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J. Biol. Chem., Vol. 275, Issue 43, 33614-33621, October 27, 2000
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From the
Received for publication, June 20, 2000, and in revised form, August 1, 2000
The detailed catalytic mechanism by which
glycosyltransferases catalyze the transfer of a glycosyl residue from a
donor sugar to an acceptor is not known. Through the multiple alignment
of all known eukaryotic glycogen synthases we have found an invariant 17-amino acid stretch enclosed within the most conserved region of the
members of this family. This peptide includes an
E-X7-E motif, which is highly conserved in four
families of retaining glycosyltransferases. Site-directed mutagenesis
was performed in human muscle glycogen synthase to analyze the roles of
the two conserved Glu residues (Glu-510 and Glu-518) of the
motif. Proteins were transiently expressed in COS-1 cells as fusions to
green fluorescence protein. The E510A and E518A mutant proteins retained the ability to translocate from the nucleus to the cytosol in
response to glucose and to bind to intracellular glycogen. Although the
E518A variant had approximately 6% of the catalytic activity shown by
the green fluorescence protein-human muscle glycogen synthase fusion
protein, the E510A mutation inactivated the enzyme. These results led
us to conclude that the E-X7-E motif is part of
the active site of eukaryotic glycogen synthases and that both
conserved Glu residues are involved in catalysis. We propose that
Glu-510 may function as the nucleophile and Glu-518 as the general
acid/base catalyst.
Glycosyltransferases and glycosidases catalyze the transfer of
glycosyl residues from a donor sugar to an acceptor. The acceptor in
glycosidases is water, the end result being hydrolysis of the glycoconjugate. For transferases the acceptor molecule is in most cases
a growing carbohydrate chain, but it can also be a protein, a lipid, or
a range of other compounds such as steroids, bilirubin, flavonones,
carotenoids, etc., that are modified by glycosylation (1).
Glycosyltransferases can be further divided into two groups depending
on whether they use a nucleotide phosphosugar (Leloir-type) or an
oligosaccharide as the glycosyl donor. In all cases, the reaction
catalyzed is a substitution at the anomeric carbon of a sugar moiety
and may occur with retention or inversion of the configuration at this
center. Accordingly, enzymes that catalyze glycosyltransfer can be
divided into retaining or inverting enzymes.
Glycogen synthase (GS)1
catalyzes the key step of glycogen formation. In mammals, two major
isoforms of the enzyme have been described, the muscle isoenzyme (2),
which is expressed in several tissues (3), and the liver form (4),
which appears to be tissue-specific (5). GS plays a crucial role in
glucose metabolism and homeostasis, and its malfunction has been
associated with several metabolic diseases such as diabetes mellitus
(6, 7) and glycogen storage disease 0 (8). Mammalian GSs
catalyze the transfer of a glucosyl moiety from UDP- Although many genes encoding glycosyltransferases have been sequenced
and expressed, no structural information from x-ray crystallography or
high resolution NMR spectroscopy is available for a retaining
glycosyltransferase. The only structures known to date are those of the
Almost all the studies of muscle and liver GS have focused on the
covalent and allosteric regulation by hormonal and metabolic stimuli
(15-17), and few attempts have been made to elucidate the catalytic
mechanism (18). The aim of this study was to identify conserved regions
and putative catalytic residues through the comparison of the amino
acid sequences of mammalian GSs with those of other known retaining
glycosyltransferases. Moreover, using site-directed mutagenesis and
human muscle glycogen synthase (HMGS) as a model, we have probed the
function of two conserved Glu residues in catalysis by this family of enzymes.
Sequence Retrieval and Analysis--
Sequences were retrieved
from the ExPASy or PubMed servers on the Web. The accession
numbers (SWISS-PROT, TrEMBL, or Entrez) of the proteins studied are
included in the figures below. BLAST and Site-directed Mutagenesis--
The plasmid pEGFP-HMGS (26),
which encodes the fusion protein GFP-HMGS, was used as a template. The
mutations in the coding sequence of HMGS were created using the
QuikChange site-directed mutagenesis kit (Stratagene). The E510A
mutation was generated with the oligonucleotide
CCTCCTACTATGcGCCaTGGGGCTACAC (the changed positions are in
lowercase) and its inverse complementary oligonucleotide, which introduced an NcoI restriction site (shown
underlined) for diagnostic purposes. Similarly, the
pEGFP-HMGS (E518A) plasmid was built with the oligonucleotide
CACACCGGCTGcaTGCACGGTTATG and its exact complement, which
introduced an SphI restriction site. The mutant plasmids
were purified by anion-exchange chromatography (Plasmid Maxi Kit,
Qiagen), and the regions encoding the fusion proteins were sequenced in
their entirety, using the ABI-PRISM DNA sequencing kit and the
ABI-PRISM 377 automatic DNA sequencer (PE Applied Biosystems), to rule
out spurious mutations.
Cell Culture and Transfection--
COS-1 cells (ATCC no.
CRL-1650) were grown on 60-mm dishes (for biochemical assays and
immunoblots) or on glass coverslips inside 35-mm dishes (for confocal
microscopy analysis) in Dulbecco's modified Eagle's medium (DMEM;
Whittaker), supplemented with 25 mM glucose, 10% fetal
bovine serum (FBS; Biological Industries), and penicillin/streptomycin
(Roche Molecular Biochemicals). Cells cultured onto 60-mm dishes were
transfected using 625 µg of DEAE-dextran (Sigma), 0.5 µmol of
cloroquine (Sigma), and 10 µg of plasmid DNA per dish in DMEM. After
a 4-h incubation, cells were treated for 2 min in DMEM containing 10%
dimethyl sulfoxide (Sigma) and 10% FBS. They were then washed with
DMEM plus 10% FBS and maintained in this medium. Cells grown on
coverslips were transfected at 70-80% of confluence using 4 µg of
liposome suspension Clonfectin (CLONTECH) and 4 µg of plasmid DNA per 35-mm dish following the manufacturer's
instructions. After transfection (4-5 h) at 37 °C in humidified 5%
CO2:95% air, cells were washed in phosphate-buffered saline (PBS) and incubated in DMEM supplemented with 25 mM
glucose and 10% FBS. Experiments were performed 48-52 h after
transfection. Cells were preincubated overnight in DMEM without
glucose, and on the day of the experiment they were incubated in DMEM
with or without 30 mM glucose for 4 h. At the end of
the incubation, cells grown on 60-mm dishes were rinsed twice with PBS
and frozen in liquid nitrogen. Cells grown on coverslips were fixed for
20 min at room temperature in PBS containing 4% paraformaldehyde (Fluka) and washed several times with PBS. Alternatively, cells were
permeabilized with digitonin (5 µg/ml) in a buffer containing 300 mM saccharose, 3 mM Hepes, 5 mM
MgCl2 (Merck), 2 mM dithiothreitol (Sigma) for
8 min and were treated or not at 37 °C with Immunocytochemistry--
Coverslips were rinsed three times with
PBS, and cells that had not been treated with digitonin were
permeabilized for 20 min with PBS containing 0.2% Triton X-100 (Sigma)
and blocked for 10 min with PBS containing 0.2% Triton X-100 and 3%
bovine serum albumina (BSA; Sigma). Alternatively, before blocking,
cells were treated for 30 min at 37 °C with Confocal Microscopy--
Fluorescence images were obtained with
a Leica TCS 4D (Leica Lasertechnik, Heidelberg, Germany) confocal
scanning laser microscope adapted to an inverted Leitz DMIRBE
microscope and 63× (numerical aperture 1.4 oil) Leitz Plan-Apo
objective. The light source was an argon/krypton laser (75 milliwatts).
Green fluorescence from GFP and GFP recombinants was excited with the
laser at 488 nm; red fluorescence of the TRITC secondary antibody was
excited at 550 nm. Optical sections (0.1 µm) were obtained.
Glycogen Synthase Activity Assays and Glycogen Content--
For
the measurement of glycogen content, cell monolayers were scraped into
30% KOH, and the extract was then boiled for 15 min and centrifuged at
5000 × g for 15 min. Glycogen was measured in the
cleared supernatants as described (28). To determine GS activity,
frozen cell monolayers from the 60-mm diameter plates were scraped
using a homogenization buffer that consisted of 10 mM
Tris-HCl (pH 7.0), 150 mM KF, 15 mM EDTA, 15 mM 2-mercaptoethanol, 10 µg/ml leupeptin, 1 mM benzamidine, and 1 mM phenylmethylsulfonyl fluoride. Cell bursting was caused by sonication. Protein concentration was measured as described by Bradford (29) using the Bio-Rad protein
assay reagent. GS activity was measured in the presence or absence of
6.6 mM Glu 6-P as described (30). The activity measured in
the absence of Glu 6-P represents the active form of the enzyme (I or a
form), whereas the activity tested in the presence of 6.6 mM Glu 6-P is a measure of total activity. The ratio of
these two activities is an estimate of the activation state of the enzyme.
Electrophoresis and Immunoblotting--
Samples from activity
assays were boiled for 2 min with gel loading buffer 5× containing 250 mM Tris-HCl (pH 6.8), 1 mM dithiothreitol, 10%
SDS, 0.5% bromphenol blue, and 50% glycerol. Electrophoresis was
performed in a 10% SDS-polyacrylamide gel as described by Laemmli (31)
in a Mini-Protean II cell (Bio-Rad) at 200 V, until the bromphenol blue
dye front reached the end of the gel. Electrotransfer of
proteins from the gel to nitrocellulose (Protran; Schleicher & Schuell)
was performed at room temperature for 1 h at 100 V (constant) in a
Bio-Rad miniature transfer apparatus, as described by Towbin et
al. (32). Nitrocellulose blots were incubated at room temperature
in blocking buffer (3% BSA, 0.05% Tween 20 (Sigma) in PBS) for 1 h, then with a rabbit antibody against GFP
(CLONTECH) for 1 h, and finally with a
secondary goat anti-rabbit horseradish peroxidase antibody for 45 min.
Immunoreactive bands were visualized on Hyperfilm (Amersham Pharmacia
Biotech) films exposed to the membrane after incubation with ECL
reagent (Amersham Pharmacia Biotech).
Sequence Analysis--
A linear multiple alignment of all known
eukaryotic GSs (human muscle (2) and liver (33), rabbit muscle (34),
rat liver (4), mouse muscle2
and brain (36), Drosophila
melanogaster3 and
Caenorhabditis elegans (38) open reading frames,
Neurospora crassa4, and
Saccharomyces cerevisiae isoforms 1 (40) and 2 (41); not
shown) revealed a 17-amino acid stretch with the sequence 507SYYEPWGYTPAECTVMG523
(the numbering corresponds to the HMGS sequence), which is
strictly conserved and is enclosed within the region where homology
among the members of this family is greatest.
Campbell et al. (24, 25) have classified
glycosyltransferases in terms of sequence similarity and the retention
or inversion of the configuration at the anomeric carbon of the
transferred sugar. Among the 43 families described, only 10 are known
to operate via a retaining mechanism (families 3, 4, 5, 6, 8, 15, 20, 32, 34, and 35), 25 are inverting transferases, whereas for the
remaining 8 families the stereochemical course of the reaction is
unknown. All eukaryotic GSs fall into family 3, whereas the NRD1
First, six to ten arbitrarily chosen sequences of each family were
retrieved from the NCBI or Swiss Protein/TrEMBL data bases and were
aligned by families using the ClustalW algorithm. The most conserved
regions were then screened for the presence of a stretch similar to the
E-X7-E motif, which was detected in four of the
ten retaining families (3, 4, 5, and 15), whereas none of the families
with unknown stereochemistry apparently possessed such a consensus
sequence. The proposed multiple linear alignment of this fragment from
representative members of families 3, 4, 5, and 15 is shown in Fig.
1. Although the overall identity among these sequences is very low, only the first Glu residue of the E-X7-E motif (which corresponds to Glu-510 in
the HMGS sequence) is invariant, similarity is much higher (~70%).
Two characteristic features of this motif, which are highly conserved
among all the proteins analyzed, are the presence of aromatic residues
at positions
To further assess the significance of this similarity, we performed
hydrophobic cluster analysis (HCA) and secondary structure prediction
of a 60-amino acid peptide spanning the E-X7-E
motif on a set of representative proteins of the aforementioned
families (Fig. 2). Again, a number of
features are conserved among the proteins analyzed, thus supporting the
hypothesis that these four families are related. Both the shape of the
hydrophobic clusters in the HCA profiles and secondary structure
prediction anticipated the presence of an The GFP-HMGS Fusion Protein Is Catalytically Active--
One way
to show that a given amino acid residue of an enzyme is essential for
catalysis consists of mutating this particular amino acid and verifying
that the mutant enzyme has a greatly decreased or null activity. This
approach requires the use of a recombinant expression system that
permits the production of active enzyme. Owing to the difficulties in
obtaining reasonable amounts of soluble and active muscle GS by
overexpression of the protein in Escherichia
coli (44),5 we decided
to use eukaryotic cells to express the chimerical protein constructed
by fusing the green fluorescent protein (GFP) at the N-terminal end of
HMGS. This system enables the ready observation of the intracellular
localization of the GFP-HMGS chimera and thus represents an adequate
means to verify the overall structural integrity of inactive mutants.
To study whether the GFP-HMGS fusion protein was catalytically active,
COS-1 cells were transiently transfected with the pEGFP-C1 and
pEGFP-HMGS plasmids, and homogenates from these cultures were assayed
for GS activity. GFP-expressing COS-1 cells displayed endogenous GS
activity, but total GS activity of cells overexpressing GFP-HMGS was
approximately 8-fold that of control cells (Table I). Roach and co-workers obtained similar
results when rabbit muscle GS was transiently expressed in COS M9 cells
(45). The activity ratio of GFP-HMGS expressed in COS-1 cells increased from 0.13 ± 0.05, when determined in homogenates from cells
incubated in a glucose-free medium, to 0.22 ± 0.09 in cells kept
in the presence of 30 mM glucose for 4 h. This result
further suggests that the fusion of GFP at the N terminus of HMGS does
not significantly interfere with the normal function of the enzyme.
The GFP-HMGS Fusion Protein Binds to Intracellular
Glycogen--
In previous studies we have shown that the intracellular
distribution of GFP-HMGS is dependent on the presence of glucose in the
incubation medium. Thus, in the absence of glucose GFP-HMGS was
concentrated in the nucleus and translocated to the cytosol in response
to the presence of the sugar. In both compartments, the fusion protein
showed a particulate pattern, and the size and the apparent complexity
of the particles in the cytosol increased as incubation with glucose
was prolonged (26), suggesting that most of the GFP-HMGS fusion protein
was bound to glycogen particles. To test this hypothesis,
immunocytochemical experiments were performed using a monoclonal
antibody that has been shown to specifically bind to glycogen from
chondrocytes, hepatocytes, and muscle cells, as well as to purified
glycogen (27). First, we checked the ability of this antibody to bind
to glycogen particles produced by COS-1 endogenous GS. Cells were
transfected with the pEGFP-C1 vector and were incubated in a
glucose-free medium. In these conditions COS-1 cultures stored
negligible amounts of glycogen, and no immunofluorescence arising from
the anti-glycogen antibody could be detected (Fig. 3A). In contrast, cells
incubated for 4 h in a medium containing 30 mM glucose
accumulated 170 ± 10 µg of glycogen/mg of protein and showed a
clear punctuate pattern in the confocal image, which was attributable
to glycogen labeling (Fig. 3B). Furthermore, treatment of
these cells with
In another set of experiments, COS-1 cells were transfected with the
pEGFP-HMGS plasmid and were also immunostained with the anti-glycogen
antibody. In the absence of glucose, transfected cells did not
accumulate measurable amounts of glycogen and no fluorescent signal
arising from glycogen immunolabeling was detected (not shown). As
previously reported (26), in these conditions green fluorescence from
GFP-HMGS was mainly found in the nucleus (not shown). After a 4-h
incubation with 30 mM glucose, GFP-HMGS was almost
exclusively found in the cytosol, mostly as round-shaped aggregates
(Fig. 4, A and D).
Surprisingly, the number of specks that were immunolabeled with the
glycogen antibody was much lower in cells overexpressing GFP-HMGS than
in non-transfected cells of the same preparation (Fig. 4, B
and E). The percentage of transfection achieved in these
experiments was always higher than 70%, and transfected and
non-transfected COS-1 cultures, when incubated for 4 h with 30 mM glucose, reached similar levels of glycogen (170 ± 10 µg of glycogen/mg of protein). Therefore, the decreased glycogen
immunolabeling could not be attributed to the accumulation of lower
amounts of the polysaccharide in the GFP-HMGS-expressing cultures.
Rather, this finding suggests that the overexpressed fusion protein
blocked the access of the antibody to glycogen particles. This
hypothesis was supported by the observation that some very large
GFP-HMGS aggregates, which were occasionally produced (Fig.
4D), were also immunolabeled with the glycogen antibody (Fig. 4E). However, the red fluorescence attributable to
glycogen staining was mainly found in the center of the large
round-shaped aggregates, whereas the green fluorescence from GFP-HMGS
was concentrated in the perimeter, and both labels appeared to be
mutually exclusive over the same particle (Fig. 4F).
To further corroborate the association between the GFP-HMGS fusion
protein and intracellular glycogen, COS-1 cells transiently expressing
GFP or GFP-HMGS were incubated in the presence of 30 mM
glucose for 4 h and were permeabilized with digitonin before fixation and observation in the confocal microscope. This treatment was
effective in releasing soluble proteins, as shown by the removal of
GFP. However, in GFP-HMGS-expressing cells the fusion protein was not
completely released by this treatment and the removal of GFP-HMGS was
only achieved when digitonin-permeabilized cells were incubated with
Characterization of the GFP-HMGS (E510A) and GFP-HMGS (E518A)
Mutant Proteins--
To test the roles of Glu-510 and Glu-518 in the
catalytic activity of HMGS, these two residues were mutated to Ala in
the plasmid pEGFP-HMGS and the resulting mutant proteins were
transiently expressed in COS-1 cells. Homogenates from these cultures
were analyzed by SDS-polyacrylamide gel electrophoresis and
immunoblotting, using an anti-GFP antibody. The mutant proteins
exhibited the expected molecular mass of ~110 kDa and were expressed
at similar levels to the wild-type protein (Fig.
5). The integrity of the GFP-HMGS (E510A)
and GFP-HMGS (E518A) proteins was further confirmed by confocal
microscopy analysis of their intracellular distribution in transiently
transfected COS-1, hepatocytes, and L6 myoblasts. In each cell type and
in both the presence and absence of glucose in the incubation media,
the two mutant enzymes exhibited an identical distribution to that of
GFP-HMGS (not shown). The size and the shape of the aggregates produced
by the mutant proteins in the presence of glucose were very similar to
those of the wild-type fusion enzyme. Moreover, glycogen immunolabeling
of COS-1 cells was also partially blocked by the overexpression of both
GFP-HMGS (E510A) and GFP-HMGS (E518A). The observation that the mutant proteins retained the ability to change their intracellular
localization in response to glucose and to bind to glycogen strongly
suggested that the mutations did not affect the overall structural
integrity of the enzyme. Thus, changes in the activity of the mutants
can be directly attributed to local disturbances at the active site machinery.
Detailed kinetic studies of the recombinant enzymes were prevented by
the presence of endogenous GS activity. However, homogenates from COS-1
cells transiently expressing the wild-type and the mutant chimerical
proteins were assayed for total GS activity. GFP-HMGS
(E518A)-expressing cultures showed a slightly higher total GS activity
than GFP-expressing cells (Table I), indicating that the E518A mutant
retained approximately 6% of the activity shown by the wild-type
GFP-HMGS enzyme in the conditions of the assay. This small increase in
GS activity over the control was consistently observed in all the
individual experiments performed. In contrast, homogenates from cells
expressing the E510A variant of HMGS did not exhibit a significant
difference in activity when compared with control cells. We conclude
that both Glu residues are involved in catalysis: Glu-510 is a critical
residue, whereas Glu-518 plays a more secondary role.
In this study we have combined bioinformatic and experimental
techniques to identify two Glu residues at the active site of eukaryotic GSs, using HMGS as a model. We have taken advantage of the
classification of glycosyltransferases into 43 families proposed by
Campbell et al. (24, 25), according to sequence similarity
and the stereochemical course of the reaction. Through the use of BLAST
searches and multiple alignments we have found an
E-X7-E motif that is highly conserved among the
members of families 3, 4, 5, and 15 of glycosyltransferases, all of
which operate with retention of configuration at the anomeric carbon. Hydrophobic cluster analysis and secondary structure prediction of this
region supported the hypothesis that these four families are related.
In eukaryotic GSs, all belonging to family 3, this motif is enclosed
within an invariant 17-amino acid stretch found roughly in the last
third of the corresponding coding sequences and in the region where
these proteins exhibit the largest degree of similarity. This conserved
core region has previously been assumed to contain the catalytic site,
in contrast to the more variable N and C termini, which harbor the
phosphorylation sites that regulate the enzyme activity (4).
The functional role of Glu-510 and Glu-518 in the
E-X7-E motif of HMGS was probed by site-directed
mutagenesis. The wild-type enzyme and two single point mutants, in
which the conserved Glu residues were replaced by Ala, were transiently
expressed in COS-1 cells as fusions to GFP. The structural integrity of
the chimerical mutant proteins was shown in several ways. They were
expressed to similar levels and showed the same molecular mass as the
wild-type. The variant proteins retained the ability to concentrate in
the nuclear compartment in the absence of glucose and translocate to
the cytosol when the monosaccharide was added (26). Finally, they were
able to bind to intracellular glycogen, as the wild-type enzyme.
However, the E518A mutant retained approximately 6% of the activity
shown by the GFP-HMGS fusion protein, whereas the E510A had
undetectable activity. This finding indicates that the catalytic
mechanism of HMGS has been impaired by the mutations.
Assuming that highly conserved regions in enzymes contain crucial
residues for catalytic activity, the E-X7-E
motif must be involved either in substrate recognition and binding or
in catalysis. However, considering the large variety of glycosyl donors
(GDP-mannose, ADP- and UDP-glucose, UDP-galactose,
UDP-N-acetylglucosamine, etc.) and acceptors (mono- and
polysaccharides, glycolipids, glycoproteins, etc.) used by the proteins
of families 3, 4, 5, and 15, only the active site would be clearly
conserved in all of them. The observation that both mutant forms of
GFP-HMGS bound to glycogen was also an indication that the
glycogen-binding site of the enzyme was not significantly disturbed by
the single point mutations. Additionally, Lys-38 of the rabbit
muscle GS has been implicated in UDP-glucose binding, suggesting that
this substrate binds to the N-terminal half of the enzyme (18). It is
therefore reasonable to assume that Glu-510 and Glu-518 are part of the
HMGS active site machinery, and by analogy, the corresponding residues
of other eukaryotic GSs play an identical role. The same may be true
for the glycosyltransferases from families 4, 5, and 15 of Campbell's
classification, although in these cases, experimental confirmation
would be required. This type of evidence has been obtained for Ace-A
(35, 46), an Enzymatic reactions that involve the substitution of a group at an
asymmetric carbon atom and yield a product with the same configuration
as the substrate generally operate by two successive displacements on
the asymmetric carbon (10). In retaining glycosidases, the first step
involves the formation of an inverted substrate-enzyme intermediate
through the coordinated attack of a nucleophile at the sugar anomeric
center and the protonation of the glycosidic oxygen by a residue acting
as a general acid catalyst. In the second step, the latter provides
general base catalytic assistance and deprotonates a water molecule,
which in turn attacks the anomeric carbon once again, thus yielding the
final product. Through site-directed mutagenesis and kinetic analysis
of the mutants, the catalytic residues of several retaining
glycosidases, always Asp or Glu residues, have been identified and
their respective roles assigned (39). Mutant enzymes in which the
nucleophile has been replaced by an Ala residue are essentially
inactive. When the acid/base catalytic residue is eliminated, the
resulting protein retains some activity with very good substrates,
i.e. those bearing good leaving groups. In this situation,
protonation of the leaving group is not crucial for catalysis (39).
Kapitonov and Yu described a domain (NRD1 Our results argue against the roles assigned to the two conserved Glu
residues by Kapitonov and Yu. First, the sequence comparisons with
selected glycosyltransferases show that, although the first Glu residue
of the motif is invariant, the second Glu is more variable and
therefore better fits the secondary role of the acid/base catalyst. It
has to be noted that, in all the enzymes analyzed in this study, the
second residue is always an amino acid whose lateral chain can
putatively act as a proton donor/acceptor. Second, the E510A mutation
in HMGS completely inactivates the enzyme, whereas the E518A mutant
maintains some residual activity. The glycosyl donor in the synthesis
of glycogen is UDP-glucose. The chemical nature of UDP dictates that
this moiety can act as a good leaving group even when it is not
protonated and thus the glycogenic reaction might still proceed at a
measurable rate in the absence of an acid catalyst. Our results are
consistent with Glu-510 being the fundamental nucleophile and with
Glu-518 providing important but not essential catalytic assistance,
possibly as the general acid/base catalyst. Further experiments
are in progress to determine the exact roles of both conserved Glu
residues of the E-X7-E motif in the catalysis by
GS.
We thank Dr. Otto Baba for providing us with
the monoclonal glycogen antibody, Susanna Castel for her skillful
technical assistance with the confocal microscope, and Tanya Yates for
assistance in preparing the English manuscript.
*
This work was supported in part by Grant PM98-0185 from
Dirección General de Enseñanza Superior (Ministerio de
Educación y Cultura, Spain), by Grant ACI 99-16 from the
Generalitat de Catalunya, and by the Juvenile Diabetes Foundation
International.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
Recipient of a doctoral fellowship from the Generalitat de
Catalunya (Comissió Interdepartamental de Recerca i
Innovació Tecnològica).
¶
Recipient of a doctoral fellowship (Formación Personal
Investigador) from the Spanish Government (Ministerio de
Educación y Cultura).
**
To whom correspondence should be addressed: Dept. de
Bioquímica i Biologia Molecular, Universitat de Barcelona,
Martí i Franquès, 1, Barcelona E-08028, Spain. Tel.:
34-93-402-1209; Fax: 34-93-402-1219; E-mail:
ferrer@sun.bq.ub.es.
Published, JBC Papers in Press, August 2, 2000, DOI 10.1074/jbc.M005358200
2
M. F. Seldin, Z. Xue, J. M. Rochelle,
R. Debry, and R. Surwit, direct submission to the GenBankTM, Accession
number AAD09457.
3
Automatic genome annotation at the Celera
Jamboree (FBrf0126705). FlyBase (1999).
4
R. de Paula, H. F. Terenzi, and M. C. Bertolini, direct submission to the EMBL/GenBankTM/DDBJ, Accession
number O93869.
5
J. C. Ferrer and J. J. Guinovart,
unpublished results.
6
P. Abdian, A. C. Lellouch, C. Gautier,
D. U. Ferreiro, L. Ielpi, and R. A. Geremia, submitted for publication.
The abbreviations used are:
GS, glycogen
synthase;
HMGS, human muscle glycogen synthase;
GFP, green fluorescence
protein;
HCA, hydrophobic cluster analysis;
DMEM, Dulbecco's modified
Eagle's medium;
FBS, fetal bovine serum;
PBS, phosphate-buffered
saline;
BSA, bovine serum albumin;
Glu 6-P, glucose 6-phosphate;
TRITC, tetramethylrhodamine isothiocyanate;
NRD1
Identification of Two Essential Glutamic Acid Residues in
Glycogen Synthase*
§,¶,
,, and**
Departament de Bioquímica i Biologia
Molecular, Universitat de Barcelona, Barcelona E-08028, Spain and
the Centre des Recherches sur les Macromolécules
Végétales, CNRS, affiliated with the Joseph Fourier
University, Grenoble F-38041, France
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucose to a
nascent chain of glycogen through an 1
4 linkage. The
stereochemistry of the resulting glycosidic bond is the same as that of
the donor sugar nucleotide, thus GS is classified as a retaining
Leloir-type glycosyltransferase. The stereochemical course of the
reaction, analogously to what has been found for retaining glycosidases (9), determines the presence of two catalytic amino acids, which allow
a double displacement mechanism. According to this model, these two
essential residues must be close within the active center of the enzyme
(10, 11).
-glucosyltransferase of T4 bacteriophage (12), the hypothetical
nucleotide-diphospho-sugar transferase SpsA from Bacillus
subtilis (13), and the catalytic domain of the bovine
1,4-galactosyltransferase T1 (14). However, all these enzymes
operate with inversion of configuration at the anomeric carbon and
presumably have different active site geometry.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-BLAST (19, 20) were
performed at the NCBI. Linear alignments were performed locally using
ClustalW (21). Hydrophobic cluster analysis (HCA) (22) plots were
obtained at the DrawHCA server. The secondary structure predictions
were performed at the Jpred server (23). The classification of
glycosyltransferases by Campbell et al. (24, 25) is
accessible on the Web also.
-amylase (22 units/ml, Sigma) and 1 mM CaCl2 in PBS for 30 min. Finally, cells were fixed with paraformaldehyde as described.
-amylase (22 units/ml, Sigma) and 1 mM CaCl2 in PBS. A
monoclonal IgM antibody against glycogen, a generous gift from Dr. Otto
Baba (27), was diluted in PBS containing 3% BSA and applied to the
cells for 45 min at room temperature. Coverslips were then washed
several times with PBS and subjected to incubation with a
tetramethylrhodamine (TRITC)-conjugated goat anti-mouse IgM secondary
antibody (Chemicon) for 30 min. Finally, coverslips were washed,
air-dried, and mounted onto glass slides using the Immuno Fluore
mounting medium (ICN Biomedicals, Inc.).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-BLAST searches using this 17-amino acid peptide showed that an E-X7-E
motif (two Glu residues separated by seven amino acids) is conserved
among other glycosyltransferases that act with retention of the
configuration at the reaction center. A similar
E-X7-E motif was described previously in a
family of retaining bacterial -mannosyltransferases (42). Through
the multiple alignment of related glycosyltransferases different to
eukaryotic GSs, Kapitonov and Yu (43) identified a conserved fragment,
arbitrarily named nucleotide recognition domain 1 (NRD1), which
was characterized by the presence of two conserved Glu residues
separated by seven amino acids.
enzymes described by Kapitonov and Yu (43) and those analyzed by
Geremia et al. (42) belong to family 4 of Campbell's
classification. Here we have extended the analysis to all the
glycosyltransferase families that operate with retaining or unknown stereochemistry.
1 and +2 from the invariant Glu residue and two almost
invariant Gly residues at positions +3 and +13. The second conserved
Glu residue in family 3 (Glu-518 in HMGS) is also present in all the members of family 4, whereas all the enzymes analyzed from family 15 possess a His residue in this position. Finally, this site is more
variable in the members of family 5, being occupied by Glu, Tyr, or His
residues.
View larger version (51K):
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Fig. 1.
Multiple sequence alignment of deduced amino
acid sequences of selected glycosyltransferases. The alignment was
performed using ClustalW and a blosum62mt matrix. Sequences were
retrieved from the Entrez-protein server (NCBI) (superscript
1) or Swiss Protein/TrEMBL (superscript 2) data
bases. The glycosyltransferase families, according to the
classification of Campbell et al. (24, 25), are indicated on
the left and the accession numbers are shown on the
right. The first aligned amino acid of each protein is
indicated between brackets. The invariant Glu residue is
shown on a black background and the conserved homologous
residues on a gray background. ORF Y46G5A.31: putative
glycogen synthase from C. elegans; ORF CG6904: putative
glycogen synthase from D. melanogaster; UGS1_HUMAN: human muscle glycogen synthase; O93869: glycogen synthase
from N. crassa; UGS1_YEAST: glycogen synthase
isoform 1 from S. cerevisiae; VIPC_SALTI: VI
polysaccharide biosynthesis protein VIPC/TVIE from Salmonella
typhi; ORF AF0045: putative mannosyltransferase A from
Archaeoglobus fulgidus; GPI3_YEAST:
N-acetylglucosaminyl-phosphatidylinositol biosynthetic
protein from S. cerevisiae; SPS_MAIZE: maize
sucrose-phosphate synthase; SUS1_MAIZE: maize sucrose
synthase 1; P78852: putative cell wall -glucan synthase Ags1 from
Schizosaccharomyces pombe; ORF PAB2292: putative glycogen
synthase from Pyrococcus abysii; GLGA_ECOLI:
glycogen synthase from E. coli; O48899: maize starch
synthase isoform zSTSII-1; BAA82346: granule-bound starch synthase I
from Phaseolus vulgaris; KRE2_CANAL: glycolipid
2--mannosyltransferase MNT1 or KRE2 from Candida
albicans; KRE2_YEAST: glycolipid
2--mannosyltransferase MNT1 or KRE2 from S. cerevisiae;
YUR1_YEAST: probable mannosyltransferase YUR1 from S. cerevisiae; KTR3_YEAST: probable mannosyltransferase
KTR3 from S. cerevisiae; O60160: putative
2--mannosyltransferase (locus SPBC19C7) from S. pombe.
-helix 12-15 amino acids
before the E-X7-E stretch. Both methods
predicted two -sheets, located 5-7 amino acids and 20-30 amino
acids after this motif, respectively. Additionally, the profiles of the
hydrophobic clusters just before the first Glu residue are compatible
with a -sheet, which is found by secondary structure prediction in
all cases but one. These observations indicate that these proteins
presumably present similarities at the level of secondary structure in
the region encompassing the E-X7-E motif and
further suggest that the invariant Glu residue plays an essential role
in the enzymatic activity of this class of enzymes. Although the second
Glu of the motif is not strictly conserved, it must be noted that in
all cases the amino acid that occupies this position can hypothetically
act as a proton donor/acceptor.
View larger version (34K):
[in a new window]
Fig. 2.
HCA alignment of the region spanning the
E-X7-E motif. The HCA plots of a
60-amino acid peptide spanning the E-X7-E motif
are presented for one protein of each glycosyltransferase family
analyzed. The regions showing similarity at the HCA level are
boxed. Circles indicate the conserved residues of
the motif. The protein sequences are written on a duplicated
-helical net, and the contour of clusters of hydrophobic residues is
automatically drawn. The standard one-letter code for amino acids is
used except for proline, glycine, serine, and threonine, which are
represented by solid star, solid diamond,
dotted square, and blank square, respectively.
The secondary structure predicted by the JnetPret algorithm is shown
below the HCA plot for each protein as a bar for an
-helix and an arrow for a -sheet. SUS1_MAIZE: maize sucrose synthase 1; UGS1_HUMAN: human
muscle glycogen synthase; GLGA_ECOLI: glycogen synthase
from E. coli; KRE2_CANAL: glycolipid
2--mannosyltransferase MNT1 or KRE2 from C. albicans.
Total GS activity in GFP and GFP-HMGS-expressing COS-1 cells
-amylase after paraformaldehyde fixation and
permeabilization completely abolished the fluorescence signal (not
shown), thus confirming the specificity of the anti-glycogen antibody.
This experiment also showed that the intracellular distribution of GFP
was insensitive to the presence of glucose in the incubation medium and
to the accumulation of glycogen (Fig. 3).
View larger version (23K):
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Fig. 3.
Glycogen immunostaining of GFP-expressing
COS-1 cells. Representative confocal images of COS-1 cells
transiently transfected with the pEGFP-C1 vector. Cells were fixed in
paraformaldehyde (48-52 h after transfection), permeabilized with
Triton X-100, and incubated with a monoclonal IgM anti-glycogen
antibody and a TRITC conjugated secondary antibody as described under
"Experimental Procedures." Both panels show the overlapped images
of the GFP and TRITC fluorescence. In A, cells were
incubated in DMEM without glucose and no immunofluorescence from
glycogen particles can be detected. B, red fluorescence
arising from glycogen granules in transfected COS-1 cells that were
incubated for 4 h in DMEM containing 30 mM glucose.
There is no redistribution of GFP from A to B,
indicating that the subcellular localization of this protein is
insensitive to the addition of glucose to the incubation medium or to
the presence of glycogen particles in the interior of the cells. The
scale bar indicates 10 µm.
View larger version (26K):
[in a new window]
Fig. 4.
Glycogen immunostaining of
GFP-HMGS-expressing COS-1 cells. Representative confocal images of
COS-1 cells transiently transfected with the pEGFP-HMGS vector. After
transfection (48-52 h), cells were incubated for 4 h in DMEM
containing 30 mM glucose, fixed in paraformaldehyde,
permeabilized with Triton X-100, and incubated with a monoclonal IgM
anti-glycogen antibody and a TRITC-conjugated secondary antibody as
described under "Experimental Procedures." A and
D show the green fluorescence from GFP-HMGS, B
and E show the red fluorescence from TRITC-labeled glycogen,
and C and F show the overlapped images.
Asterisks (C and F) indicate the
position of the nuclei. Open arrows (F) point to
the large crown-shaped GFP-HMGS aggregates, which are also labeled with
the anti-glycogen antibody. The scale bar indicates 10 µm.
-amylase to degrade glycogen before fixation (not shown). We
conclude that the particulate pattern shown by the GFP-HMGS chimera is
due to its close association with the glycogen particles produced when
COS-1 cells are incubated in the presence of glucose.
View larger version (48K):
[in a new window]
Fig. 5.
Immunodetection of GFP-HMGS fusion proteins
expressed in COS-1 cells. COS-1 cells were transiently transfected
with the pEGFP-HMGS, pEGFP-HMGS (E510A), and pEGFP-HMGS (E518A)
vectors. After transfection (48 h), aliquots from cell homogenates
containing 10 µg of total protein were subjected to
SDS-polyacrylamide electrophoresis on a 10% gel and transferred to a
nitrocellulose membrane. Immunoblotting with a rabbit anti-GFP
polyclonal antibody and a horseradish peroxidase-linked anti-rabbit
antibody was performed as described under "Experimental
Procedures." As shown, the mutant proteins were expressed in
comparable amounts to the wild-type fusion enzyme and moved similarly
in the gel, indicating no gross rearrangement due to the
mutations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-mannosyltransferase that belongs to family 4. Geremia
et al. (42) found an E-X7-E motif
similar to that described here in a group of prokaryotic
-mannosyltransferases and proposed that both conserved Glu residues
were important for catalysis. The replacement by Ala residues of
Glu-287 or Glu-295 in Ace-A (equivalent to Glu-510 and Glu-518 in HMGS,
respectively) led to the same changes in enzymatic activity as those
observed in HMGS. The E287A variant was inactive, whereas Ace-A (E295A)
showed very little activity.6
Very recently, Nichols et al. (37) have shown that Glu-391 of maize starch synthase IIb-2, a glycosyltransferase from family 5, is
essential for activity. According to our alignments, this residue
corresponds to the indispensable Glu-510 in HMGS.
), present in several
members of the glycosyltransferases of family 4 different from the
-mannosyltransferases analyzed by Geremia et al. (42), which also contained an E-X7-E segment. The
authors arbitrarily proposed, by analogy with the mechanism of
retaining glycosidases, that the first conserved Glu residue was be the
general acid/base catalyst, while the second one acted as the
nucleophile (43). However, these assumptions were not supported experimentally.
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
, nucleotide
recognition domain 1;
ORF, open reading frame.
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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