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:
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
MS/MS analysis. Unlike a-amylase the amyloglucosidase
used cleaved oligoglucans attached to Tyr232. Probably, the
enzyme also hydrolyzes the linkage between the residual
glucosyl residue and T yr232. A similar mode of action has
been described f or isoamylase but not for pullulanase [29]. It
should be noted that in the current study the use of both
a-amylase and amyloglucosidase allowed t he unambiguous
identification of the glucosylation sites in Glg2p. However,
the different modes of action o f the two hydrolases result i n
no obvious physiological implications.
The degree to which the t wo sites a re glucosylated can be
estimated on the basis o f peak a reas in the c hromatograms
of Fig. 3. Peak 19 (Fig. 3A) represents approximately
5–10% of all p eaks containing P

2
glucopeptides. T herefore,
approximately 5–10% of the protein molecules bear glucose
on Tyr232 only. The p eak containing the doubly glucosyl-
ated peptide P
2
(Fig. 3B) represents approximately 3 0% of
all P
2
-related peaks. Henc e, in at least 30% of the Glg2p
molecules t he two sites are occupied. H owever, this value is
probably largely underestimated. In 40% of the molecules
the gluco se was totally removed b y t he amyloglucosidase
treatment ( fraction 18) a nd therefore their initial g lucosy-
lation state is unknown. The monoglucosylated peptide
eluting i n fraction 15 accoun ts for a pproximately 20%. T he
corresponding MS/MS spectrum revealed that the glucose is
mainly attached to T yr230, however, a deglucosylation of
Tyr232 by the action of the amyloglucosidase cannot be
excluded. In summary: a pproximately 10% of the Glg2p
protein is glucosylated o n T yr232 only, 30% of the protein
is glucosylated on both t yrosine r esidues a nd the remaining
60% are glucosylated either on both residues or o n Tyr230
only.
As revealed by Q-TOF MS/MS a major proportion of
the Glg2p molecules is simultaneously g lucosylated at both
Tyr230 and Tyr232 (Fig. 5 and Table 2). This feature,
which has not been described for other glycogenins, is
remarkable as it implies that two glucan chains can be
synthesized simultaneously by a single g lycogenin molecule.

It probably e xplains t he unusually high number of glucosyl
residues attached to G lg2p. The dual g lucosylation of both
Tyr230 and Tyr232 within the same Glg2p molecule, as
demonstrated in this study, i s consistent with the failure to
detect both residues during Edman degradation [10].
Although Glg2p is effectively glucosylated in the pro-
karyotic cells, i t was not found to stimulate the bacterial
glycogen accumulation (Fig. 6). In fact, we consistently
observed that t he Glg2p-expressing cells contained approxi-
mately 30% less glycogen. This effect was observed under a
variety of experimental conditions, such as raising the
glucose content to 5% ( w/v) or nitrogen starvation (data not
shown). When the glucose content in the (NH
4
Cl contain-
ing) modified M9 medium was lowered t o 0 .1% (w/v) both
the Glg2p expressing cells and the control culture accumu-
lated very little glycogen but no difference in the glycogen
content was detectable (data not shown).
The yeast glycogenin Glg2p utilizes UDPglucose and
exhibits very little activity with ADPglucose. In in vitro
experiments performed with Glg2p the self-glucosylation
rate observed with
14
C-labeled A DPglucose was less than
1% of that obtained with UDPglucose ( data not shown).
Therefore, it is highly unlikely t hat the Glg2p-related
glucosyl transfer reactions compete with the prokaryotic
glycogen synthase for ADPglucose. This conclusion concurs
with the fact that E. coli cells deficient in UDPglucose

pyrophosphorylase activity form a carbohydrate-free gly-
cogenin [30]. Because in our study essentially all the Glg2p
extracted from t he bacterial cells was f ound to be glucos-
ylated, 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, s uch
as the galactose or trehalose metabolism, and also in t he
biosynthesis of membrane-derived oligosaccharides [31,32].
Thus, it seems that the eukaryotic initiator of glycogen
biosynthesis, although functional, is not compatible with the
prokaryotic path of glycogen formation. This conclusion is
supported by the fact that the size distribution of the
glycogen molecules is essentially unaffected by the produc-
tion of the eukaryotic glycogenin (Fig. 6). The dual function
of the g lycogen synthase recently proposed for Agrobacte-
rium tumefaciens [18] is consistent with this assumption.
Furthermore, it concurs with some enzymatic measure-
ments performed with glycogen accumulating E. coli cells.
The G lg2p-producing bacterial cells d id not differ notice-
ably from the control in v arious glycogen-related enzymes,
such as phosphorylase, soluble glycogen synthase or e ndo-
amylase (data not shown). Because of t he lower glycogen
content and the unchanged size distribution, the f requency
of an effective initiation of glycogen b iosynthesis 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 extract-
able maltodextrins is significantly altered (Fig. 7). As the
most prominent change, the level of a relatively small

maltodextrin is increased more than threefold that, as
judged from HPAEC, has a DP of approximately 5. It i s
remarkable th at the size of this m altodextrin i s s ignificantly
lower than t hat of the Glg2p -associated glucans. Possibly,
the Glg2p-derived oligoglucans are subjected to an extended
maltodextrin metabolism. External maltose a nd maltodex-
trins appear to be metabolized by a complex pathway that
includes the action of amylomaltase ( malQ), maltodextrin
phosphorylase ( malP) and maltodextrin g lucosidase ( malZ)
[33]. It is not inconceivable that a similar oligoglucan
metabolism utilizes the Glg2p-derived glucans and thereby
counteracts the Glg2p-dependent initiation of glycogen
biosynthesis.
Acknowledgements
Financial s upport by t he D eutsche Forschungsgemeinschaft (DFG ) is
gratefully acknowledged (SFB 429 T P B2). TA is grateful to t he Land
Brandenburg for a scholarship. We also thank PJ Roach and WA
Wilson (Indiana U niversity, Indianapolis, I N, U SA) for p roviding us
with the yeast strain EG328–1 A.
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