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 issimultaneously glucosylated at two vicinal tyrosine residues but results in a reduced bacterial glycogen accumulation Tanja Albrecht

Yeast glycogenin (Glg2p) produced in Escherichia coli is

simultaneously glucosylated at two vicinal tyrosine residues but

results in a reduced bacterial glycogen accumulation

Tanja Albrecht1, Sophie Haebel2, Anke Koch1, Ulrike Krause1, Nora Eckermann1and Martin Steup1

1

Institute of Biochemistry and Biology and2Interdisciplinary Center for Mass Spectrometry of Biopolymers, University of Potsdam, Potsdam-Golm, Germany

Saccharomyces cerevisiaepossesses two glycogenin isoforms

(designated as Glg1p and Glg2p) that both contain a

con-served tyrosine residue, Tyr232 However, Glg2p possesses

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 of flight mass

spectrometry Glg2p, carrying a C-terminal (His6) tag, was

produced in Escherichia coli and purified By tryptic

diges-tion and reversed phase chromatography a peptide (residues

219–246 of the complete Glg2p sequence) was isolated that

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 phase

chromatography, mono- and diglucosylated peptides having

the sequence PNYGYQSSPAM were generated MS/MS

spectra revealed that glucosyl residues were attached to both Tyr232 and Tyr230 within the same peptide The formation

of the highly glucosylated eukaryotic Glg2p did not favour the bacterial glycogen accumulation Under various experi-mental conditions Glg2p-producing cells accumulated approximately 30% less glycogen than a control trans-formed with a Glg2p lacking plasmid The size 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 bacterial cells expressing the functional eukaryotic transgene was significantly altered Thus, the 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-linked polyglucans that can be utilized if other reduced

carbon compounds are insufficiently available Both

auto-trophic and heteroauto-trophic prokaryotes synthesize glycogen

as fungi and animals do [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 also the substrate of the

various eukaryotic starch synthases), whereas the polyglucan

synthases from both fungi and animals rely on UDPglucose

[3] The transfer of glucosyl residues from either UDPglucose

or ADPglucose to the nonreducing end of an a-glucan-like primer, as catalyzed by glycogen synthases, results in an elongation of a linear oligoglucan or a polysaccharide 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 be initiated by the action of another UDPglucose-dependent glucosyltransferase, designated as glycogenin (EC 2.4.1.186) [6,7] This homodimeric protein is thought to comprise several distinct enzymatic activities: First, in an autocatalytic intersubunit reaction it transfers a glucosyl moiety to a tyrosin 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 differ 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 Steup, Institute of Biochemistry and Biology,

Plant Physiology, University of Potsdam, Karl-Liebknecht-Str 24–25,

Building 20, D-14476 Potsdam-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 laser 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 synthase (EC 2.4.1.11); branching enzyme

(EC 2.4.1.18); phosphorylase (EC 2.4.1.1); glycogenin (EC 2.4.1.186).

(Received 15 June 2004, revised 10 August 2004, accepted 16 August

2004)

C-chains of glycogen [13] Furthermore, a protein family

has been recently identified that interacts with the

mamma-lian glycogenin and thereby enhances the self-glucosylating

activity [14,15]

In prokaryotes the initiation of glycogen biosynthesis has

not yet been elucidated The occurrence of proteins

covalently bound to glycogen has been described for

Escherichia coli [16,17], but until now no glycogenin

orthologues have been identified in prokaryotic genomes

[7] Recently, it has been proposed that in Agrobacterium

tumefaciensglycogen synthase catalyzes both an

ADPglu-cose-dependent autoglycosylation and an

ADPglucose-dependent glucan elongation, suggesting that it functionally

replaces glycogenin [18] Similarly, the initial reactions of the

eukaryotic amylopectin and/or starch granule formation are

not known yet In the genome of Arabidopsis thaliana L., at

least seven glycogenin orthologues have 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 appear to be

functionally equivalent are known This assumption is

based on experiments in which a yeast mutant deficient in

both functional 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 identity Both

Glg1p and Glg2p contain one conserved tyrosine residue,

Tyr232, which presumably corresponds to the single

auto-glucosylation site of the rabbit skeletal glycogenin, Tyr194

[20] Unlike Glg1p, Glg2p possesses another tyrosine

residue, Tyr230 located in 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 were replaced by phenylalanine

[10] These data suggest that Glg2p possesses two

self-glucosylation sites However, when the yeast mutant

defi-cient in both Glg1p and Glg2p was complemented with the

doubly mutated Glg2p glycogen biosynthesis was, to some

extent, restored and the glycogen content of the

comple-mented cells was approximately 10% of that of the wild-type

control A complete loss of the in vivo function of Glg2p was

achieved when the mutant was complemented with a triply

mutated Glg2p lacking Tyr230, Tyr232 and the 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 two

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 this approach, the following questions were

addressed: Does Glg2p utilize two vicinal glucosyl acceptor

sites? Does glucosylation of these sites result in a glycogenin

molecule that contains two covalently bound

oligosaccha-ride chains? Is Tyr362 an additional site of glucosylation?

Does the expression of the functional eukaryotic glycogenin

enhance the bacterial glycogen accumulation and/or affect

the size distribution of the bacterial glycogen molecules?

Based on various mass spectrometric techniques we provide direct evidence for a dual glucosylation of Glg2p at Tyr230 and Tyr232, whereas no glucosylation was 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 cerevisiaestrain EG328–1A (MATa trp1 leu2 ura3–52) was used Prior to total RNA preparation cells were grown for 24 h at 30C in

comple-mented with amino acids Total RNA was isolated by using the RNeasy midi kit (Qiagen, Hilden, Germany)

For RT-PCR the 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 RNaseH–RT (Invitrogen, Karlsruhe, Germany) and High Fidelity Expand Poly-merase (Roche, Mannheim, Germany) For expression experiments, the cDNA was subcloned into pET101/ D-TOPO (Invitrogen) providing a C-terminal (6xHis) tag The cDNA was confirmed by complete sequencing (AGOWA, Berlin, Germany)

Production of Glg2p inE coli and purification For heterologous expression, the E coli strain BL21 Star DE3 (Invitrogen) was used Cells containing a Glg2p expression construct were grown on

medium containing 100 lg ampicillin per mL at 30C until exponential growth phase was reached After induction by isopropyl thio-b-D-galactoside (IPTG; final concentration 0.1 mM) cultivation was continued for 90 min at 30C Cells were harvested by centrifugation (12 min at 3000 g; 4C), resuspended in lysis buffer (50 mM NaH2PO4, 300 mM

NaCl, pH 8.0) complemented with 10 mM 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; 4C) 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 slurry; Qiagen) under gentle agitation on ice After loading the slurry onto a mini column 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 of imidazole (pH 8.0), dissolved

in lysis buffer: 3· 50 mM imidazole, 1· 75 mM, and

1· 250 mM Most of the Glg2p protein was released by the last elution step

Western blotting Buffer-soluble proteins were separated by SDS/PAGE and were then transferred to nitrocellulose (Protean, 0.2 lm pore size; Schleicher & Schuell) for 16 h at 20 V The transfer buffer contained 50 m Tris, 150 m glycine, 0.02% (w/v)

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 colicells were pelleted, washed in deionized water

resuspended in a medium containing 50 mM HEPES/

NaOH pH 7.5, 1 mM EDTA, 5 mM 1,4-dithioerythritol

10% (v/v) glycerol, 0.5 mMphenylmethanesulfonyl fluoride,

and 2 mMbenzamidine Cells were broken by sonification

for 90 s on ice and the homogenate was centrifuged (12 min

at 20 000 g; 4C) The supernatant was used for the

enzyme activity assays Total phosphorylase (EC 2.4.1.1)

activity was determined at 30C according to [22]

Glyco-gen synthase (EC 2.4.1.11) activity was monitored using

14C-labeled ADPglucose [23] Endoamylase activity was

estimated by SDS/PAGE following renaturation [24]

Autoglycosylation assay

Following purification the recombinant Glg2p was dialyzed

against 50 mMHepes/NaOH pH 7.5 (1 h; 4C) For in vitro

autoglycosylation the protein (250 lgÆmL)1) was incubated

at 30C in a mixture that contained, in a final volume of

180 lL, 5 mM UDPglucose, 5 mM MnCl2 and 50 mM

HEPES/NaOH pH 7.5 At intervals, aliquots (30 lL) of

the reaction mixture were withdrawn Following the

addi-tion of 15 lL SDS containing sample buffer the protein was

denatured (5 min at 95C) and used for SDS/PAGE

Proteinin-gel digestion and extraction of peptides

Following SDS/PAGE and Coomassie blue staining,

pro-tein bands (approximately 7.5 lg propro-tein each) were treated

as described in [25]

Protein cleavage by cyanogen bromide

Recombinant Glg2p (20 lg) was dissolved in 40 lL of a

cyanogen bromide solution (20 mgÆmL)1 in 70% [v/v]

trifluoroacetic acid)

temperature in darkness Subsequently, the reaction mixture

was lyophilized, redissolved in 100 lL H2O (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 cyanogen 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 lL of an

a-amylase solution (144 U in 1 mL 50 mMsodium acetate

pH 4.8) and incubated for 14–16 h at 37C

Amyloglucosidase treatment

Following RP-HPLC (see below) selected fractions were

lyophilized and the glycopeptides were further

deglucosyl-ated by a commercial amyloglucosidase (from Aspergillus niger; Roche, Germany) The fractions were dissolved in

20 lL of an amyloglucosidase solution (14 U in 1 mL

50 mM sodium acetate pH 4.8) and incubated for 30–120 min at 56C

Reversed phase high performance liquid chromatography (RP-HPLC)

The peptides generated by either in-gel 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 performed using a Reflex II MALDI-TOF instrument (Bruker-Daltonik, Bremen, 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 applied to the target followed by the addition of 1 lL of matrix solution and drying under a gentle stream of air To determine the 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 Sciex, Toronto, Canada) 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 collision with Argon and analyzed in the TOF mass analyzer Glycogen extraction and quantification (procedure A) Bacterial cells (E coli strain BL21 star DE3) were grown in TY-medium until the exponential growth phase was reached After induction by IPTG (final concentration 0.1 mM) for 90 min, culture was continued in modified M9 minimal medium [96 mM Na2HPO4, 44 mM KH2PO4,

15 mM NaCl, 35 mM NH4Cl, 0.1 mM CaCl2, 2 mM

MgSO4, 1% (w/v) glucose, and 0.1 mM IPTG] Under these conditions the E coli cells accumulate glycogen and the expression of the transgene continues At intervals aliquots of the cell suspension (25 mL each) were with-drawn and glycogen was extracted according to [26] Subsequently, the glucose content of the glycogen fraction was determined enzymatically using the starch kit (r-biopharm, Darmstadt, Germany) Alternatively, glyco-gen was extracted from the bacterial cells as described below (procedure B) For nitrogen starvation, NH4Cl was omitted from the medium

Glycogen extraction and size distribution (procedure B)

For the determination of 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, 4C), resuspended in

8 mL deionized water and sonicated for 90 s at 4C The

homogenate was centrifuged for 12 min at 20 000 g The

supernatant containing low molecular mass glycans,

glyco-gen, nucleic acids, and soluble proteins was heated (5 min at

100C) Denatured proteins were removed by centrifugation

(10 min at 10 000 g) High molecular mass nucleic acids were

degraded by adding both DNase (Roche, Germany) and

RNase (Macherey/Nagel, Du¨ren, Germany; 10 lgÆmL)1

supernatant each) and incubation for 4 h at 37C

Subse-quently, the nucleases were inactivated by heating (5 min at

100C) and the denatured protein 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 0.1Msodium nitrate and 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., Salt 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 cross 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 Barbara, USA) and

Optilab DSP Interferometric Refractometer (WTC),

respect-ively The molecular mass distribution was calculated from

light scattering and RI data by using theASTRAsoftware

(version 4.75, WTC; extrapolation by Debye, first order)

Maltooligosaccharide patterns

Bacterial cells (100 mL suspension) were pelleted by

centrifugation (12 min at 3000 g) and washed with

deionized water Maltooligosaccharides were extracted with

10 mL 80% (v/v) aqueous ethanol for 15 min at 95C

Following extraction insoluble compounds were removed

by centrifugation (10 min at 20 000 g) and the supernatant

containing the soluble carbohydrates was lyophilized The

residue was resuspended in 4 mL deionized water and

proteins were removed from the aqueous phase by

treat-ment with an equal volume of chloroform 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 and used

for high performance anion exchange chromatography with

pulsed amperometric detection (HPAEC-PAD, Dionex

BioLC) using a CarboPac PA-100 column Following

sample injection (90 lL each) the column was equilibrated

for 10 min with 5 mMsodium acetate in 100 mM NaOH

Analytes were eluted using a linear gradient of sodium

acetate (5–500 mM) in 100 mMNaOH (30 min; flow rate:

1 mLÆmin)1)

Results

Glucosylation of recombinant Glg2p The recombinant Glg2p carrying a C-terminal His6tag was purified close to homogeneity and incubated with UDPglu-cose At intervals (0, 5, 10, 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 of the recombinant glycogenin (Fig 1) For a more detailed analysis, the dominant protein band from the patterns obtained at the beginning (Glg2p0) and from the end of the incubation period (Glg2p20) were excised and digested with trypsin in the gel The 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 assigned

to tryptic peptides derived from the Glg2p sequence However, peptides containing Tyr230 and Tyr232 were detected in neither the nonglucosylated nor the glucosylated form In contrast, several nonglucosylated tryptic peptides representing residues 360–370, 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 mass increment of 162 Da, or a multiple of it, were detectable 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 Glg2p0and Glg2p20were separated by RP-HPLC For both mixtures, essentially the same HPLC chromatograms were obtained (data not shown) All collected fractions were analyzed by MALDI-TOF MS For both the trypsinated Glg2p0 and Glg2p20glucosylated 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 MnCl 2 at 30 C After 0 (lane a), 5 (b), 10 (c), 15 (d), and 20 (e) min an aliquot (7.5 lg protein 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 for MALDI-TOF MS analysis (Fig 2).

compounds whose m/z values differ by 162 Da Despite

some overlapping, analytes from HPLC fraction 12

con-tained more covalently bound hexosyl residues than those

from fraction 13

In Glg2p, both Tyr230 and Tyr232 are potential glucosylation sites As trypsin does not cleave between the two vicinal tyrosine residues, all the glucosylated peptides obtained by trypsination are expected to share the same

Fig 2 MALDI-MS analysis of tryptic peptides of Glg2p For in vitro autoglucosylation recombinant purified Glg2p was incubated with UDP-glucose for 0 (Glg2p 0 ) or 20 (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 containing eluate fractions were identified by MALDI-TOF MS For both samples Glg2p 20

and Glg2p 0 mass spectra of the HPLC fractions 12 and 13 are shown.

amino acid sequence (residues 219–246) designated as P1 In

the nonglucosylated state the molecular mass of P1 is

calculated to be 3352.8 Da Taking into account that the

two methionine residues are likely to be oxidized during

analyte processing the actual mass of the (nonglucosylated)

peptide P1is assumed to be 3384.8 Da By using this m/z

value and the data shown in Fig 2 it is estimated that 4–25

glucosyl residues are covalently bound to P1 This implies

that Glg2p is significantly glucosylated during production in

E coli Glucosylation in E coli has also been observed with

the rabbit muscle glycogenin [20] In the latter study 1–8

glucosyl residues were found to be linked to Tyr194

Following self-glycosylation for 20 min, the glucosylation

of Glg2p is even more complex At least 30 m/z signals

originating from the differently glucosylated P1peptide were

detected (Fig 2) In similar experiments, up to 40 glucosyl

residues attached to P1 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 Fig 2 clearly indicate a high degree of

glucosylation of the Glg2p-derived peptide P1 However,

they do not permit the determination of the actual

glucosy-lation site(s) within the 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 given: First, the glucopeptide to be

fragmented must be suitable in size Second, the fragment

pattern obtained must be dominated by the fragmentation

of the peptide backbone (and not by that of the glucosidic

bonds) For fragmentation studies, Glg2p was treated with

cyanogen bromide rather than with trypsin as the chemical

cleavage results in smaller peptides Cyanogen bromide (and

also trypsin) does not cleave between Tyr230 and Tyr232

and therefore a mixture of peptides is obtained one of which

contains both tyrosine residues Following chemical

clea-vage, the peptide mixture was incubated with a hydrolase

(such as a-amylase) and was then separated by HPLC

Enzymatic deglucosylation was found to be essential as

fragmentation of highly glucosylated peptides occurs most

frequently by cleavage of glycosidic linkages whereas peptide

backbone fragments are suppressed The latter are, however,

relevant for the 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 P2) but differing in the degree of

glucosylation In fraction 19, P2 was observed in the

monoglucosylated form designated as G-P2a However, the

majority of the peptide P2 occurs in higher glucosylated

forms as revealed by MALDI-TOF analysis of fractions

11–15 These glucopeptides were resistant to a prolonged or

repeated a-amylase treatment indicating an exhaustive

a-amylase action

For a more effective deglucosylation of the higher

glucosylated forms of P, a second enzymatic treatment

was included: RP-HPLC fractions 11–15 (Fig 3A) were pooled, lyophilized and incubated with amyloglucosidase Subsequently, the peptides were resolved by a second RP-HPLC run (Fig 3B)

In the elution profile of the 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 were analyzed by MALDI-TOF MS The mass spectra obtained show that the number of hexosyl residues attached to P2was reduced

to 0, 1 or 2 The completely deglucosylated P2was recovered

in fraction 18 (Fig 3B) The occurrence of the nonglucos-ylated peptide strongly 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 P2was recovered in fraction

15 of the second RP-HPLC run (Fig 3B) and was designated as G-P2b The diglucosylated P2 was detected

in fraction 12 (Fig 3B) and is referred to as G2-P2b The two mono-glucosylated P2samples, G-P2aand G-P2b and the diglucosylated peptide, G2-P2b were analyzed by fragmentation using both Q-TOF MS/MS and MALDI-TOF PSD Fragmentation often results from cleavage of the peptide backbone Fragments obtained are classified as a-, b- or y-type fragments [28] Both a- and 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 therefore both types differ by one CO group, i.e a mass of 28 Da All y-type fragments contain the C-terminus of the peptide Many fragments show a satellite peak at )17 Da This peak is due to a loss of NH3, presumably from an asparagine residue The fragments observed by Q-TOF MS/MS for the two mono-glucosyl-ated P2 conjugates, G-P2a and G-P2b 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-P2a and G-P2b fragmentation of the singly charged peptides gives rise to a strong series of b type ions ranging from b2 to b8 The relative molecular mass difference between two consecutive b-ions corresponds to the mass of 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 MS/MS spectra from G-P2a (Fig 4A) 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 the large (M+H+)-162 peak However, the occurrence of both b3 and b4 exclusively in the nonglucosylated state indicates that in G-P2a, Tyr230 does not carry a glucosyl residue and therefore Tyr232 must be the glucosylated residue The fragment pattern obtained with G-P2bdiffers signi-ficantly (Table 1 and Fig 4B) All fragments from b3 to b8 were recovered both in the glucosylated and in the nonglucosylated form As the two fragments b3 and b4

contain Tyr230 but not Tyr232 it is obvious that in G-P2b

the Tyr230 is a glucosyl acceptor

In summary, the MS/MS analysis of G-P2a and G-P2b

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 of the singly charged glucopeptide G2-P2b

(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 the diglucosylated form is undetectable In contrast, fragments b5 to b8 occur in the diglucosylated as well as in the mono- and in the nonglucosylated form This is exactly the fragmentation pattern to be predicted if both Tyr230 and Tyr232 bear one glucosyl residue each

Expression of the eukaryotic glycogenin and bacterial glycogen accumulation

The data shown in Figs 3–5 clearly indicate that the transformed E coli cells produce the recombinant Glg2p

in a functional 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 separated by RP-HPLC (A) Eluate fractions were analyzed by MALDI-TOF MS as indicated Eluate fractions 11–15 were pooled, incubated with amyloglucosidase and then subjected to a second RP-HPLC (B) Eluate fractions were analyzed by MALDI-TOF MS as indicated Eluate fractions marked with * were used for further investigations (see Figs 4 and 5).

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 tryptone–yeast extract medium

E colicells formed only insignificant amounts of glycogen irrespective of a proceeding transformation with the GLG2 gene or an induction of the transgene expression (data not shown) In contrast, bacterial cells accumulated glycogen following a transfer to the modified M9 minimal medium Therefore, we chose the following growth and induction protocol: after growth in tryptone–yeast extract medium, the production of Glg2p 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 As revealed by Western blotting using an anti-(His)5IgG

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 Procedure B yields an essentially unmodified polysaccharide fraction as revealed by control experiments using a commercial glyco-gen preparation By applying both methods we consistently observed that throughout the culture in the modified M9 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 letters For each b-type fragment the relative amount of the

glucosylated species is given in percentage For nomenclature of the

fragment ions see [28].

G-P 2a

Sequence

G-P 2b

b ions

b ions

b ions

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 for G-P 2a and G-P 2b (Fig 3) are shown in Fig 4A and B, respectively Both a- and b-type fragments contain the N-terminus of the peptide, they 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 Fragments bearing a glucosyl moiety are marked with an asterisk (*) A summary of the glucosylation state of the observed b-type fragments is given in Table 1.

control cells In Fig 6B, the cellular glycogen content, as

determined by procedure A, was followed over 20 h At the

end of this period of time, the protein-based glycogen level

of the Glg2p expressing bacterial cells was 70.6 ± 7.2% of

that of the control cells (average of five independent

experiments) The glycogenin producing E coli cells did

not differ from the control with respect 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 the absence of the eukaryotic glycogenin was determined As revealed by Western blotting experiments performed with buffer soluble proteins, the recombinant GlG2 gene was expressed throughout the entire period of glycogen accumulation (Fig 6A) E coli cells that had been transformed with the plasmid lacking the GlG2 gene were cultivated and harvested using precisely the same conditions From all six cell samples glycogen was prepared and analyzed by FFF-MALLS-RI The molecular mass distribution of the glycogen averages from 4· 107 to 1.5· 108gÆmol)1 for both Glg2p-producing cells and for the control For clarity, only the onset of glycogen accumulation (0 h) and 20 h are shown in Fig 6C Purity

of the glycogen preparations was ensured by acid hydrolysis and subsequent monosaccharide analysis (data not shown) Bacterial maltodextrin patterns

The oligosaccharide patterns from both glycogenin produ-cing cells and the control cells are complex and contain more than 30 compounds Most of the oligosaccharides were eluted during 0–15 min indicating a DP of < 12 In the Glg2p-forming cells, the by far dominant oligosaccharide peak eluted between DP 5 and DP 6 of a maltodextrin standard This compound is present in the oligosaccharide pattern of the control cells as well but in the glycogenin-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 spectrum of the Glg2p-derived diglucosylated peptide G 2 -P 2b For details see Figs 3 and 4 Fragments bearing one or two glucosyl moieties are marked with a single asterisk (*) and double asterisks (**), respectively A summary of the glucosylation state of the observed b-type fragments is given in Table 2.

Table 2 List of the b-type fragments of the Glg2p-derived

monoglu-cosylated peptide G 2 -P 2b obtained by Q-TOF MS/MS analysis

Frag-ments observed in the spectrum (Fig 5) are printed in bold letters For

nomenclature of the fragment ions see [28].

G 2 -P 2b

b ions b ions + 162 b ions + 324 Sequence

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 of 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 peptide

(designated as P1) was isolated that contains covalently

bound glucosyl residues covering a wide range of DP For

P, analyte ions having more than 23 different m/z values

were observed Following self-glucosylation for 20 min under in vitro conditions, for P1 at least 30 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 P1 peptide This is an unexpectedly high glucose content of the peptide that, to the best of our knowledge, has not yet been reported for 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 glucosylation sites, Tyr230 and Tyr232, self-glucosylation of Glg2p is expected

to give rise to covalently bound chains that possess at least 17–20 glucosyl residues, provided that both Tyr230 and Tyr232 are occupied within the same glycogenin molecule Peptide P2(PNYGYSSPAM) was obtained by chemical cleavage and allowed the identification of the glucosylation sites (Fig 3) Following heterologous expression in E coli,

we observed this peptide in a nonglucosylated form only following treatment with both a-amylase and amyloglucos-idase Thus, it seems that in 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 MS/MS analysis we provide direct evidence that both Tyr230 and Tyr232 act as glucosylation sites of Glg2p Discrimination between the two glucosylated tyrosine residues was achieved by taking advantage of a selectivity of the a-amylase When P2was 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 In 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 Bacterial

cells were grown for 20 h in modified M9 medium As a control, 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 analyzed by HPAEC-PAD As a standard,

a commercial maltodextrin sample (Dextrin 15, Fluka, Germany) was

used Data from a single experiment are shown The maltodextrin

patterns were confirmed in two additional independently performed

experiments.

Fig 6 Glycogen content and glycogen size distribution in E coli cells following Glg2p expression (A) Western blotting of buffer-soluble proteins extracted from E coli cells after 20 h growth in modified M9 medium Lane a: Control (bacterial cells 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 probed using an anti-His

Ig (B) E coli cells transformed with a plasmid containing (dark columns) or lacking (control; white columns) the GlG2 gene were transferred to modified M9 medium At intervals, aliquots were withdrawn and the glycogen content was monitored (procedure A; see Materials and methods) 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 (t¼ 0 h and 20 h) Glycogen was prepared using procedure B (see Materials and methods) All other experimental

conditions as in Fig 6A: , Glg2p (0 h); m, Glg2p (20 h); , control (0 h); and , control (20 h) Data from a single experiment are shown Three independently performed experiments yielded essentially the same results.