Tài liệu Báo cáo khoa học: Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation docx

Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation Tanja Albrecht 1 , Sophie Haebel 2 , Anke Koch 1 , Ulrike Krause 1 , Nora Eckermann 1 and Martin Steup 1 1 Institute of Biochemistry and Biology and 2 Interdisciplinary Center for Mass Spectrometry of Biopolymers, University of Potsdam, Potsdam-Golm, Germany Saccharomyces cerevisiae possesses two glycogenin isoforms (designated as Glg1p an d Glg2p) that both contain a con- served tyrosine residue, Tyr232. Howe ver, Glg2 p posse sse s an additional tyrosine residue, Tyr230 and therefore two potential autoglucosylation sites. Glucosylation of Glg2p was studied using both matrix-assisted laser desorption ionization and electrospray quadrupole time o f flight mass spectrometry. Glg2p, carrying a C-terminal (His 6 ) tag, was produced in Escherichia coli and purified. B y tryptic diges- tion and r eversed phase chromatography a peptide (residues 219–246 of the complete Glg2p sequence) was isolated t hat contained 4–25 glucosyl residues. Following incubation of Glg2p with UDPglucose, more than 36 glucosyl residues were covalently bound to this peptide. Using a combination of cyanogen bromide cleavage of the protein backbone, enzymatic hydrolysis of glycosidic bonds and reversed p hase chromatography, mono- and diglucosylated peptides h aving the sequence P NYGYQSSPAM were generated. MS/MS spectra revealed that glucosyl re sidues were attached to both Tyr232 and T yr230 within the same peptide. The formation of the h ighly glucosylated eukaryotic G lg2p did not favo ur the bacterial glycogen accumulation. Under v arious experi- mental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control trans- formed with a Glg2p lacking p lasmid. T he siz e distribution of the glycogen and extractable activities of several glycogen- related enzymes were essentially unchanged. As revealed by high performance anion exchange chromatography, the intracellular maltooligosaccharide pattern of the b acterial cells expressing the functional eukaryotic transgene was significantly altered. Thus, t he eukaryotic glycogenin appears to be incompatible with the bacterial initiation of glycogen biosynthesis. Keywords: glycogenin; glycogen metabolism; self-glucosyla- tion; glucosylation sites; maltodextrins. Almost all organisms possess the capacity to accumulate a-linke d polyglucans that can be utilized if other r educed carbon compounds are i nsufficiently available. Both auto- trophic and heterotrophic prokaryotes synthesize glycogen as fungi and anim als d o [1,2], whereas almost all plastid- containing organisms synthesize starch particles [3]. The prokaryotic glycogen synthases ( EC 2.4.1.11) use ADPglu- cose as the glucosyl donor (which is als o the substrate of the various eukaryotic starch synthases), whereas the polyglucan synthases from both fungi a nd animals rely on UDPglucose [3]. The transfer o f glucosyl residues from either UDPglucose or ADPglucose to the nonreducing e nd of an a-glucan-like primer, as catalyzed by glycogen synthases, results in an elongation of a linear oligoglu can or a polysacc h aride chain and, in conjunction with the branching enzyme (EC 2.4.1.18), in the formation of a glycogen-like molecule [4,5]. However, at least in eukaryotes the cooperation of these two enzymes does not permit the de novo synthesis of a glucan. Both in fungi and in animals glycogen biosynthesis appears to b e initiated by the action of another U DPglucose- dependent glucosyltransferase, designated as glycogenin (EC 2.4.1.186) [6,7]. This homodimeric protein is thought to comprise several distinct enzymatic activities: F irst, in an autocatalytic intersubunit reaction it transfers a glucosyl moiety to a t yrosin residue forming a glucose 1-O-tyrosyl linkage [8]. Second, several glucosyl residues are sequentially transferred to the glucosylglycogenin resulting in an oligo- glucan chain that is covalently bound to the glycogenin. It is possible that these glucosylation reactions (or at least some of them) are due to an intramonomer glucosyl transfer and therefore d iffer mechanistically from the initial glucosylation step(s) [9]. Third, glycogenin is capable of transferring glucosyl residues to unbound acceptors such as free oligo- glucans or oligoglucan derivatives [10,11]. Glycogenin has been found to occur either associated with glycogen synthase [7,12] or covalently linked to Correspondence to M. Steu p, Institute of Biochemistry a nd Biology, Plant Physiology, University of Potsdam, Karl-Liebknecht-Str. 24–25, Building 20, D-14476 Pot sdam-Golm, Germany. Fax: +49 331 9772512, Tel.: +49 331 9772651, E-mail: msteup@rz.uni-potsdam.de Abbreviations: DP, degrees of polymerization; FFF-MALLS-RI, field flow fractionation with multi-angle l aser light scattering and refractive index device; Q, quadrupole; HPAED -PAD, high performance anion exchange chromatography with pulsed amperometric detection; IPTG, isopropyl thio-b- D -galactoside. Enzymes: glycogen s ynthase (EC 2.4.1.11); b ranching enzy me (EC 2.4.1.18); phosphorylase (EC 2.4.1.1); glycogenin (EC 2.4.1.186). (Received 1 5 June 2004, revised 10 August 2004, accepted 1 6 August 2004) Eur. J. Biochem. 271, 3978–3989 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04333.x C-chains of glycogen [13]. Furthermore, a protein f amily has b een recently identified that interacts with the mamma- lian glycogenin and thereby enhances the self-glucosylating activity [14,15]. In prokaryotes the initiation of glycogen b iosynthesis has not yet been elucidated. The occurrence of proteins covalently boun d t o glycogen has been described f or Escherichia coli [16,17], but until now no glycogenin orthologues have been identified in prokaryotic genomes [7]. Recen tly, it has been proposed that in Agrobacterium tumefaciens glycogen synthase catalyzes both an A DPglu- cose-dependent autoglycosylation and an ADPglucose- dependent glucan elongation, suggesting that it f unctionally replaces glycogenin [18]. Similarly, the i nitial reactions of the eukaryotic amylopectin and/or starch granule formation are not known yet. In t he genome of Arabidopsis th aliana L., at least seven glycogenin orthologues h ave been identified but the biochemical functions (and the intracellular locations) of the products of all these genes remain to be defined. In Saccharomyces cerevisiae, two glycogenin isoforms (designated as Glg1p and Glg2p) that a ppear to be functionally equivalent are known. This assumption is based o n e xperiments in which a yeast mutant deficient in both f unctional glycogenin genes was transformed with either the GLG1 or the GLG2 gene and each transformation restored glycogen biosynthesis [19]. In the N-terminal domains (which contain the autoglucosylation region) Glg1p and Glg2p possess a 55% sequence i dentity. Both Glg1p and Glg2p c ontain one conserved t yrosine residue, Tyr232, which presumably corresponds to the single auto- glucosylation site of the rabbit skeletal g lycogenin, Ty r194 [20]. Unlike Glg1p, Glg2p possesses another tyrosine residue, Tyr230 located i n close vicinity to the conserved Tyr232. In in vitro assays performed with Glg2p, mutation of either Tyr230 or Tyr232 resulted in a partial loss of the autoglucosylation activity which was completely abolished when both tyrosine residues w ere replaced by phenylalanine [10]. These data suggest that Glg2p possesses two self- glucosylation sites. H owever, when t he yeast mutant d efi- cient i n both Glg1p an d G lg2p was complemented w ith the doubly mutated Glg2p glycogen biosynthesis was, to some extent, restored and the g lycogen content of the comple- mented cells was a pproximately 10% of that of the wild-type control. A complete loss of the in vivo function of Glg2p was achieved when the m utant was complemen ted w ith a t riply mutated Glg2p lacking Tyr230, Tyr232 and t he C-terminal Tyr362. Glycogen was undetectable in these transformants [10]. Thus, the precise function of the multiple tyrosine residues remains to be clarified. In this communication, we have expressed one of the t wo yeast glycogenins, Glg2p, in E. coli. Functionality of the transgene product was ensured both by monitoring the glycogenin-catalyzed glucosylation reactions and by mass spectrometric analysis of glycogenin-derived glycopeptides. By using t his approach, the following questions were addressed: Does Glg2p utiliz e t wo vicinal glucosyl acceptor sites? Does glucosylation o f these sites r esult in a glycogenin molecule that contains two covalently bound oligosaccha- ride chains? Is T yr362 an additional site o f glucosylation? Does the expression of the functional e ukaryotic glycogenin enhance the bacterial glycogen accumulation and/or affect the size distribution of the bacterial glycogen molec ules? Based on various mass spectrometric techniques we provide direct evidence for a dual glucosylation of Glg2p at Tyr230 and Tyr232, whereas no glucosylation w as observed at Tyr362. The bacterial glycogen accumulation was, however, not stimulated by the production of the functional eukaryotic glycogenin, Glg2p. Materials and methods Cloning of Glg2p S. cerevisiae strain EG328–1A (MATa trp1 leu2 ura3–52) was used. Prior to total RNA preparation cells were grown for 24 h at 30 °Cin 1 yeast nitrogen b ase medium c omple- mented with amino a cids. Total RNA was isolated by using the RNeasy midi kit (Qiagen, Hilden, Germany). For R T-PCR t he following two primers were designed according to the cDNA sequence of Glg2p (accession number U25436) 5¢-ATGGCCAAGAAAGTTGCCATC TGT; 3¢-TCAGGTATCAGGCTTTGGGAATGC. RT- PCR was performed using SSII R NaseH – RT (Invitrogen, Karlsruhe, Germany) and High Fidelity Expand Poly- merase (Roche, Mannheim, Germany). For expression experiments, the cDNA was subcloned i nto pET101/ D-TOPO (Invitrogen) providing a C-terminal (6xHis) tag. The cDNA was confirmed by complete sequencing (AGOWA,Berlin,Germany). Production of Glg2p in E. coli and purification For h eterologous expression, the E. coli s train BL21 Star DE3 (Invitrogen) was used. Cells con taining a Glg2p expression construct were grown on 2 tryptone–yeast extract medium containing 100 lg ampicillin per mL a t 30 °C until exponential growth phase was reached. After induction by isopropyl thio-b- D -galactoside (IPTG; final concentration 0.1 m M ) cultivation was continued for 90 min at 30 °C. Cells were harveste d by centrifugation (12 min at 3000 g;4°C), resuspended in lysis buffer (50 m M NaH 2 PO 4 , 300 m M NaCl, pH 8.0) complemented with 10 m M imidazole (8 mL per 1 g fresh weight of pelleted cells) and broken by sonication for 90 s on ice. Following centrifugation (12 min at 20 000 g;4°C) the supernatant was passed through a nitrocellulose membrane filter (0.45 lm pore size; Schleicher & Schuell, Dassel, Germany). The filtrate was incubated for 60 min with Ni-NTA agarose ( 8 mL filtrate per 1 mL agarose s lurry; Qiagen) un der g entle a gitation on ice. After loading the slurry onto a mini c olumn the Ni-NTA agarose was washed with lysis buffer (10 mL per 8 mL filtrate). Ni-bound proteins were released from the agarose gel by five successive elution steps (1 mL each) using increasing concentrations o f imidazole ( pH 8.0), dissolved in lysis buffer: 3 · 50 m M imidazole, 1 · 75 m M ,and 1 · 250 m M .MostoftheGlg2pproteinwasreleasedby the last e lution step. Western blotting Buffer-soluble proteins were separated by SDS/PAGE and were then transferred to n itrocellulose (Protean, 0 .2 lm pore size; Schleicher & Schuell) for 16 h at 20 V. The transfer buffer c ontained 5 0 m M Tris, 150 m M glycine, 0.02% (w/v) Ó FEBS 2004 Expression of yeast glycogenin (Glg2p) in E. coli (Eur. J. Biochem. 271) 3979 SDS, 20% (v/v) methanol [21]. The His-tagged Glg2p was detected using a primary anti-(His) 5 IgG (Qiagen) and a secondary anti-mouse immunoglobulin coupled to alkaline phosphatase (Promega, Madison, USA). Glycogen-related enzyme activities E. coli cells were pelleted, washed in deionize d water 3 and resuspended i n a medium containing 50 m M HEPES/ NaOH pH 7.5, 1 m M EDTA, 5 m M 1,4-dithioerythritol 4 , 10% (v/v) glycerol, 0.5 m M phenylmethanesulfonyl fluoride, and 2 m M benzamidine. Cells w ere broken by sonification for 90 s on ice a nd the homogenate was centrifuged (12 min at 20 000 g;4°C). The supernatant was used for the enzyme activity assays. Total phosphorylase (EC 2.4.1.1) activity was determined at 30 °C according to [22]. Glyco- gen synthase (EC 2.4.1.11) activity was monitored using 14 C-labeled ADPglucose [23]. Endoamylase activity was estimated by SDS/PAGE following renaturation [24]. Autoglycosylation assay Following purification the r ecombinant Glg2p was dialyzed against 50 m M Hepes/NaOH pH 7.5 (1 h; 4 °C). For in vitro autoglycosylation the protein ( 250 lgÆmL )1 ) was i ncubated at 30 °C in a mixture t hat contained, in a final volume o f 180 lL, 5 m M UDPglucose, 5 m M MnCl 2 and 50 m M HEPES/NaOH pH 7.5. At intervals, aliquots (30 lL) of the reaction m ixture were withdrawn. Following the addi- tion of 15 lL SDS containing sample buffer the protein was denatured (5 min at 95 °C) and used for SDS/PAGE. Protein in-gel digestion and extraction of peptides Following SDS/PAGE and Coomassie blue staining, pro- tein bands (approximately 7 .5 lg protein each) were t reated as describe d in [25]. Protein cleavage by cyanogen bromide Recombinant Glg2p (20 lg) was d issolved in 40 lLofa cyanogen bromide solution (20 mgÆmL )1 in 70% [v/v] trifluoroacetic acid) 5 and incubated f or 4 h at room temperature in darkness. Subsequ ently, t he reaction mixture was lyophilized, redissolved in 100 lLH 2 O (bidest) and dried again. During cleavage methionine is converted to homoserine lactone ()48 Da) which subsequently slowly forms homoserine ()30 Da). a-Amylase treatment The c yanogen bromide-derived peptide mixture was hydro- lyzed using a commercial a-amylase preparation (from Bacillus amyloliquefaciens; Roche, Mannheim, Germany). The lyophilized peptides were dissolved in 40 lLofan a-amylase solution (144 U in 1 mL 50 m M sodium acetate pH 4.8) and incubated for 14–16 h at 37 °C. Amyloglucosidase treatment Following RP-HPLC (see below) selected fractions were lyophilized and the glycopeptides were further deglucosyl- ated by a c ommercial a myloglucosidase (from Aspergillus niger; Roche, Germany). The fractions were dissolved in 20 lL of an amyloglucosidase solution (14 U in 1 mL 50 m M sodium acetate pH 4 .8) and incubated for 30–120 min at 56 °C. Reversed phase high performance liquid chromatography (RP-HPLC) The peptides generated by either in-ge l trypsination or by cyanogen bromide cleavage and subsequent a-amylase treatment were separated by RP-HPLC (SMART system, Pharmacia, Uppsala, Sweden) on a Pharmacia C2/C18 SC 2.1/10 column using a linear 0–50% (v/v) acetonitrile gradient containing 0.1% (v/v) trifluoroacetic acid. A constant flow rate of 100 lLÆmin )1 was applied. In the eluate, absorbance was monitored at 214 nm. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry MALDI-TOF analyses were p erformed using a Reflex II MALDI-TOF instrument (Bruker-Daltonik, B remen, Ger- many). All spectra were recorded in the reflector mode. As matrix 2,5-dihydroxybenzoic acid (20 mg DHB in 1 mL 20% (v/v) aqueous methanol) was used. Aliquots of the eluate fractions of interest (2–3 lL each) were ap plied to the target followed by t he addition of 1 lL of matrix solution and drying under a gentle stream of air. To d etermine t he glucosylation sites, mono-glucosylated peptides purified by RP-HPLC were subjected to post source decay (PSD) analysis. Nanoelectrospray quadrupole time of flight (NanoESI Q-TOF) mass spectrometry MS/MS spectra were recorded using a API QSTAR pulsar I (Applied Biosystems/MDS S ciex, Toronto, Canad a) hybrid mass spectrometer equipped with a nanoelectrospray ion source. The ion of interest was selected in the Q1 quadrupole. Fragments were generated in the collision cell by c ollision with Argon a nd analyzed in the TOF mass analyzer. Glycogen extraction and quantification (procedure A) Bacterial c ells (E. coli strain BL21 s tar DE3) were grown in TY-medium until the exponential growth phase was reached. After induction by IPTG (final concentration 0.1 m M ) for 90 min, culture w as continued in modified M 9 minimal m edium [96 m M Na 2 HPO 4 ,44m M KH 2 PO 4 , 15 m M NaCl, 3 5 m M NH 4 Cl, 0 .1 m M CaCl 2 ,2m M MgSO 4 , 1% (w/v) glucose, and 0.1 m M IPTG]. Under these conditions the E. coli cells accumulate glycogen a nd the expression of the transgene continues. At intervals aliquots of the cell suspension (25 mL each) were with- drawn a nd g lycogen was extracted according to [ 26]. Subsequently, the glucose content of the glycogen fraction was determined enzymatically using t he starch kit (r-biopharm, Darmstadt, Germany). Alternatively, glyco- gen was extracted from the bacterial cells as described below (procedure B ). For nitrogen starvation, NH 4 Cl was omitted from the medium. 3980 T. Albrecht et al. (Eur. J. Biochem. 271) Ó FEBS 2004 Glycogen extraction and size distribution (procedure B) For the determination o f the size distribution of glycogen molecules an alternative extraction procedure was devel- oped. Bacterial cells (100 mL suspension) were pelleted by centrifugation (12 min at 3000 g,4°C), resuspended in 8 mL deionized water and sonicated f or 90 s at 4 °C. The homogenate was centrifuged for 1 2 m in at 20 000 g.The supernatant containing low molecular mass glycans, glyco- gen, nucleic acids, and s oluble proteins was heated (5 min at 100 °C). Denatured proteins were removedby centrifugation (10 min at 10 000 g). High molecular mass nuc leic acids were degraded by adding both DNase (Roche, Germany) and RNase (Macherey/Nagel, Du ¨ ren, Germany; 10 lgÆmL )1 supernatant e ach) and incubation for 4 h at 37 °C. Subse- quently, the nucleases were inactivated by heating (5 min at 100 °C) and the denatured p rotein was removed by centrif- ugation. The supernatant was concentrated by filtration using an Amicon filter (cut-off 10 kDa; Millipore, Eschborn, Germany) and the retentate was transferred into a mixture containing 0.1 M sodium nitrate a nd 0.05% (w/v) sodium azide. This mixture served as eluent for the field flow fractionation multi-angle laser light scattering refractive index (FFF-MALLS-RI) device [27]. After centrifugation (5 min at 14 000 g; pellet discharged) the samples were injected into a symmetrical FFF instrument (F-1000 equipped with a regenerated cellulose membrane, cut off 10 kDa; FFFractionation Inc., S alt Lake City, UT, USA). After an equilibration period of 2 min analytes were separated using a constant channel flow (1 mLÆmin )1 )and a linear c ross flow gradient (0–5 min: 3 mLÆmin )1 , 30–45 min: 0.2 mLÆmin )1 ). Light scattering and concentra- tion were detected with a multiangle DAWN DSP laser photometer (He-Ne-laser; WTC, Santa Barbar a, USA) and Optilab DSP Interferometric Refractometer (WTC), respect- ively. The molecular mass distribution w as calculated fr om light scattering and R I data by using the ASTRA software (version 4.75 , WTC; e xtrapolation by D ebye, first order) . Maltooligosaccharide patterns Bacterial cells (100 mL suspension) were p ellete d by centrifugation (12 min at 3000 g)andwashedwith deionized water. Maltooligosaccharides were extrac ted with 10 mL 80% (v/v) aqueous ethanol for 15 min at 95 °C. Following extraction insoluble c ompounds were removed by centrifug ation ( 10 min at 2 0 000 g) a nd the s upernatant containing the soluble carbohydrates was lyophilized. The residue was resuspended in 4 mL deionized water and proteins were removed from the aqu eous phase by treat- ment with an equal volume o f c hloroform. Deproteinization was repeated three times. Subsequently, the aqueous phase was passed through a 10-kDa membrane (Millipore, Germany) and the filtrate was lyophilized. Finally, the residue was dissolved in 200 lL deionized water a nd used for high performance anion exchange chromatography w ith pulsed a mperometric d etection (HPAEC-PAD, D ionex BioLC) using a CarboPac PA-100 column. Following sample injection (90 lL e ach) the column was equilibrated for10minwith5m M sodium acetate in 1 00 m M NaOH. Analytes were eluted using a linear gradient of sodium acetate (5–500 m M ) in 100 m M NaOH (30 min; flow rate: 1mLÆmin )1 ). Results Glucosylation of recombinant Glg2p The r ecombinant Glg2p carryin g a C-terminal His 6 tag w as purified clo se to homogeneity and incubated with UDPglu- cose. At i ntervals (0, 5, 1 0, 15 and 20 min), aliquots of the incubation mixture were withdrawn and denatured. As revealed by SDS/PAGE, the mobility of the dominant protein band slightly decreased with incubation time suggesting a progressive glucosylation o f the recombinant glycogenin (Fig. 1). For a more detailed a nalysis, the dominant protein band from the patterns obtained a t the beginning (Glg2p 0 ) and from the end of the incubation period (Glg2p 20 ) were excised and digested with trypsin in the gel. T he resulting peptide mixtures were eluted from the gel pieces and analyzed by MALDI-TOF mass spectro- metry. All major peptides of both samples could be a ssigned to tryptic peptides d erived from the Glg2p sequence. However, peptides containing Tyr230 and Tyr232 were detected in neither the nonglucosylated nor the g lucosylated form. In co ntrast, several nonglucosylated tryptic peptides representing residues 3 60–370, 345–370, and 340–370 that all contain the C-terminal Tyr362 were observed as major peaks (data not shown). No traces of peaks with a mas s increment of 162 Da, o r a multiple of it, were d etectable. Thus, it appears that Tyr362, although essential for the functionality of glycogenin, is not glucosylated. In order to detect Glg2p-derived glucopeptides, the two peptide mixtures generated by trypsination of Glg2p 0 and Glg2p 20 were separated by RP-HPLC. For both mixtures, essentially the same H PLC chromatograms were obtained (data not shown). All collected fractions were analyzed by MALDI-TOF MS. For both th e trypsinated Glg2p 0 and Glg2p 20 glucosylated peptides were observed in fractions 12 and 13 (Fig. 2). Glucopeptides were detected as a series of Fig. 1. SDS/PAGE of recombinant Glg2p. Purified recombinant Glg2p was incubated with UDPglucose and M nCl 2 at 3 0 °C. After 0 (lane a), 5 (b), 10 (c), 15 (d), and 20 (e) min an aliquot (7.5 lgprotein each) was denatured and applied to a slab gel. Lane M: relative molecular mass markers. The Glg2p containing Coomassie-stained bands from lanes a (Glg2p 0 ) and e (Glg2p 20 ) were cut out, digested with trypsin and used fo r MALDI-TOF MS analysis (Fig. 2). Ó FEBS 2004 Expression of yeast glycogenin (Glg2p) in E. coli (Eur. J. Biochem. 271) 3981 compounds whose m/z values differ by 162 Da. Despite some overlapping, analytes from HPLC fraction 12 con- tained more covalently bound hexosyl r esidues than those from fraction 13. In Glg2p, both Tyr230 a nd Tyr232 are potential glucosylation s ites. As t rypsin does not cleave between the two vicinal tyrosine residues, all t he glucosylated peptides obtained by trypsination are expected to share the same Fig. 2. MALDI-MS ana lysis o f t ryptic peptides o f Glg 2p. For in vitro autoglucosylation r ecomb inant purified Glg2p w as incubated with U DP- glucose for 0 (Glg2p 0 )or20(Glg2p 20 ) min (Fig. 1 ). Following SDS/PAGE, both protein bands were digested with trypsin and the resulting peptide mixtures were separated by RP-HPLC. Glycopeptide c ontaining eluate fractions were identified by MALDI-TOF MS. For both samples G lg2p 20 and Glg2p 0 mass spectra o f the HPLC f ractions 12 a nd 13 are s hown. 3982 T. Albrecht et al. (Eur. J. Biochem. 271) Ó FEBS 2004 amino acid sequence (residues 219–246) designated as P 1 .In the nonglucosylated state the molecular mass of P 1 is calculated to be 3352.8 Da. Taking into account that the two methionine residues are likely to be oxidized during analyte p rocessing the actual mass of the (nonglucosylated) peptide P 1 is assumed to be 3384 .8 Da. B y using this m/z value a nd the data shown in Fig. 2 it is estimated t hat 4–25 glucosyl residues are covalently bound to P 1 . This implies that Glg2p i s significantly g lucosylated during p roduction in E. coli. Glucosylation in E. coli has also been observed with the rabbit muscle glycogenin [20]. I n the latter study 1–8 glucosyl residues were found to be linked to Tyr194. Following se lf-glycosylation for 20 min, the glucosylation of Glg2p is even more complex. At least 30 m/z signals originating from the differently g lucosylated P 1 peptide were detected (Fig. 2). In similar experiments, up to 40 glucosyl residues attached to P 1 were observed (data not shown). Following over night incubation of Glg2p with UDPglu- cose, a series of free oligosaccharides ranging from degrees of polymerization (DP) 7–25 was also detected (data not shown). Identification of glucosylation sites The data shown in F ig. 2 clearly indicate a high degree of glucosylation of the Glg2p-derived peptide P 1 .However, they d o not permit the determination of the actual g lucosy- lation site(s) w ithin t he peptide. The amino acid residue(s) that is/are covalently bound to glucosyl moieties can be identified by mass spectrometry of Glg2p-derived fragments if two prerequisites are g iven: First, the glucopeptide to be fragmented must be suitable in size. Second, the fragment pattern obtained must be do minated by the fragmentation of the p eptide backbone (and not by that of the glucosidic bonds). For fragmentation studies, Glg2p was treated with cyanogen bromide rather t han with t rypsin as the c hemical cleavage results in smaller p eptides. Cyanogen bromide (and also trypsin) does not cleave between Tyr230 a nd Tyr232 and therefore a mixtu re of peptides is obtained one of which contains both tyrosine residues. Following chemical clea- vage, the peptide mixture was incubated with a hydro lase (such as a-amylase) and was then separated by HPLC. Enzymatic deglucosylation was found to be essential as fragmentation of highly glucosylated pep tides o ccurs most frequently by cleavage of glycosidic linkages w hereas peptide backbone fragments are suppressed. The latter are, however, relevant for t he identification of glucosylation sites. The HPLC chromatogram of a Glg2p-derived peptide mixture, as obtained by cyanogen bromide and a-amylase treatment, is shown in Fig. 3A. As revealed by MALDI- TOF analysis, several eluate fractions contained an 11-mer peptide having the sequence PNYGYQSSPAM (residues 228–238; designated as P 2 ) but differing in the degree of glucosylation. In fraction 19, P 2 was observed in the monoglucosylated form d esignated as G -P 2a . However, the majority of the peptide P 2 occurs in higher glucosylated forms as revealed by MALDI-TOF analysis of fractions 11–15. These g lucopeptides were resist ant to a prolonged or repeated a-amylase treatment i ndicating an exhaustive a-amylase action. For a more effective deglucosylation of the higher glucosylated forms of P 2 , a second enzymatic treatment was included: RP-HPLC fractions 11–15 (Fig. 3A) were pooled, l yophilized and incubated w ith a myloglucosidase. Subsequently, the peptides were resolved by a second RP-HPLC run (Fig. 3B). In the elution profile of th e second chromatogram, peaks in the original region (fractions 11–15; Fig. 3A) disappeared and new peaks (fraction 12, 15 and 18; Fig. 3B) were detected indicating that the amyloglucosidase treatment was effective. These RP-HPLC fractions we re analyzed by MALDI-TOF MS. The mass spectra obtained show that the number of h exosyl residues attached t o P 2 was reduced to 0, 1 or 2. The completely deglucosylated P 2 was recovered in fraction 18 (Fig. 3B). The occurrence of the nonglucos- ylated peptide s trongly suggests that the amyloglucosidase is capable of cleaving both the interglucose bonds and the glycosidic linkage between a tyrosine residue and a glucose moiety. The mono-glucosylated P 2 was recovered in fraction 15 of the second RP-HPLC run (Fig. 3B) and was designated as G-P 2b . The diglucosylated P 2 was detected in fraction 12 (Fig. 3B) and is referred to as G 2 -P 2b . The two mono-glucosylated P 2 samples, G-P 2a and G-P 2b and the diglucosylated peptide, G 2 -P 2b were analyzed by fragmentation using both Q -TOF MS/MS a nd MALDI- TOF P SD. F ragmentation often results from cleavage of the peptide backbone. Fragments obtained are classified as a-, b- or y-type fragments [28]. B oth a- a nd b-type fragments contain the N-terminus of the peptide. Unlike a -type fragments, b-fragments are generated by breakage of the peptide bond and t herefore both types differ by one CO group, i.e. a mass of 28 Da. All y-type fragments contain the C-terminus of the p eptide. Many fragments show a satellite peak at )17 Da. This peak is due to a loss of NH 3 , presumably from an asparagine residue. The fragments observed b y Q -TOF MS/MS for the two mono-glucosyl- ated P 2 conjugates, G-P 2a and G-P 2b are compiled in Table 1 . The corresponding spectra are shown in Fig. 4. Essentially the same results were obtained by MALDI-TOF PSD (results not shown). For both G-P 2a and G-P 2b fragmentation of the singly charged peptides gives rise to a strong series of b type ions ranging from b2 to b8. The relative molecular m ass d ifference b etween two c onsecutive b-ions corresponds to the mass o f the amino acid residue at that position in the sequence. An additional relative molecular mass increment of 162 Da reveals that a hexose is attached to the corresponding amino acid. In the M S/ MS spectra from G -P 2a (Fig. 4 A) the fragments b3 and b4, which both contain Tyr230, were observed without a glucosyl residue being attached. In contrast, the fragment b5 containing both Tyr230 and Tyr232 was recovered both in the nonglucosylated and in the glucosylated form. Presumably, the formation of a nonglucosylated b5 fragment is due to a simultaneous backbone fragmentation and loss of glucose. The lability of the glycosidic bond is also indicated by t he large (M+H + )-162 peak. However, the occurrence of both b3 and b4 exclusively in the nonglucosylated state indicates that in G-P 2a , T yr230 does not carry a glucosyl residue and therefore Tyr232 must be the glucosylated residue. The fragment p attern obtained with G-P 2b differs signi- ficantly ( Table 1 and Fig. 4B). All fragments from b3 to b8 wererecoveredbothintheglucosylatedandinthe nonglucosylated form. As the two fragments b3 and b4 Ó FEBS 2004 Expression of yeast glycogenin (Glg2p) in E. coli (Eur. J. Biochem. 271) 3983 contain Tyr230 but not Tyr232 it is obvious that in G-P 2b the Tyr230 is a glucosyl acceptor. In summary, the MS/MS analysis of G -P 2a and G -P 2b clearly shows that following production in E. coli,Glg2p possesses two functional glucosylation sites, Tyr230 and Tyr232. Simultaneous glucosylation of the two vicinal tyrosine residues occurs. This conclusion was reached by MS/MS analysis o f the sin gly charged g lucopeptide G 2 -P 2b (Fig. 3B). The Q-TOF MS/MS spectrum is shown in Fig. 5 and the fragments observed are listed in Table 2. The fragmentation spectrum is again dominated by the strong series of b fragments. As expected, b2 is observed only in the nonglucosylated form. Fragments b3 and b4 are observed in the mono-glucosylated and in the nonglucosylated state whereas t he diglucosylated form is undetectable. I n contrast, fragments b5 to b8 occur i n t he diglucosylated as well as in the mono- and in the nonglucosylated form. This is exactly the fragmentation pattern to b e p redicted if both Tyr230 and Tyr232 bear one glucosyl residue each. Expression of the eukaryotic glycogenin and bacterial glycogen accumulation The data s hown in Figs 3–5 clearly indicate t hat the transformed E. coli cells produce the recombinant Glg2p in a f unctional and highly glucosylated state. As the Fig. 3. RP-HPLC and MALDI-MS analyses of Glg2p-derived peptides (cyanogen bromide cleavage). Recombinant purified Glg2p (20 lg) was incubated with cyanogen bromide and subsequently with a-amylase. The partially deglucosylated peptides were separat ed by RP-HPLC (A). Eluat e fractions were analyzed by MALDI-TOF MS as indicated. Eluate fractions 11–15 were pooled, inc ubated with amyloglucosidase and then subjected to a second RP-HPLC ( B). Eluate fractions were analyzed by MALDI-TOF M S a s i ndicated. Eluate fractions marked w ith * were used for further investigations (see Figs 4 and 5). 3984 T. Albrecht et al. (Eur. J. Biochem. 271) Ó FEBS 2004 initiation of the prokaryotic glycogen biosynthesis is still incompletely understood, we investigated whether or not the expression of the functional eukaryotic glycogenin supports the bacterial glycogen accumulation. During growth in the trypto ne–yeast extract medium E. coli cells formed only insignificant amounts of glycogen irrespective of a proceeding transformation with the GLG 2 gene or an induction of the transgene expression (data not shown). In contrast, bacterial cells accumulated glycogen following a transfer t o t he modified M9 minimal medium. Therefore, we chose the following growth and induction protocol: after growth in tryptone–yeast extract m edium, the production of G lg2p was induced by IPTG under other- wise unchanged conditions. Ninety minutes later cells were transferred to modified M9 minimal medium. At intervals, aliquots of the suspension were withdrawn and the cellular glycogen content was monitored. As a control, E. coli cells containing the plasmid without the insert were kept under exactly the same conditions. A s revealed by Western blotting using an a nti-(His) 5 IgG 9 , Glg2p was detectable during the entire period of glycogen accumulation (Fig. 6A). Bacterial glycogen was determined by either of two methods (see Materials and methods). Procedure A does not require homogenization of the cells but presumably results in a partial hydrolysis of the polyglucan. P rocedure B yields an ess entially unmodified polysaccharide fraction as revealed by control experiments using a commercial glyco- gen p reparation. By applying both m ethods we co nsistently observed t hat throughout the culture in the modified M 9 minimal medium the glycogenin producing E. coli cells did accumulate approximately 30% less glycogen than the Table 1. List of the b-type fragments of the Glg2p-derived mono-glu- cosylated peptides G-P 2a and G-P 2b obtained by Q-TOF MS/MS analysis. All fragments observed in the spectrum (Fig. 4) are printed in bold le tters. F or each b-type frag menttherelativeamountofthe glucosylated species is given in percenta ge. For nomenclatu re of the fragment ions se e [28]. G-P 2a Sequence G-P 2b b ions b ions + 162 % b ions b ions + 162 % 98.1 260.1 P 98.1 260.1 212.1 374.1 PN 212.1 374.1 375.2 537.2 PNY 375.2 537.2 34 432.2 594.2 PNYG 432.2 594.2 32 595.3 757.3 36 PNYGY 595.3 757.3 38 723.3 885.3 39 PNYGYQ 723.3 885.3 40 810.3 972.3 39 PNYGYQS 810.3 972.3 44 897.4 1059.4 42 PNYGYQSS 897.4 1059.4 41 994.4 1156.4 PNYGYQSSP 994.4 1156.4 1065.5 1227.5 PNYGYQSSPA 1065.5 1227.5 1148.5 1310.5 PNYGYQSSPAX 1148.5 1310.5 Fig. 4. Nanoelectrospray Q-TOF MS/MS spectra of the Glg2p-derived mono-glucosylated peptides. Part of the fragmentation spectra obtained f or G-P 2a and G -P 2b (Fig. 3) are shown in F ig. 4 A and B, respectively. Both a - and b-ty pe fragments c ontain the N -termin us of th e peptide, th ey differ by one CO group, i.e. a mass of 28 Da. y-Type fragments are C-terminal [28]. Probably due to a loss of NH 3 from asparagine, many fragments show a satellite peak at )17 Da. F ragments bearing a glucosyl moie ty are marked w ith an a sterisk (*). A su mmary of the g lucosylation state of the observed b-type fragments i s given in Table 1. Ó FEBS 2004 Expression of yeast glycogenin (Glg2p) in E. coli (Eur. J. Biochem. 271) 3985 control cells. In F ig. 6B, the cellular glycogen content, as determined by procedure A, was followed o ver 20 h. At t he end of this period of time, the protein-based glycogen level of the G lg2p expressing bacterial cells was 70.6 ± 7.2% of that of the c ontrol cells (average of five i ndependent experiments). The glycogenin producing E. coli cells did not differ from the control with r espect to growth rate and the content of buffer soluble proteins (data not shown). The size distribution of the bacterial glycogen formed either in the presence or t he absence o f the eukaryotic glycogenin was determined. As revealed by Western blotting experiments performed with buffer soluble proteins, the recombinant GlG2 gene was expressed throughout the e ntire period of glycogen accumulation (Fig. 6A). E. coli c ells that had b een transformed with t he plasmid lacking the GlG2 gene were cultivated a nd harvested using precisely the same conditions. From all six cell s amples glycogen was prepared and analyzed by FFF-MALLS-RI. The molecular mass distribution of the glycogen averages from 4 · 10 7 to 1.5 · 10 8 gÆmol )1 for both G lg2p-producing cells and for the control. For clarity, only the onset of glycogen accumulation (0 h) and 20 h are shown in Fig. 6C. P urity of the glycogen preparations was ensured by acid hydrolysis and s ubsequent monosaccharide analysis (data not shown). Bacterial maltodextrin patterns The oligosaccharide patterns from both g lycogenin produ- cing cells and the control cells are complex and contain more than 30 compounds. Most of the oligosaccharides were eluted during 0–15 m in indicating a DP of < 12. In the Glg2p-forming cells, t he by far dominant oligosaccharide peak eluted between DP 5 and DP 6 of a maltodextrin standard. This c ompound is present in the oligosaccharide pattern o f t he control cells as well but in the g lycogenin- producing cells it is significantly increased. Following acid hydrolysis, the relative glucose content of both oligosaccharide fractions exceeded 95% (data not shown). Fig. 5. Nanoelectrospray Q-TOF MS/MS spe ctrum of the Glg2p-derived diglucosylated peptide G 2 -P 2b . For details see Figs 3 and 4. Fragments bearing one or two g lucosyl m oietie s are mark ed wi th a single asterisk (*) and double asterisks (**), respectiv ely. A summ ary of the glucosylation state of th e observed b -type fragments i s given in T able 2. Table 2. List of the b-type fragments of the Glg2p-derived monoglu- cosylated peptide G 2 -P 2b obtained by Q -TOF M S/MS ana lysis. Frag- ments o bse rved in the spectrum (Fig. 5) are printed in bold lette rs. For nomenclature of the fragm ent ions s e e [28]. G 2 -P 2b b ions b ions + 162 b ions + 324 Sequence 98.1 260.1 422.1 P 212.1 374.1 536.1 PN 375.2 537.2 699.1 PNY 432.2 594.2 756.1 PNYG 595.3 757.3 919.2 PNYGY 723.3 885.3 1047.3 PNYGYQ 810.3 972.3 1134.3 PNYGYQS 897.4 1059.4 1221.3 PNYGYQSS 994.4 1156.4 1318.4 PNYGYQSSP 1065.5 1227.5 1389.4 PNYGYQSSPA 1148.5 1310.5 1472.5 PNYGYQSSPAX 3986 T. Albrecht et al. (Eur. J. Biochem. 271) Ó FEBS 2004 It is therefore reasonable to assume that the vast majority of the compounds resolved by HPAEC (Fig. 7) are homoglucans. Discussion In this communication, we have studied glucosylation of one o f the two yeast glycogenins, Glg2p, under both in vivo and in vitro conditions. Following production in E. coli, purification and trypsin treatment, a Glg2p-derived pep tide (designated as P 1 ) was isolated that contains covalently bound glucosyl residues covering a wide range of DP. For P 1 , analyte ions having more than 23 different m/z values were observed. Following self-glucosylation for 20 min under in vitro conditions, for P 1 at least 3 0 different m/z values were detected (Fig. 2). From the data shown in Fig. 2, we calculated that up to 35 (or, in other experiments, even up to 40) glucosyl residues are covalently linked to the Glg2p derived P 1 peptide. This is an unexpectedly high glucose content of the peptide that, to the best of our knowledge, has n ot yet been reported fo r glycogenins. For the rabbit muscle glycogenin, approximately 10 glucosyl residues have been observed to be linked to a single glucosylation site [20]. As Glg2p contains two putative g lucosylation sites, Tyr230 and Tyr232, self- glucosylation of Glg2 p is e xpected to give rise to covalently boun d chains that possess at least 17–20 glucosyl residues, provided that both Tyr230 and Tyr232 are occupied within the same glycogenin molecule. Peptide P 2 (PNYGYSSPAM) was obtained by chemical cleavage and a llowed the identificat ion of the glucosylation sites (Fig. 3). Following heterologous expression in E. coli , we observed t his peptide in a nonglucosylated form only following tre atment with both a-amylase and amyloglucos- idase. Thus, it seems t hat i n E. coli the eukaryotic glyco- genin is almost quantitatively glucosylated. As deduced from Fig. 2, the minimum number of glucosyl residues attached to Glg2p is four. By combining protein backbone cleavage, enzymatic hydrolysis of glycosidic bonds and M S/MS analysis we provide direct evidence that both Tyr230 and Tyr232 a ct as glucosylation sites of Glg2p. Discrimination between the two glucosylated tyrosine residues was achieved by taking advantage of a se lectivity of the a-amylase. When P 2 was reacted with a-amylase, glucosyl residues linked to Tyr232 were removed with the exception of the glucose that is covalently bound to the amino acid residue. I n contrast, the glucan chain linked to Tyr230 was incompletely hydrolyzed even after prolonged incubation. It is likely that a-amylase acts effectively on the glucans bound to Tyr232 only if Tyr230 is not glucosylated. Whilst the reason for this selective a-amylase action is unknown, it is useful for the generation of glucosylated peptides that are accessible to Fig. 7. Maltodextrin pattern of Glg2p-producing E. coli cells. Bacte rial cells were grown for 2 0 h in modified M9 medium. As a c ontrol, E. coli cells transformed with a plasmid lacking the GlG2 gene were grown simultaneously. Following the extraction in 80% (v/v) ethanol, the deproteinized extracts were analy zed by H PAEC-PAD. As a standard, a commercial maltodextrin sample (Dextrin 15, Fluka, Germany) was used. Data f rom a single e xperiment are shown. The maltod extrin patterns were con firmed in two additional independently performed experiments. Fig. 6. G l yco gen content a nd glycogen size dist ribut ion in E. coli cells foll owing G lg2p e xpression. (A) Western blottin g of buffer-soluble p roteins extracted from E. coli cells after 2 0 h growth in modified M9 medium. Lane a: Control (bacterial c ells transformed with a plasmid lacking the GlG2 gene); lane b: Glg2p producing cells. Per lane 50 lg protein was applied. Following transfer to nitrocellulose, proteins were p robed using an anti-His Ig. (B) E. coli ce lls transformed with a p lasm id containing (dark columns) or lacking (control; white c olumns) the GlG2 gene were transferred to modified M9 medium. At inte rvals, aliquots were w ithdrawn a nd the glyco gen content was monitored (procedure A; see Materials and metho ds). Glycogen was quantified as glucose equivalents and based on the buffer-soluble proteins. Data from a single experiment are shown. In four additional independently performed experiments similar data were obtained. (C) Size distribution of glycogen prepared from Glg2p producing E. coli cells and control (t ¼ 0 h and 20 h). G lycogen was pre pared using proc edure B (see Materials a nd methods). All other experimental conditionsasinFig.6A: ,Glg2p(0h);m,Glg2p(20h); , con trol (0 h); a nd , c on trol (20 h). Data from a single experiment are shown. T hree independently performed experiments yielded essentially the s ame results. Ó FEBS 2004 Expression of yeast glycogenin (Glg2p) in E. coli (Eur. J. Biochem. 271) 3987 [...]... is increased more than threefold that, as judged from HPAEC, has a DP of approximately 5 It is remarkable that the size of this maltodextrin is significantly lower than that of the Glg2p-associated glucans Possibly, the Glg2p-derived oligoglucans are subjected to an extended maltodextrin metabolism External maltose and maltodextrins appear to be metabolized by a complex pathway that includes the action... proteoglycogen by isoamylase and is able to autoglucosylate Biochem Biophys Res Commun 305, 811–814 14 Skurat, A. V., Dietrich, A. D., Zhai, L & Roach, P.J (2002) GNIP, a novel protein that binds and activates glycogenin, the selfglycosylating initiator of glycogen biosynthesis J Biol Chem 277, 19331–19338 15 Zhai, L., Dietrich, A. , Skurat, A. V & Roach, P.J (2004) Structure-function analysis of GNIP, the glycogenin- interacting... treatment (fraction 18) and therefore their initial glucosylation state is unknown The monoglucosylated peptide eluting in fraction 15 accounts for approximately 20% The corresponding MS/MS spectrum revealed that the glucose is mainly attached to Tyr230, however, a deglucosylation of Tyr232 by the action of the amyloglucosidase cannot be excluded In summary: approximately 10% of the Glg2p protein is glucosylated. .. glycogen: Agrobacterium tumefaciens glycogen synthase is involved in glucan initiation and elongation Proc Natl Acad Sci USA 100, 10659–10663 19 Cheng, C., Mu, J., Farkas, I., Huang, D., Goebl, M.G & Roach, P.J (1995) Requirement of the selfglucosylating initiator proteins Glg1p and Glg2p for glycogen accumulation in Saccharomyces cerevisiae Mol Cell Biol 15, 6632–6640 20 Cao, Y., Mahrenholz, A. M., DePaoli-Roach,... that two glucan chains can be synthesized simultaneously by a single glycogenin molecule It probably explains the unusually high number of glucosyl residues attached to Glg2p The dual glucosylation of both Tyr230 and Tyr232 within the same Glg2p molecule, as demonstrated in this study, is consistent with the failure to detect both residues during Edman degradation [10] Although Glg2p is effectively glucosylated. .. glucosylated on Tyr232 only, 30% of the protein is glucosylated on both tyrosine residues and the remaining 60% are glucosylated either on both residues or on Tyr230 only As revealed by Q-TOF MS/MS a major proportion of the Glg2p molecules is simultaneously glucosylated at both Tyr230 and Tyr232 (Fig 5 and Table 2) This feature, which has not been described for other glycogenins, is remarkable as it... The discovery of glycogenin and the priming mechanism for glycogen biogenesis Eur J Biochem 200, 625–631 7 Lomako, J., Lomako, W.M & Whelan, W.J (2004) Glycogenin: the primer for mammalian and yeast glycogen synthesis Biochim Biophys Acta 1673, 45–55 8 Lin, A. , Mu, J., Yang, J & Roach, P.J (1999) Self-glucosylation of glycogenin, the initiator of glycogen biosynthesis, involved an inter-subunit reaction... 11 Manzella, S.M., Roden, L & Mezan, E (1994) A biphasic radiometric assay of glycogenin using the hydrophobic acceptor n-dodecyl -a- maltoside Anal Chem 216, 383–391 12 Tavridou, A & Agius, L (2003) Phosphorylase regulates the association of glycogen synthase with a proteoglycogen substrate in hepatocytes FEBS Lett 551, 87–91 13 Romero, J.M & Curtino, J .A (2003) C-chain-bound glycogenin is released... of an effective initiation of glycogen biosynthesis appears to be even lower in the presence of the eukaryotic glycogenin It should, however, be noted that the turnover of the bacterial glycogen has not yet been analyzed In Glg2p-producing E coli cells, the pattern of extractable maltodextrins is significantly altered (Fig 7) As the most prominent change, the level of a relatively small maltodextrin is. .. essentially all the Glg2p extracted from the bacterial cells was found to be glucosylated, the autoglucosylation of the glycogenin is unlikely to be limited by the cellular levels of UDPglucose In E coli, UDPglucose plays a central role in various pathways, such as the galactose or trehalose metabolism, and also in the biosynthesis of membrane-derived oligosaccharides [31,32] Thus, it seems that the eukaryotic . Yeast glycogenin (Glg2p) produced in Escherichia coli is simultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial. mass o f the amino acid residue at that position in the sequence. An additional relative molecular mass increment of 162 Da reveals that a hexose is attached