. Whelan, Substrate specificity of the autocatalytic protein that primes glycogen synthesis, FEBS Lett. 264 (1990) 13–16. [8] S.M. Manzella, L. Rodén, E. Meezan, A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-b-D-maltoside, Anal. Biochem. 216 (1994) 383–391. [9] Y. Cao, L.K. Steinrauf, P.J. Roach, Mechanism of glycogenin self-glucosylation, Arch. Biochem. Biophys. 319 (1995) 293–298. [10] M.D. Alonso, J. Lomako, W.M. Lomako, W.J. Whelan, Catalytic activities of glycogenin additional to its autocatalytic self-glucosylation, J. Biol. Chem. 270 (1995) 15315–15319. [11] T.D. Hurley, S. Stout, E. Miner, J. Zhou, P.J. Roach, Requirements for catalysis in mammalian glycogenin, J. Biol. Chem. 280 (2005) 23892–23899. [12] L. Roden, S. Ananth, P. Campbell, S. Manzella, E. Meezan, Xylosyl transfer to an endogenous renal acceptor, J. Biol. Chem. 269 (1994) 11509–11513. [13] M.D. Alonso, J. Lomako, W.M. Lomako, W.J. Whelan, J. Preiss, Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity, FEBS Lett. 352 (1994) 222–226. [14] E. Viskupic, Y. Cao, W. Xhang, C. Cheng, A.A. Depaoli-Roach, P.J. Roach, Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli, J. Biol. Chem. 267 (1992) 25759–25763.
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[15] Y. Cao, A.M. Mahrenholz, A.A. DePaoli-Roach, P.J. Roach, Characterization of rabbit skeletal muscle glycogenin: Tyrosine194 is essential for function, J. Biol. Chem. 268 (1993) 14687–14693. [16] S. Bazán, J.A. Curtino, The size of the C-chain maltosaccharide of glycogen: evidence for the presence of only a single branch, Glycobiology 15 (2005) 14C–18C. [17] T.D. Hurley, C. Walls, J.R. Bennett, J.P. Roach, W. Wang, Direct detection of glycogenin reaction products during glycogenin initiation, Biochem. Biophys. Res. Commun. 348 (2006) 374–378. [18] M.E. Carrizo, M.C. Miozzo, A. Goldraij, J.A. Curtino, Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity
of polysaccharide-free and -bound glycogenin, Glycobiology 7 (1997) 571– 578. [19] V. Lin, J. Mu, J. Yang, P.J. Roach, Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction, Arch. Biochem. Biophys. 363 (1999) 163–170. [20] B.J. Gibbons, P.J. Roach, T.D. Hurley, Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin, J. Mol. Biol. 319 (2002) 463– 477. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
The intramolecular autoglucosylation of monomeric glycogenin Soledad Bazán 1, Federico M. Issoglio 1, María E. Carrizo, Juan A. Curtino * Centro de Investigaciones en Química Biológica de Córdoba (CIQUIBIC), UNC-CONICET, Departamento de Química Biológica, Facultad de Ciencias Químicas, Haya de la Torre y Medina Allende, Ciudad Universitaria, X5000HUA Córdoba, Argentina
a r t i c l e
i n f o
Article history: Received 4 April 2008 Available online 23 April 2008
Key words: Glycogenin Proteoglycogen Autoglucosylation Autoxylosylation Reaction mechanism Molecular size
a b s t r a c t The ability of monomeric glycogenin to autoglucosylate by an intramolecular mechanism of reaction is described using non-glucosylated and partially glucosylated recombinant glycogenin. We determined that monomer glycogenin exists in solution at concentration below 0.60–0.85 lM. The specific autoglucosylation rate of non-glucosylated and glucosylated monomeric glycogenin represented 50 and 70% of the specific rate of the corresponding dimeric glycogenin species. The incorporation of a unique sugar unit into the tyrosine hydroxyl group of non-glucosylated glycogenin, analyzed by autoxylosylation, occurred at a lower rate than the incorporation into the glucose hydroxyl group of the glucosylated enzyme. The intramonomer autoglucosylation mechanism here described for the first time, confers to a just synthesized glycogenin molecule the capacity to produce maltosaccharide primer for glycogen synthase, without the need to reach the concentration required for association into the more efficient autoglucosylating dimer. The monomeric and dimeric interconversion determining the different autoglucosylation rate, might serve as a modulation mechanism for the de novo biosynthesis of glycogen at the initial glucose polymerization step. Ó 2008 Elsevier Inc. All rights reserved.
Proteoglycogen [1], the glycogenin-bound glycogen of eukaryotic organisms, is the final product of the de novo biosynthesis of the polysaccharide. This process begins with the autoglucosylation of the 38 kDa protein glycogenin to produce the primer for the full polymerization and branching by glycogen synthase and branching enzyme [2–6]. Using UDP-glucose as substrate donor and acting as substrate acceptor and reaction product, glycogenin catalyzes two chemically different autoglucosylation reactions, the glucosylation of a tyrosine hydroxyl group and the formation of a-1,4 glucosidic linkages by subsequent glucosylations. Glycogenin also catalyzes the transglucosylation of exogenous acceptors [7–9], the hydrolysis of UDP-glucose [10,11] and it can use UDP-xylose as an alternative sugar donor [12,13]. Recombinant glycogenin expressed in wild-type Escherichia coli is partially autoglucosylated within the bacterial cell from endogenous UDP-glucose [14] to acquire four to seven attached glucose residues [15,16]. Expressed in an E. coli mutant defective in UDPglucose synthesis, the recombinant protein is produced as the non-glucosylated form apo-glycogenin [13,17]. Native glycogenin released from purified muscle proteoglycogen [18] and recombinant glycogenin are truncated at the C-terminal end by protease
Abbreviations: IPTG, isopropylthiogalactopyranoside; TLCK, N-p-tosyl-L-lysine chloromethyl ketone. * Corresponding author. Fax: +54 351 4334074. E-mail address: [email protected] (J.A. Curtino). 1 S.B. and F.M.I. contributed equally to this work. 0006-291X/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2008.04.076
activity to be converted into a more stable 31 kDa species without loss of activity. The protease activity responsible for truncation of the purified enzyme preparations escaped conventional protease inhibition mixtures (Bazán and Curtino, unpublished result). The incubation of recombinant glycogenin with UDP-[14C]glucose in the presence of Mn2+ ions results in auto[14C]glucosylation of the protein. Glycogenin containing labeled maltosaccharides of 6–13 glucose units is produced after cessation of the autoglucosylation reaction [16]. A first order reaction kinetics, independent of glycogenin concentration, was described for the autoglucosylation of glycogenin, which supported an intramolecular mechanism of reaction [3,15,19]. Gel filtration studies showed that full length glycogenin as well as the enzyme species truncated at residue 270, existed as 95% dimer with less than 5% tetramer [11]. Based on the kinetic results and the gel filtration data, the authors concluded that autoglucosylation might occur by intradimer intersubunit glucosylation [11,19]. The concentration dependence, second order rate autoglucosylation of glycogenin was also reported [10]. The activity–concentration curve pointed to pass the origin at low protein concentrations, as it would be the case if the dimeric form of the enzyme was the only active autoglucosylating species. Resolving the reaction mechanism by which glycogenin autoglucosylates imply to consider, in addition to the specific autoglucosylation rate at different enzyme concentrations, the oligomerization state of the enzyme, particularly at the low concentrations at which the specific rate is measured. The 3D structure
S. Bazán et al. / Biochemical and Biophysical Research Communications 371 (2008) 328–332
of glycogenin, showing enzyme protomers forming dimers, was resolved using 210–450 lM glycogenin, and studies indicating the enzyme existed in solution mainly as dimer, were carried out by gel filtration of 17–20 lM glycogenin [11]. These protein concentrations are well above the 0.05–2.5 lM range at which the protein concentration dependence of autoglucosylation has been studied [15,19]. The autoglucosylation kinetics of apo-glycogenin has not been studied and that of the already glucosylated form was measured without considering the substrate donor waste produced during autoglucosylation by the unproductive hydrolysis of UDPglucose. In addition to lowering the UDP-glucose concentration, the hydrolytic release of glucose would produce a concomitant increase in the concentration of UDP which might compete with UDP-glucose for binding into the active site. In the present work, we analyze the oligomeric state of non-glucosylated and glucosylated glycogenin at low- and high-protein concentrations, and the enzyme concentration dependence of the specific autoglucosylation rate at 0.05–10 lM of both glycogenin species. The specific autoxylosylation rate of non-glucosylated and glucosylated glycogenin was also analyzed. Materials and methods Materials. Escherichia coli strain ER 2566 was from New England Biolabs. Vector pET15b was from Novagen and the E. coli strain CGSC 4997, a galactose-minus strain lacking UDP-glucose pyrophosphorylase activity, was obtained from the E. coli Genetic Stock Center, Department of Molecular, Cellular, and Developmental Biology, Yale University. The rabbit muscle 38-kDa glycogenin clone used as PCR template was a gift of Dr. P. Roach (Indiana University School of Medicine). UDP-[14C]glucose (320 mCi/mmol) was purchased from Instituto de Investigaciones Bioquímicas Fundación Leloir (Buenos Aires, Argentina) and UDP-[14C]xylose (264 mCi/mmol) was from Perkin-Elmer Life Sciences, Inc. Chelating-Sepharose was from Amersham Biosciences. Except as indicated, all other reagents came from Sigma. Glycogenin expression and purification. In order to obtain the truncated forms of glycogenin at residue 270, primers (forward primer, 50 -GGTGGTCTCGAGACAGATC AGGCCTTTGTG-30 ; reverse primer 50 -GGTGGTGGATCCCTAGGTATCTTGGACAAG-30 ) containing an XhoI site (upstream) and a BamHI site (downstream) were used to polymerase chain reaction amplify the fragment of interest. The resultant cDNA was in frame inserted in the vector pET15b, downstream the His6-tag encoding sequence of the plasmid. For the preparation of the truncated already glucosylated species the plasmid was transformed into the E. coli strain ER2566. The truncated non-glucosylated species was prepared by expression in the CGSC 4997 E. coli cell-line, previously lysogenized for site-specific integration of a T7 RNA polymerase gene with a kDE3 lysogenization kit (Novagen) according to the manufacturer guidelines. For expression of the recombinant proteins, one-liter culture grown to A600 = 0.6–0.8 was induced with 0.3 mM IPTG for 14 h at 20 °C. The cells were collected by centrifugation, resuspended in 20 mM Tris–HCl buffer, pH 8.0, containing 0.5 M NaCl, 0.1% Triton X-100, 20 lM PMSF and 0.14 mM TLCK, sonicated and centrifuged in order to collect the clarified lysate containing the protein of interest. For purification of the His6-glycogenins wt-31 and apo-31, the clarified lysate was passed over a 10 ml Ni2+-column equilibrated with Ni-column buffer (20 mM Tris–HCl, pH 8.0, containing 0.5 M NaCl and 10 mM imidazole). The column was washed to give baseline UV absorbance with the same buffer containing 50 mM imidazole and the protein was eluted rising the imidazole content up to 200 mM. Fractions containing glycogenin were dialyzed against 20 mM Tris–HCl buffer, pH 7.5, containing 0.24 M sucrose, and stored at 20 °C. Molecular mass determination by gel filtration. The molecular mass of glycogenin in solution was determined on a Superdex-75 column (24 ml) equilibrated in 0.1 M phosphate buffer pH 7.4 containing 0.1 M NaCl. The column, with a void volume of 8.0 ml, was standardized using blue dextran, alcohol dehydrogenase (150 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (29 kDa) and chymotrypsin (25 kDa). Glycogenin was subjected to autoglucosylation by incubation with 80 lM UDP-[14C]glucose for 30 min as indicated below and 0.1 ml of 20 lM or 0.5 lM of the labeled enzyme was loaded into de column. The column was operated at 0.5 ml/min and 0.3-ml-fractions were collected and radioactivity counted in a liquid scintillation counter. Where mentioned, the elution of unlabeled glycogenin from the column was monitored by UV absorbance. Autoglycosylation assays. Glycogenin autoglucosylation was assayed by incubation at 30 °C with 80 lM UDP-[14C]glucose for 3 min or 20 lM UDP-[14C]glucose for 5 min, in the presence of 0.1 M Mes buffer, pH 7.0, containing 5 mM MnSO4 and 1 mg/ml of BSA. Where indicated, 80 lM UDP-[14C]xylose replaced UDP[14C]glucose and the incubation time was 5 min. Aliquots were removed from the reaction mixture and the labeled protein precipitated by addition of trichloroacetic acid, washed and counted as elsewhere described [18].
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HPTLC. The radioactive components of the incubated mixtures were separated on HPTLC plates in the running solvent ethanol/1.0 M ammonium acetate/1.0 M acetic acid (150:8:52, by vol) as elsewhere described [16]. After running the plates were air-dried and subjected to autoradiography. The radioactive signals were detected using a Fujifilm FLA-3000 Imaging System and the images were quantified with the ImageGauge Software. Other methods. Protein concentration was determined by the method of Bradford [21] using BSA as standard.
Results and discussion The aggregation state of glycogenin The determination of the molecular size of the enzyme by gel filtration of low concentration of protein was facilitated by the use of the auto[14C]glucosylated glycogenin. Gel filtration studies carried out by loading 17–20 lM protein solutions into the column showed that the glucosylated and non-glucosylated glycogenin species truncated at residue 270, respectively, existed mainly (95%) as dimer of molecular mass 58 kDa [11]. Coincident with this result, the passage through a Superdex-75 column of a 20-lM solution of the non-glucosylated truncated species apo-31 resulted in elution of a 59 kDa protein, corresponding to the size of the dimer form. However, when loaded at 0.5 lM, elution of the protein was shifted to that of a monomer of molecular mass 34 kDa (Fig. 1A). The gel filtration of the glucosylated truncated species wt-31, loaded at 20 lM and 0.5 lM (Fig. 1B), and of 20 lM unlabeled
Fig. 1. Gel filtration of glycogenin. The 20 lM (h) and 0.5 lM (s) solutions of apo31 (A) and wt-31 (B) were loaded into a Superdex-75 column after auto[14C]glucosylation from UDP-[14C]glucose. The inserts correspond to the eluted fractions extended to the void volume of the column. For details see Materials and methods.
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31 kDa glycogenin (not shown), showed the same elution patterns of the apo-enzyme for the dimer and monomer species. No labeled species of higher size was observed at neither of the concentration analyzed (see inserts in Fig. 1A and B). Kinetics and mechanism of autoglucosylation Taking into account the hydrolytic activity of glycogenin toward UDP-glucose [10,11], it was important to carry out the present kinetic studies assuring saturation with the donor substrate throughout the entire range of enzyme concentration analyzed. This was accomplished by incubation with 80 lM UDP-glucose for three minutes instead of five minutes with 20 lM UDP-glucose which was the experimental condition previously used to analyze the protein concentration dependence of the reaction rate [3,15,19]. The hydrolytic activity toward UDP-glucose was reported to be higher for the non-glucosylated than the partially glucosylated enzyme [11]. Analyzing the apo-31 species, the activity–concentration curves resulting from incubation under both of the abovementioned conditions were quite different (Fig. 3A and B). The analysis by HPTLC of the incubated reaction mixtures, clearly indicated that the hydrolytic release of glucose, expressed as percentage of the total radioactive compounds resolved in the chromatogram, was higher for the assay system containing the lower UDP-glucose concentration (Fig. 2). Exhaustion of the sugar nucleotide was observed at the highest enzyme concentration when the 20 lM UDP-glucose containing incubation system was used. Analyzing the autoglucosylation time-course of apo-glycogenin by mass spectrometry, it was determined that maltosaccharides of
Fig. 2. HPTLC analysis of the reaction products of autoglucosylation. Aliquots from each incubated enzyme concentration of the activity–concentration curves of apo-31 in Fig. 3A and B, corresponding to the autoglucosylations with 80 lM UDP[14C]glucose for 3 min (A), and 20 lM UDP-14C]glucose for 5 min (B) were subjected to HPTLC and the autoradiographic signals of UDP-[14C]glucose (s) and [14C]glucose (h) were quantified as indicated in Materials and methods. The percentage of UDP-glucose and glucose was calculated as the corresponding radioactive product divided by the total radioactive products shown in each line of the autoradiogram (insert). GN, glycogenin.
four to eight glucose units are formed at four minutes reaction [17]. Thus, measuring the autoglucosylation rate of apo-glycogenin by a short incubation with UDP-[14C]glucose allowed us to describe the dependence of the reaction kinetics on the enzyme concentration for incorporation into the tyrosine hydroxyl group of the first glucose units, up to the formation of about three to six or seven a1,4 glycosidic linkages. The apo-glycogenin used was the species truncated at residue 270, apo-31, which showed to be more stable to proteolytic degradation than the particularly susceptible full length apo-38 species. Truncation of recombinant glycogenin at residue 270 resulted in a fully active form, with similar activities to the intact enzyme [11]. The specific autoglucosylation rate of apo-31 was partially dependent on the enzyme concentration (Fig. 3A), showing half reduction at the low concentrations at which apo-31 existed as monomer (Fig. 1A). For a reaction requiring a dimeric form of the enzyme as the unique active species, dissociation at low concentration should result in an activity–concentration curve passing through the origin, the characteristic behavior of a second order
Fig. 3. Specific rate of autoglucosylation against glycogenin concentration. Apo-31 (A,B) and wt-31 (C) were incubated at the indicated concentrations with 80 lM UDP-[14C]glucose for 3 min (A,C) and 20 lM UDP-[14C]glucose for 5 min (B), and the [14C]glucosylated protein was measured as indicated in Materials and methods. The results represent mean values of three independent experiments for each concentration and error bars show the standard deviation.
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reaction. Contrarily, the specific reaction rate curve of apo-31 leveled at the lower glycogenin concentrations without been affected by further dilution. Thus, the activity–concentration curve revealed first order kinetics occurring at two levels of specific reaction rate, according to the concentrations at which the enzyme existed as monomer or dimer. The first order rate observed at high concentrations is consistent with the proposed intra-dimer intersubunit glucosylation of dimeric glycogenin, while that occurring at low concentrations is consistent with an intramolecular glucosylation of the monomeric enzyme. From half the specific rate difference between the monomeric and dimeric enzyme forms (Fig. 3A), an apparent dissociation constant of 0.85 lM was calculated. As above mentioned, recombinant glycogenin is expressed in wild-type E. coli as a partially glucosylated enzyme containing four to seven glucose units. Accordingly, it was possible to study the rate of autoglucosylation of glycogenin after having acquired an intermediate grown maltosaccharide, by measuring the specific rate of auto[14C]glucosylation of the already glucosylated recombinant form. The wt-31 species showed a pattern in the activity–concentration curve similar to apo-31, with one third reduction of the dimeric specific autoglucosylation rate at the concentrations corresponding to the monomeric form of the enzyme (Fig. 3C). An apparent dissociation constant of 0.6 lM, calculated from the activity–concentration curve, indicated a slightly higher dimerization affinity for glucosylated than non-glucosylated glycogenin. The overall conclusion drawn from the results in Fig. 3A and C is that both, the dimeric and monomeric forms of non-glucosylated and partially glucosylated glycogenin are able to catalyze the autoglucosylation by an intramolecular mechanism of reaction. The comparison of the autoglucosylation rate of monomeric and dimeric glycogenin was extended to the xylosylation reaction. As it was reported before [13], the autoxylosylation of apo-glycogenin and already glucosylated glycogenin results in the incorporation of a unique sugar unit into the hydroxyl group of the tyrosine residue or the C40 -hydroxyl group of glycogenin-linked glucose, respectively. In a difference with the autoglucosylation rate, which represents the average of a variable number of glucose units transferred per glycogenin molecule, the autoxylosylation rate refers to the kinetics for the transfer of one molecular proportion of xylose. Autoxylosylation might thus reveal how the rate of incorporation of a sugar unit into the tyrosine hydroxyl is, related to the incorporation into the glucose hydroxyl. The specific reaction rate for the incorporation of xylose into apo-31 and wt-31 was about 20% lower for monomeric than dimeric glycogenin (Table 1). Independently of the aggregation state, autoxylosylation of the apo-enzyme occurred at half the specific rate of the glucosylated enzyme. In spite of the sugar difference, this result might explain the low rate of diminution of apo-glycogenin relative to the increase in polymerization degree of the glucosylated species, reported during the autoglucosylation time-course analyzed by mass spectrometry [17]. Crystallographic studies of glycogenin showed that the hydroxyl group on the tyrosine residue implicated in the binding of glucose, is at least 15 Å from the C1” position of the glucose residue on the UDP-glucose molecule bound in the same glycogenin monomer, a distance which would not permit an intramonomer reaction
Table 1 Specific autoxylosylation rate of apo-31 and wt-31 Glycogenin (lM)
3 0.3
Specific rate (pmol/min/pmol) 102 apo-31
wt-31
3.19 ± 0.10 2.51 ± 0.02
6.18 ± 0.01 4.83 ± 0.07
Results are means ± SD of three independent experiments.
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for initiation of the autoglucosylation [11,20]. However, a conformational change permitting the necessary motion to reduce the 15 Å distance appear quite plausible considering the dynamics of a protein in solution. This would bring the tyrosine residue into direct contact with UDP-glucose, allowing formation of the covalent intermediate between the active site nucleophile and the donor for the intramonomer glucosylation. In conclusion, in the present work, we described by the first time the existence of monomer glycogenin in solution at concentration below 0.60–0.85 lM, the protein concentration independent autoglucosylation rate of the enzyme at the dimer and monomer states, and the lower reaction rate for autoglycosylation of the tyrosine than glucose hydroxyl group. The results provide the most direct evidence to date that besides dimeric glycogenin, the monomeric form of the enzyme is able to accomplish the incorporation of the first and subsequent glucose units by efficient intramolecular autoglucosylation. Referring to which intramolecular reaction mechanism, intramonomer or intradimer glucosylation, would actually occur in vivo, it is conceivable that just after synthesized, a molecule of glycogenin is able to initiate its autoglucosylation at least for acquiring the first(s) sugar unit(s), without the need to reach the concentration required for dimerization. Whenever dimerization occurs, its autoglucosylation rate will be two fold higher than the glucosylation rate of monomer glycogenin. Thus, the monomer and dimer conversion determining the different rate of autoglucosylation would allow glycogenin to modulate the de novo biosynthesis of proteoglycogen at the initial step of the glucose polymerization. Acknowledgments We thank Drs. J. M. Romero and G. Montich for carefully and critically reading the manuscript. This work was supported by grants from CONICET, FONCyT and SECyT-UNC. S.B. was a Fellow of CONICET. M.E.C. and J.A.C. are Career Investigators of CONICET. References [1] M.A. Aon, J.A. Curtino, Evidence for the glycoprotein nature of retina glycogen, Eur. J. Biochem. 140 (1984) 557–566. [2] J. Pitcher, C. Smythe, D.G. Campbell, P. Cohen, Identification of the 38-kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin, Eur. J. Biochem. 169 (1987) 497–502. [3] J. Pitcher, C. Smythe, P. Cohen, Glycogenin is the priming glucosyltransferase required for the initiation of glycogen biogenesis in rabbit skeletal muscle, Eur. J. Biochem. 176 (1988) 391–395. [4] J. Lomako, W.M. Lomako, W.J. Whelan, A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis, FASEB J. 2 (1988) 3097–3103. [5] C. Smythe, P. Cohen, The discovery of glycogenin and the priming mechanism for glycogen biogenesis, Eur. J. Biochem. 200 (1991) 625–631. [6] C. Cheng, J. Mu, I. Farkas, D. Huang, M.G. Goebel, P.J. Roach, Requirement of the self-glucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae, Mol. Cell. Biol. 15 (1995) 6632–6640. [7] J. Lomako, W.M. Lomako, W.J. Whelan, Substrate specificity of the autocatalytic protein that primes glycogen synthesis, FEBS Lett. 264 (1990) 13–16. [8] S.M. Manzella, L. Rodén, E. Meezan, A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl-b-D-maltoside, Anal. Biochem. 216 (1994) 383–391. [9] Y. Cao, L.K. Steinrauf, P.J. Roach, Mechanism of glycogenin self-glucosylation, Arch. Biochem. Biophys. 319 (1995) 293–298. [10] M.D. Alonso, J. Lomako, W.M. Lomako, W.J. Whelan, Catalytic activities of glycogenin additional to its autocatalytic self-glucosylation, J. Biol. Chem. 270 (1995) 15315–15319. [11] T.D. Hurley, S. Stout, E. Miner, J. Zhou, P.J. Roach, Requirements for catalysis in mammalian glycogenin, J. Biol. Chem. 280 (2005) 23892–23899. [12] L. Roden, S. Ananth, P. Campbell, S. Manzella, E. Meezan, Xylosyl transfer to an endogenous renal acceptor, J. Biol. Chem. 269 (1994) 11509–11513. [13] M.D. Alonso, J. Lomako, W.M. Lomako, W.J. Whelan, J. Preiss, Properties of carbohydrate-free recombinant glycogenin expressed in an Escherichia coli mutant lacking UDP-glucose pyrophosphorylase activity, FEBS Lett. 352 (1994) 222–226. [14] E. Viskupic, Y. Cao, W. Xhang, C. Cheng, A.A. Depaoli-Roach, P.J. Roach, Rabbit skeletal muscle glycogenin. Molecular cloning and production of fully functional protein in Escherichia coli, J. Biol. Chem. 267 (1992) 25759–25763.
332
S. Bazán et al. / Biochemical and Biophysical Research Communications 371 (2008) 328–332
[15] Y. Cao, A.M. Mahrenholz, A.A. DePaoli-Roach, P.J. Roach, Characterization of rabbit skeletal muscle glycogenin: Tyrosine194 is essential for function, J. Biol. Chem. 268 (1993) 14687–14693. [16] S. Bazán, J.A. Curtino, The size of the C-chain maltosaccharide of glycogen: evidence for the presence of only a single branch, Glycobiology 15 (2005) 14C–18C. [17] T.D. Hurley, C. Walls, J.R. Bennett, J.P. Roach, W. Wang, Direct detection of glycogenin reaction products during glycogenin initiation, Biochem. Biophys. Res. Commun. 348 (2006) 374–378. [18] M.E. Carrizo, M.C. Miozzo, A. Goldraij, J.A. Curtino, Purification of rabbit skeletal muscle proteoglycogen: studies on the glucosyltransferase activity
of polysaccharide-free and -bound glycogenin, Glycobiology 7 (1997) 571– 578. [19] V. Lin, J. Mu, J. Yang, P.J. Roach, Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involves an inter-subunit reaction, Arch. Biochem. Biophys. 363 (1999) 163–170. [20] B.J. Gibbons, P.J. Roach, T.D. Hurley, Crystal structure of the autocatalytic initiator of glycogen biosynthesis, glycogenin, J. Mol. Biol. 319 (2002) 463– 477. [21] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein–dye binding, Anal. Biochem. 72 (1976) 248–254.