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J. Biol. Chem., Vol. 277, Issue 2, 1158-1165, January 11, 2002
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From the
Received for publication, July 16, 2001, and in revised form, October 30, 2001
Glutamate-cysteine ligase (GCL) plays an
important role in regulating glutathione homeostasis. In mammals, it
comprises a catalytic (GCLC) and modifier (GCLM) subunit. The existence
of a modifier subunit in invertebrates has not been described to date.
We now demonstrate that GCL from Drosophila melanogaster has a functional modifier subunit (DmGCLM). A putative DmGCLM was
obtained as an expressed sequence tag with 27% identity to human GCLM at the amino acid level. D. melanogaster GCLC
(DmGCLC) and the candidate DmGCLM were expressed separately in
Escherichia coli, purified, mixed, and then subjected to
gel filtration, where they eluted as an ~140-kDa complex. DmGCLC
co-immunoprecipitated with DmGCLM from S2 cell extracts, suggesting
that they also associate in vivo. Enzyme kinetic analyses
showed that DmGCLC has a Km for glutamate of 2.88 mM, but when complexed with DmGCLM, the Km for glutamate is 0.45 mM. Inhibition
of DmGCLC activity by glutathione was found to be competitive with
respect to glutamate (Ki = 0.03 mM),
whereas inhibition of the GCL complex was mixed (Ki = 0.67 mM), suggesting allosteric effects. In accordance
with this, DmGCLC and DmGCLM have the ability to form reversible
intermolecular disulfide bridges. A further mechanism for control of
D. melanogaster GCL was found to be induction of DmGCLC by
tert-butylhydroquinone in S2 cells. DmGCLM levels were, however, unaffected by tert-butylhydroquinone.
Cellular injury from reactive oxygen species and electrophilic
agents is inhibited by glutathione, an abundant and essential tripeptide thiol. Despite the critical importance of glutathione for
virtually all aerobically respiring cells, relatively little is known
about how cells regulate glutathione homeostasis. It is apparent,
however, that a key player in the control of glutathione synthesis is
glutamate-cysteine ligase
(GCL,1 EC 6.3.2.2,
Native and recombinant GCLs from rat and human have been
extensively characterized (1-4). It has been shown that GCL from these
species comprises an ~73-kDa catalytic subunit and an ~31-kDa regulatory subunit. GCLC has all of the catalytic activity, but in vitro experiments with rat kidney GCL suggested that,
unless complexed with GCLM, it has exquisite sensitivity to feedback inhibition by glutathione and a high Km (~18
mM) for glutamate (2). Combination with GCLM caused the
Ki for glutathione to be substantially increased and
the Km for glutamate to be reduced by 13-fold. The
presence of reducing agents caused the Km for
glutamate to increase by ~2-fold and increased the extent of
inhibition by the glutathione analog ophthalmic acid, suggesting that
GCLM may regulate GCLC activity in response to the immediate redox
environment. Based on the in vitro data, it has been
proposed that rat GCLC would not be functional in vivo
without the presence of GCLM (1).
Although GCLM seems to have an important role in regulating glutathione
synthesis, to date, only GCL from vertebrates has been shown to have a
functional regulatory subunit. GCL from Escherichia coli is
distinct from the eukaryotic forms described so far and is a monomer
with a subunit molecular mass of 58 kDa (5). Interestingly, the
E. coli GCL amino acid sequence has a greater level of
identity to GCLM than to GCLC (1). Catalytic subunits of GCL from
Saccharomyces cerevisiae and Caenorhabditis
elegans have been identified (6, 7), and although they are
structurally related to human GCLC (32 and 54% identities at the amino
acid level, respectively), there are no convincing candidate genes in
the data base with comparable levels of sequence identity to GCLM. GCL
from Trypanosoma brucei has been cloned and its enzyme
activity rigorously characterized (8, 9). Although T. brucei
GCL shares 45% sequence identity at the amino acid level with
mammalian GCLC, the kinetic data are highly suggestive that T. brucei GCL would be fully functional in vivo without
the necessity for a regulatory subunit.
Recently, GCL from Drosophila melanogaster was cloned by
functional complementation of an S. cerevisiae gsh1 mutant
(10). This was previously called D. melanogaster
The genetic tractability of Drosophila makes it a highly
attractive organism to study the regulation of GCL activity in
vivo. Identification of common regulatory mechanisms for
glutathione synthesis in Drosophila and man would make this
an important model to aid our understanding of the functions of
glutathione and the factors that regulate its homeostasis. In this
study, we have identified a candidate GCL regulatory subunit in
Drosophila and demonstrate that it regulates DmGCL activity
in a manner similar, but not identical, to that of human or rat
GCLM.
Construction of DmGCL Constructs--
The open reading frame
(ORF) of the gcl gene was amplified from pDmGCS4.3.3
(10) by PCR using upstream (5'-GGGAATTCATATGGGTCTACTGAGCGAGGGC-3') and
downstream (5'-GCCTTAACTCGAGTCATTTCTCCTCGCAGCAGCC-3') oligonucleotides designed to insert EcoRI and NdeI sites at the
5'-end and an XhoI site at the 3'-end of the ORF. The
amplified fragment was cloned into the EcoRI and
XhoI sites of pBluescript SK
The DmGCLM cDNA was obtained as an expressed sequence tag (GH01757)
from ResGen Genomics Resources (a subsidiary of Invitrogen), and
upstream (5'-GGGAATTCCATATGATACCGACCATAACG-3') and downstream (5'-GGAATTCCTCGAGCTAAACGCTCGACCTCG-3') primers were used to amplify the
ORF by PCR. The oligonucleotides were designed to insert
EcoRI and NdeI sites at the 5'-end and an
XhoI site at the 3'-end of the ORF. The amplified fragment
was subcloned into the EcoRI and XhoI sites of
pBluescript SK Expression and Purification of Recombinant DmGCL
Proteins--
pETDmGCLC and pETDmGCLM were expressed separately in
E. coli strain BL21(DE3). Transformed cells were grown at
37 °C with shaking in Terrific Broth (Sigma) containing ampicillin
(100 µg/ml) to an A600 nm of 0.5, and protein
expression was induced by the addition of
isopropyl- Gel Filtration Chromatography--
Recombinant protein samples
were dialyzed overnight against 3 liters of Buffer A (20 mM
Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM
L-glutamate, and 5 mM MgCl2)
containing 10 mM glutathione. Protein was precipitated by
the addition of ammonium sulfate to 90% saturation and pelleted by
centrifugation. Precipitated protein was solubilized in 0.5 ml of
Buffer A containing 10 mM glutathione and applied to a
Sephacryl S-200 16/60 Hi-prep gel filtration column (Amersham
Biosciences, Inc.) pre-equilibrated with Buffer A containing 0.2 mM dithiothreitol. Gel filtration was carried out by fast
protein liquid chromatography (Amersham Biosciences, Inc.) at a flow
rate of 0.5 ml/min, and fractions were collected at 1-min intervals.
The eluate was monitored by continuous absorption at 280 nm, and
protein concentrations in the eluted fractions were determined by the
method of Bradford (11). The column was calibrated using protein
molecular mass standards (Amersham Biosciences, Inc.).
Analysis of GCL Activity--
GCL activity was determined at
25 °C by a modification of the method of Seelig et al.
(12) using cysteine as substrate instead of
L- S2 Cell Culture--
Drosophila S2 cells were a
generous gift from Dr. W. Whitfield (School of Biological Sciences,
University of Dundee, Dundee, UK) and were maintained in serum-free
medium (Invitrogen) routinely supplemented with 2 mM
L-glutamine and 5% (v/v) fetal bovine serum at room temperature.
Cells were seeded 24 h prior to treatment at a density of 1 × 106 cells/ml and were treated with
tert-butylhydroquinone (tBHQ) for 18 h. tBHQ was
prepared as a 100 mM stock in Me2SO and was diluted to the appropriate concentration in the medium. Control cells
were treated with the equivalent concentration of Me2SO. After treatment, cells were harvested and resuspended in Buffer B (50 mM Tris-HCl (pH 7.2), 20 mM sodium phosphate
buffer (pH 7.0), 150 mM NaCl, and 1% (v/v) Igepal).
Insoluble material was removed by centrifugation, and the soluble
fractions were retained for analysis.
Immunoblotting and Immunoprecipitation--
Antisera against
purified recombinant DmGCLC or DmGCLM were raised in sheep using
standard procedures (Scottish Diagnostics, Carluke, UK). Western
blotting was performed by the method of Towbin et al. (14),
and the even transfer of samples was ensured by staining with Ponceau S
as described previously (15). As an additional control for even protein
loading and transfer, blots were also probed with a mouse monoclonal
antibody raised against Drosophila
For immunoprecipitation experiments, cleared S2 cell lysates (250 µl,
0.5 mg/ml protein) were incubated with 50 µl of a protein G-agarose
bead slurry (Amersham Biosciences, Inc.) and 1.5 µl of anti-DmGCLM
antiserum for 1 h at 4 °C with gentle agitation. The beads were
collected by centrifugation and washed three times with Buffer B,
followed by a single wash with phosphate-buffered saline.
Immunoprecipitates were analyzed by SDS-PAGE and Western blotting.
Statistical Analyses--
Statistical analyses were performed
using Student's paired t test.
Identification of a Putative DmGCLM Subunit--
The amino acid
sequence of DmGCLC is 57% identical to human GCLC. Phylogenetic
alignment of DmGCLC with other GCL sequences shows that DmGCL has
greater identity to mammalian GCL than to any other GCL subunit so far
identified (10). We therefore hypothesized that DmGCL may be
functionally similar to mammalian GCL, which requires a modifier
subunit for regulatable and maximal activity. To test this hypothesis,
we searched the Drosophila genome data base (16) for a GCLM
candidate gene using the human GCLM cDNA sequence as a probe. The
BLAST search identified a single gene within a bacterial
artificial chromosome clone (BACF23F10,
GenBankTM/EBI Data Bank accession number AC009846).
The cDNA predicted from the genomic sequence encodes a 285-amino
acid polypeptide with an estimated molecular mass of 31.5 kDa. The
genomic sequence was used to search the Drosophila expressed
sequence tag data base (17), and an expressed sequence tag (GH01757,
accession number AI062531) with a nucleotide sequence identical to the genomic sequence was identified. The gene does not appear to have any
introns over this area of the cDNA. Comparison of the predicted amino acid sequence from the putative DmGCLM cDNA with human GCLM revealed that the proteins share 27% identity and 42% similarity. The
greatest level of identity is within the C-terminal region (Fig.
1).
Physical Characterization of DmGCL Subunits--
The expressed
sequence tag (GH01757) was used as a template to amplify the ORF of the
putative DmGCLM cDNA by PCR. A single PCR product of 850 bp was
obtained and subcloned into pBluescript SK
The cDNA encoding DmGCLC was isolated recently by functional
complementation of an S. cerevisiae GCL mutant (10). In the present study, the 2160-bp ORF was cloned into pET15b and used to
express DmGCLC as a recombinant His-tagged protein. This was purified
from soluble E. coli extracts using nickel-agarose affinity chromatography, and ~20 mg of DmGCLC was obtained from an 800-ml culture. Analysis of purified recombinant DmGCLC by SDS-PAGE showed that it comprises a single polypeptide with an approximate molecular mass of 80 kDa (data not shown). This is in good agreement with the
predicted molecular mass of 81 kDa from its amino acid sequence (719 amino acids).
The Putative DmGCLM Subunit Interacts with DmGCLC in
Vitro--
Human GCLM associates with GCLC to form a holoenzyme with
an estimated size of 114 kDa (4). To determine whether the putative DmGCLM subunit identified here associates with DmGCLC to form a
complex, purified DmGCLM (10 mg of protein) was mixed with DmGCLC (10 mg of protein) and subjected to gel filtration. The existence of an
enzyme complex should be evident by an increase in the molecular mass
of the eluted protein, relative to DmGCLC or DmGCLM alone.
Analysis of DmGCLC (10 mg of protein) alone by gel filtration (Fig.
2) revealed that it eluted as a single
peak with an estimated molecular mass of ~80 kDa. DmGCLM alone eluted
as a single peak with an estimated molecular mass of ~30 kDa (Fig.
2). Resolution of the DmGCLC/DmGCLM mixture by gel filtration showed
that there was one major protein peak with an estimated molecular mass
of ~140 kDa and a minor protein peak with an estimated molecular mass
of 30 kDa corresponding to uncomplexed DmGCLM. From these findings, we
concluded that a new protein complex of a higher molecular mass is
generated when DmGCLC and DmGCLM are mixed, inferring that they are
interacting with each other in vitro. Enzyme activity was
determined with the fractions collected from gel filtration of DmGCLC,
DmGCLM, or the mixture of DmGCLC and DmGCLM. Fig. 2B shows
that the peaks corresponding to DmGCLC and the putative DmGCLC·DmGCLM
complex both have GCL activity.
To determine the composition of the 140-kDa protein, samples were
analyzed by SDS-PAGE under reducing conditions and compared with
purified DmGCLC and DmGCLM. Fig. 3 shows
that polypeptides with molecular masses of 80 kDa (corresponding to
DmGCLC) and 30 kDa (corresponding to DmGCLM) are present in the 140-kDa
protein peak. Reversible disulfide bridges can form between GCLC and
GCLM in the mammalian holoenzyme (12). To determine whether disulfide bonds can form between DmGCLM and DmGCLC, we dialyzed the peak fractions from the 140-kDa complex to remove dithiothreitol and subjected the protein to SDS-PAGE analysis under nonreducing conditions (Fig. 3). This showed that a higher molecular mass complex formed under
nonreducing conditions with an approximate molecular mass of 140 kDa.
The 140-kDa band occurred only when DmGCLC and DmGCLM were together and
was absent when identically treated DmGCLM or DmGCLC samples were
analyzed separately in the same way (Fig. 3). The 140-kDa band was
notably absent in the corresponding samples analyzed under reducing
conditions. These data are therefore highly suggestive that reversible
disulfide linkages are involved in the association of DmGCLC with the
putative DmGCLM subunit.
It is interesting to note that, like the complex eluted from the gel
filtration column, the estimated molecular mass of the DmGCLC·DmGCLM
disulfide complex is higher than the expected 110-kDa size of a
homodimer. The estimated molecular mass of the DmGCL complex is more in
keeping with a heterotrimeric structure. Molecular mass estimations by
gel filtration and SDS-PAGE can, however, be subject to confounding
factors, and further work will be required to determine the subunit
stoichiometry of DmGCL.
The DmGCLM Subunit Interacts with DmGCLC in Vivo--
To confirm
that DmGCL exists as a complex containing DmGCLC and DmGCLM in
vivo and that our data did not result from an artifact of in
vitro mixing, we determined whether the two subunits would co-immunoprecipitate from Drosophila S2 cell extracts.
Soluble extracts from S2 cells were incubated with anti-DmGCLM
antiserum and protein G-agarose beads, and the precipitate was analyzed by Western blotting using antiserum raised against DmGCLC. As shown in
Fig. 4, a unique band was identified with
an approximate molecular mass of 80 kDa when the immunoprecipitate was
probed with antiserum raised against DmGCLC. This band was absent when the immunoprecipitation experiment was performed with the preimmune serum. The ability of endogenous DmGCLM to coprecipitate with endogenous DmGCLC from S2 cell extracts implies that DmGCLM and DmGCLC
form a complex in vivo as well as in vitro.
DmGCLM Modulates DmGCLC Activity--
Evidence suggests that
mammalian GCLM can enhance the catalytic efficiency of GCLC by
increasing its affinity for its substrate L-glutamate (1,
2). To determine whether DmGCLM has a similar effect on DmGCLC
activity, we compared the activities of DmGCLC and the 140-kDa DmGCL
holoenzyme after purification by gel filtration.
DmGCLC and the DmGCL holoenzyme were found to have specific activities
of 244 and 569 nmol/min/mg, respectively (calculated with respect to
the amount of DmGCLC in the assay mixture) when measured using standard
assay conditions with 10 mM L-glutamate. Activity was not detected in the absence of L-glutamate or
L-cysteine. When L-
Km and Vmax values for
L-glutamate and L-cysteine were determined. The
apparent Km of the DmGCL holoenzyme for
L-glutamate is 0.45 mM, considerably lower than
that determined for DmGCLC, which is 2.88 mM (Table I).
Km values for cysteine were found to be 6.55 and
5.53 mM for the DmGCL holoenzyme and DmGCLC, respectively.
These values are substantially higher than the corresponding values for
human GCL, for which values of between 0.1 and 0.8 mM have
been reported (3, 4). We also examined the possibility that other amino
acids could substitute for cysteine in the enzyme assay and tested a
range of amino acid substrates, including methionine, alanine, serine,
glycine, leucine, and lysine, in place of cysteine. We were unable to
detect any GCL activity over background levels (data not shown),
suggesting that these amino acids cannot be utilized instead of
cysteine by DmGCL.
The kcat/Km values of the
holoenzyme for L-cysteine and L-glutamate are
significantly higher than those of DmGCLC alone (Table I). These
findings indicate that the holoenzyme is catalytically more efficient
than DmGCLC and show that DmGCL is catalytically similar to mammalian
GCL in that GCLM influences GCLC enzyme activity.
DmGCLM Alters the Susceptibility of DmGCLC to
Inhibition--
Mammalian GCL activity can be inhibited by glutathione
(2). The nature and extent of inhibition are modulated by the presence of the regulatory subunit. To determine whether a similar mechanism of
regulation of GCL activity exists for DmGCL, we first examined the
effect of increasing concentrations of glutathione on GCL activity
under standard assay conditions (Fig. 5).
DmGCLC activity was reduced by 60% in the presence of 1 mM
glutathione and by 93% in the presence of 2 mM
glutathione. By contrast, the holoenzyme was less susceptible to
inhibition by glutathione, with 1 and 2 mM glutathione
lowering activity by 32 and 58%, respectively. Higher concentrations
of glutathione (10 mM) almost completely abolished the
activities of both DmGCLC and the DmGCL holoenzyme. The kinetics of
glutathione inhibition were studied by measuring DmGCL reaction
velocities with increasing concentrations of glutamate in the presence
of fixed glutathione concentrations (0.1, 0.25, and 0.5 mM). DmGCLC was inhibited competitively by glutathione when
glutamate was the variable substrate. By contrast, the holoenzyme was
subjected to mixed inhibition by glutathione. The apparent inhibition
constant (Ki) values for glutathione were 0.03 mM for DmGCLC and 0.67 mM for the DmGCL
holoenzyme (Table I). These findings show that DmGCLM significantly
reduces the sensitivity of the catalytic subunit to inhibition by
glutathione, possibly by generating a conformational change preventing
access of glutathione to the active site in a manner similar to the
model proposed by Meister and co-workers (2).
BSO is an inhibitor of GCL and is phosphorylated by the enzyme to form
an intermediate that is tightly and irreversibly bound at the active
site (18, 19). It was of interest to examine the effect of BSO on the
DmGCLC subunit and the DmGCLC·DmGCLM complex. Incubation of DmGCLC
with 1.5 µM BSO for 10 min prior to kinetic analysis
caused a 47% reduction in GCL activity, whereas DmGCL holoenzyme
activity was reduced by 90% (Table II),
showing that the DmGCLC·DmGCLM complex is more readily inhibited by
BSO compared with DmGCLC.
Mammalian GCL has also been shown to be susceptible to inhibition by
cystamine, which is thought to inhibit GCL activity by binding a
cysteine residue in or around the active site (20-22). Inhibition can
be reversed by treatment with dithiothreitol. Incubation of DmGCLC for
10 min prior to kinetic analysis with 1.5 µM cystamine reduced DmGCLC activity by 34%, suggesting that the DmGCLC active site
may be structurally similar to mammalian GCL with a cysteine residue
near the active site (Table II). Curiously, the DmGCL holoenzyme was
unaffected by the presence of 1.5 µM cystamine. It is
possible that free thiol groups on the DmGCLM subunit may preferentially form disulfides with cystamine, sparing the cysteine that is close to the active site. This raises the possibility that the
GCL regulatory subunit may have an additional function to protect the
catalytic subunit from thiol-reactive agents.
DmGCL Subunit Levels Are Modulated by Oxidative Stress in S2
Cells--
GCLC and GCLM protein levels have been shown to increase in
response to agents capable of generating sublethal oxidative stress in
human cell lines (23-25). We wished to determine whether induction of
the DmGCL subunits constitutes part of the adaptive response to
oxidative stress in Drosophila and treated S2 cells with the redox cycling agent tBHQ to examine this possibility.
Western blot analyses of S2 cells treated with increasing
concentrations of tBHQ showed that tBHQ caused a
concentration-dependent increase in intracellular DmGCLC
protein levels (Fig. 6A).
Treatment with 50 or 100 µM tBHQ caused increases in
DmGCLC of ~3- and 4-fold, respectively. By contrast, DmGCLM protein
levels did not appear to be substantially increased by treatment with
tBHQ (Fig. 6B).
GCL activity in mammals is subject to intricate regulation
involving both post-translational and transcriptional control
mechanisms (19). It was unknown whether these regulatory features are
conserved in invertebrates, and in this study, we have demonstrated a
parallelism in the regulation of GCL activity in mammals and
Drosophila. We have shown that DmGCL is composed of a
catalytic subunit and at least one regulatory subunit that enhances the
affinity of DmGCLC for glutamate and reduces its susceptibility to
inhibition by glutathione. Furthermore, as has been shown for human
GCLC, DmGCLC protein levels can be up-regulated in cell lines by agents
that generate oxidative stress.
Non-mammalian GCL has been described for a wide variety of eukaryotic
species, including yeast, Nematoda, Protozoa, and Insecta, and has been
isolated as a single chain polypeptide with similarity to human GCL
(6-8, 10, 26-28). By contrast, Arabidopsis thaliana GCL
has no significant identity (15-19%) to other GCL forms (29, 30).
Until now, functional non-mammalian GCLM homologs have not been
described in the literature. It has been suggested that certain lower
eukaryotic species do not require a GCLM subunit, as enzyme kinetic
studies showed that they are likely to be active in vivo
without the requirement for a regulatory subunit (8). The lack of
documentation about GCLM homologs in lower eukaryotes does not,
however, confirm that they are absent. It is possible that, in certain
lower eukaryotes, GCL enzymes contain functional GCLM subunits, but
their presence has been overlooked due to the method of GCL isolation.
Very few GCL enzymes have been isolated from invertebrates by
purification of native proteins. Instead, direct cloning or functional
complementation of mutants has been used to identify cDNAs encoding
proteins with GCL activity. As GCLM is not required for activity
in vitro, a requirement for its presence in vivo
may not have been noted. The possibility that GCL may comprise
catalytic and modifier polypeptides in invertebrates other than
Drosophila is strengthened by work by Hussein and Walter (31), who purified GCL from the nematode Ascaris suum.
Purification of A. suum GCL by gel filtration showed the
presence of two protein peaks with GCL activity with molecular masses
of 100 and 70 kDa. Although the 100-kDa protein was not characterized,
the possibility that it contains catalytic and regulatory subunits
would be interesting to investigate further.
Although we have raised the possibility that GCL may comprise catalytic
and regulatory subunits in other invertebrates, we do not suggest that
this would necessarily occur in all cases. Indeed, GCL from T. brucei has a Km for glutamate of 0.24 mM and a Ki for glutathione of 1.1 mM, which are similar to those obtained for the
DmGCLC·DmGCLM complex. The kinetics of T. brucei GCL
activity suggest that it may not require further activation by a GCLM
subunit (8). It remains to be determined whether the regulation of GCL
activity by a modifier subunit is a common feature in different
invertebrates and when, in evolutionary terms, it became an important
regulator of glutathione synthesis.
DmGCLM reduces the Km of DmGCLC for glutamate and
raises the Ki for glutathione in a manner similar to
that of its mammalian counterparts. GCLM-mediated changes in the
kinetic efficiency of mammalian GCLC are thought to be due to a
conformational change in GCLC favoring a glutamate-binding site with
high affinity and specificity for L-glutamate; the high
affinity glutamate-binding site is also less accessible to glutathione
(2). This conformational change is thought to result, in part, from the
formation of intersubunit disulfide bridges between GCLM and GCLC.
These covalent linkages are susceptible to changes in the reducing
environment and are proposed to modify GCL activity in response to
changes in intracellular glutathione concentrations (2). Our kinetic
studies on inhibition of DmGCL by glutathione show that similar
regulatory mechanisms for GCL activity may exist in
Drosophila. DmGCLC inhibition by glutathione is competitive
with respect to glutamate, whereas inhibition of the holoenzyme by
glutathione can be classed as mixed. These findings are in keeping with
the hypothesis that reduction of disulfide bridges between DmGCLC and
DmGCLM by glutathione facilitates access of glutathione to the active
site, where it could compete with glutamate. When DmGCLM is absent, the
active site of DmGCLC appears to be accessible for competitive
inhibition by glutathione. Our hypothesis that inhibition of the DmGCL
holoenzyme by glutathione is likely to involve reduction of disulfide
linkages between the subunits is supported by the demonstration that
the DmGCLC·DmGCLM complex can form reversible disulfide linkages when subjected to SDS-PAGE under nonreducing conditions (Fig. 3). Comparison of the amino acid sequences of DmGCLM and human GCLM shows that there
are two conserved cysteine residues (Fig. 1, asterisks). It
is possible that one or both of these residues mediate the covalent
interactions with DmGCLC, and we are currently investigating this further.
In addition to examining the effect of the modifier subunit on the
catalytic efficiency of GCLC and its inhibition by glutathione, we also
investigated whether GCLM influenced susceptibility to inhibition by
BSO or cystamine. DmGCLM appears to protect DmGCLC against inhibition
by cystamine, as DmGCLC was inhibited by cystamine, whereas the DmGCL
holoenzyme was unaffected under the assay conditions used. The
mechanism for this protection is unclear, but it is possibly due to
DmGCLM buffering the local cystamine concentrations by providing
additional thiol-binding sites. Cystamine forms mixed disulfides with
GCLC and binds a cysteine residue close to the active site, blocking
substrate access (9, 21). It is possible that surface sulfhydryl
residues on DmGCLM become preferentially bound by cystamine, thus
protecting a critical cysteine at or near the active site of DmGCLC. By
contrast, the DmGCL holoenzyme was found to be more sensitive to
inhibition by BSO compared with DmGCLC. It has been shown that
phosphorylation of BSO by GCL generates an intermediate that binds
tightly and irreversibly to the glutamate- and cysteine-binding sites
of GCL (32). Although we have not investigated the mechanism for the
differential inhibition by BSO, it is possible that the conformational
change imposed upon the active site of DmGCLC by DmGCLM generates a
binding pocket with greater affinity for BSO, thus making it a more
potent inhibitor for the holoenzyme than the catalytic subunit alone.
Alternatively, as BSO is a time-dependent inhibitor, the
differential inhibition could reflect an increase in the rate of BSO
phosphorylation in the presence of the regulatory subunit.
The apparent size of the DmGCL holoenzyme complex appears to be larger
than that described for rat or human GCL. We estimate the DmGCL complex
formed in vitro to be ~140 kDa. This suggests that the
reconstituted DmGCL holoenzyme may, in fact, comprise one catalytic
subunit and two regulatory subunits. Studies with GCL from rat kidney
led Sekura and Meister (33) and Seelig et al. (12) to
suggest that it exists as a heterodimer. Other studies with recombinant
human GCL are also consistent with a heterodimeric structure (3, 4). It
has, however, been documented that the amount of GCLM associated with
GCLC may vary between different enzyme preparations (34).
Interestingly, GCL purified from rat liver by Davis et al.
(35) was estimated by gel filtration to be 138 kDa, which would be
consistent with a heterotrimeric structure. It is possible that the
molar ratios of the catalytic and modifier subunits may be subject to a
degree of variation in vivo.
Elevated intracellular glutathione levels and glutathione-metabolizing
enzymes have been observed in mammalian cells and yeast as part of an
adaptive response to oxidative stress (23, 24). Glutathione-depleting
agents, heavy metals, redox cycling chemicals, inflammatory cytokines,
chemotherapeutic drugs, and ionizing radiation have been shown to
modify intracellular glutathione and GCL protein levels. This appears
to be due to induction of GCL transcription as well as mRNA
stabilization. The redox cycling agent tBHQ has been used in many
mammalian cell lines to generate oxidative stress and has been shown to
elevate GCL subunit mRNA levels by increasing gene transcription,
leading to increased GCL protein levels, GCL activity, and elevated
glutathione levels (23, 24). In this study, we found that DmGCLC
protein levels were up-regulated in Drosophila S2 cells in
response to tBHQ. Although we did not determine whether transcriptional
activation of DmGCLC mRNA occurred in this study, it is likely that
the elevation of DmGCLC protein levels reflects increased DmGCLC gene
transcription. We have identified several putative enhancer elements in
the genomic DNA sequence (16) upstream from the DmGCLC ORF. These
include AP-1- and nuclear factor- In contrast to our findings for DmGCLC, DmGCLM protein levels were only
marginally enhanced in response to tBHQ treatment. In mammals and
mammalian systems, GCLC and GCLM are also subject to a degree of
differential regulation, although both subunits are usually increased
in response to oxidative stress (23, 24).
As has been shown for the human and mouse GCL genes (38), the DmGCL
genes are on separate chromosomes. The DmGCLC gene is on the X
chromosome (polytene map position 7CD) (10), whereas the DmGCLM gene
maps to 94C on the third chromosome. One of the advantages of working
with the Drosophila system is the existence of mutant stocks
created by P-element mobilization (39, 40). Stocks are often in
existence with mutations in or near the gene of interest. We have
obtained the P-element-induced recessive lethal l(3)L0580,
which contains a P-element insertion in the 5'-noncoding region of the
DmGCLM gene. Although we have yet to establish that the P-element
insertion is responsible for the lethality, there is a good possibility
that DmGCLM is essential for glutathione synthesis in vivo,
as proposed by Meister and co-workers (1) for rat GCL from their
in vitro studies. Targeted deletion of the GCLC gene in the
mouse is lethal, showing that glutathione is essential for normal
embryonic development (41, 42). To date, corresponding models for the
GCLM gene have not been described. If, as we suspect, loss of DmGCLM
gene function is lethal, mutants will provide valuable genetic models
with which to study the regulation of GCL activity in
vivo.
We thank Dr. David Sheehan (University College
Cork, Cork, Ireland) for invaluable help with analysis of enzyme
kinetic data.
*
This work was supported in part by Grants 94/G15091 and
108/G15090 from the Biotechnology and Biological Sciences Research Council and by Royal Society Research Grant 21511.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.
§
Supported by Biotechnology and Biological Sciences Research Council
Studentship 98/A/C/04147.
Published, JBC Papers in Press, November 6, 2001, DOI 10.1074/jbc.M106683200
2
www.chem.qmw.ac.uk/iubmb/enzyme/EC6/3/2/2.html.
The abbreviations used are:
GCL, glutamate-cysteine ligase;
GCLM, glutamate-cysteine ligase modifier
subunit;
GCLC, glutamate-cysteine ligase catalytic subunit;
DmGCL, D. melanogaster glutamate-cysteine ligase;
ORF, open reading
frame;
BSO, L-buthionine-(SR)-sulfoximine;
tBHQ, tert-butylhydroquinone.
Drosophila melanogaster Glutamate-Cysteine Ligase
Activity Is Regulated by a Modifier Subunit with a Mechanism of Action
Similar to That of the Mammalian Form*
§,
Biomedical Research Center, University of
Dundee, Ninewells Hospital and Medical School, Dundee DD1 9SY, United
Kingdom and the ¶ Department of Biological Sciences, Open
University, Walton Hall, Milton Keynes MK7
6AA, United Kingdom
ABSTRACT
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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INTRODUCTION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
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-glutamylcysteine synthetase), which catalyzes the first reaction in
the two-step pathway for de novo synthesis. The ultimate
enzyme in the pathway, glutathione synthetase, does not appear to play
such an important part in the fine-tuning of cellular redox status. GCL
activity seems to be regulated by cellular thiol antioxidant balance,
and in mammals, this control mechanism is principally mediated by a
non-catalytic polypeptide (GCLM) that dimerizes with the catalytic
subunit of GCL (GCLC) (1, 2).
-glutamylcysteine synthetase, but in the present study, the
nomenclature has been changed to DmGCL in accordance with the
recommendations of IUBMB.2
DmGCL was found to have 57% amino acid sequence identity to human GCLC, and its expression in the S. cerevisiae gsh1 mutant
partially restored the glutathione deficiency in this strain. The
levels of glutathione in the complemented yeast strain were, however, only 8% of those in the parental strain. GCL activity of the expressed protein was not characterized in that study, but based on the high
level of amino acid sequence identity to human GCLC and the fact that
complementation of the gsh1 mutant resulted in such a modest
restoration of glutathione levels, it seemed to us highly plausible
that DmGCL may require a regulatory subunit for efficient activity
in vivo.
EXPERIMENTAL PROCEDURES
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(Stratagene),
whereupon a 1880-bp AccIII/XhoI fragment was
replaced with the corresponding fragment from pDmGCS4.3.3 to minimize
the introduction of PCR errors. Plasmid DNA was isolated, and the insert was sequenced to confirm that it was identical to the predicted ORF of pDmGCS4.3.3. The DmGCLC ORF was subcloned into the
NdeI and XhoI sites of pET15b (Novagen) to
generate plasmid pETDmGCLC.
and sequenced before subcloning into the
NdeI and XhoI sites of pET15b to generate plasmid pETDmGCLM.
-D-thiogalactopyranoside (1 mM). The temperature was lowered to 30 °C, and growth was allowed to continue for ~4 h before harvesting the cells. Bacterial cell pellets
were resuspended in 20 mM Tris-HCl (pH 7.9), 500 mM NaCl, 5 mM imidazole, and 0.1% (v/v)
Igepal, and recombinant proteins were purified using nickel-agarose
chromatography (QIAGEN Inc.) according to the manufacturer's instructions.
-aminobutyrate and adapted for use on a Cobas Fara
centrifugal analyzer (Roche Molecular Biochemicals,
Hertfordshire, United Kingdom). Reaction mixtures (0.21 ml)
contained 10 mM L-glutamate, 10 mM
L-cysteine, 2 mM phosphoenolpyruvate, 0.4 mM NADH, 1 unit of lactate dehydrogenase, and 1 unit of
pyruvate kinase. The reaction was initiated by the addition of ATP to a
final concentration of 5 mM. Km and
Vmax values were determined by measuring initial
reaction rates at glutamate and cysteine concentrations of between 0 and 60 mM. Hyperbolic regression analysis software (43) was
used to fit the Michaelis-Menten parameters using the Hanes plot. For
inhibition studies with
L-buthionine-(SR)-sulfoximine (BSO), GCL samples
were incubated at 25 °C with 1.5 µM BSO in 100 mM Tris-HCl (pH 8.2), 20 mM MgCl2,
and 5 mM ATP for 10 min prior to kinetic analyses. GCL
inhibition studies with cystamine (2,2'-dithiobis(ethylamine)) were
performed by incubating GCL samples with 1.5 µM cystamine
in 100 mM Tris-HCl (pH 8.2) for 10 min at 25 °C prior to
kinetic analyses. For inhibition studies with glutathione, reduced
glutathione was included in the standard reaction mixture using
concentrations of between 0.1 and 10 mM. Inhibition
constants were estimated using different concentrations of glutathione
(0.1, 0.25, and 0.5 mM) with glutamate as the variable substrate. Nonlinear regression analysis of data was performed using
Hyper software (13), and Ki values were estimated using Enzpack (Biosoft). Lineweaver-Burk plots were used to assess the
mechanisms of inhibition.
-tubulin (a gift from
Dr. W. Whitfield). The antisera raised against the DmGCL subunits were
each used at a dilution of 1:1000.
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View larger version (32K):
[in a new window]
Fig. 1.
Sequence alignment of DmGCLM with human
GCLM. Black boxes show amino acid sequence
identity. Gray boxes show amino acid sequence similarity.
The cysteine residues conserved between the Drosophila and
human (hGCLM) proteins are indicated with asterisks.
. The sequence
was verified before the insert was subcloned into pET15b, and the
resulting plasmid was used to express recombinant His-tagged DmGCLM in
E. coli as described under "Experimental Procedures."
Recombinant DmGCLM was purified from bacterial cell lysates using
nickel-agarose affinity chromatography. Approximately 40 mg of
recombinant protein was obtained from a 400-ml culture. Analysis of
purified recombinant DmGCLM by SDS-PAGE showed that it comprises a
single polypeptide with an approximate molecular mass of 31 kDa (data
not shown). This is in agreement with the molecular mass calculated
from the predicted amino acid sequence.
View larger version (30K):
[in a new window]
Fig. 2.
Gel filtration of DmGCL subunits and the
DmGCL holoenzyme. Recombinant DmGCL holoenzyme was generated by
mixing 10 mg of purified DmGCLM with 10 mg of DmGCLC and concentrated
by ammonium sulfate precipitation as described under "Experimental
Procedures." The DmGCL holoenzyme ( 
) and the uncomplexed DmGCLM
(
) and DmGCLC (
) subunits were separately subjected to gel
filtration chromatography. Eluted fractions were analyzed for protein
concentration (A) and GCL activity (B).
View larger version (38K):
[in a new window]
Fig. 3.
Analysis of the subunit composition of the
DmGCL holoenzyme. The peak fractions from gel filtration
chromatography of DmGCLC, DmGCLM, or the DmGCL holoenzyme (2 µg of
each) were analyzed by SDS-PAGE in the presence (lanes 2-4)
or absence (lanes 6-8) of 2-mercaptoethanol. Lanes
1 and 5, molecular mass markers; lanes 2 and
6, DmGCLM; lanes 3 and 7, DmGCLC;
lanes 4 and 8, DmGCL holoenzyme.
View larger version (22K):
[in a new window]
Fig. 4.
Immunoprecipitation of endogenous DmGCLC from
S2 cell extracts. Immunoprecipitation of DmGCLM from S2 cell
extracts with antiserum raised against recombinant DmGCLM was performed
as described under "Experimental Procedures." Immunoprecipitated
proteins were analyzed by Western blotting using antiserum raised
against recombinant DmGCLC. S, supernatant fraction
remaining after immunoprecipitation; IP,
immunoprecipitate.
-aminobutyrate was used
as substrate in place of cysteine, activity was comparatively low, even
when high concentrations were used. Km and
Vmax values were determined for
L--aminobutyrate, and the Km was
found to be between 4- and 12-fold higher (Table
I) than that reported for human GCL (3,
4). The catalytic efficiency of DmGCL
(kcat/Km) with
L--aminobutyrate as substrate is 0.53 min1
mM1, implying that
L--aminobutyrate is a poor substrate for DmGCL.
Kinetic constants for Drosophila GCL
-aminobutyrate.
View larger version (13K):
[in a new window]
Fig. 5.
Inhibition of recombinant DmGCL activity by
glutathione. The specific activities of the DmGCL
holoenzyme ( 
) and DmGCLC () were measured using standard assay
conditions in the presence of increasing concentrations of glutathione.
Activity is expressed as a percentage of the control activity in the
absence of glutathione (mean ± S.E.).
Effect of BSO or cystamine on DmGCL activity
View larger version (41K):
[in a new window]
Fig. 6.
Effect of tBHQ on DmGCL subunit levels in S2
cells. S2 cells were cultured in the presence of tBHQ for 18 h, and cell lysates (30 µg of protein) were analyzed by Western
blotting using antibodies raised against DmGCLC (A), DmGCLM
(B), or Drosophila -tubulin (C).
The lanes were loaded with extracts from cells that had been treated as
follows. Lane 1, no treatment; lane 2,
Me2SO; lane 3, 25 µM tBHQ;
lane 4, 50 µM tBHQ; lane 5, 75 µM tBHQ; lane 6, 100 µM tBHQ.
The relative intensities of cross-reactive bands were examined by
scanning densitometry.
DISCUSSION
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EXPERIMENTAL PROCEDURES
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DISCUSSION
REFERENCES
B-binding sites and could
potentially be involved in increasing DmGCLC transcription in response
to tBHQ. Oxidative stress is thought to induce transcription of
mammalian glutathione-associated genes by triggering signaling cascades
that activate various transcription factors such as Nrf2, AP-1,
and nuclear factor-B. Homologs of mammalian c-Jun and c-Fos
as well as nuclear factor-B exist in Drosophila (D-Jun,
D-Fos, and the Rel family, respectively) (36, 37), but their role in
adaptation to oxidative stress within Drosophila has not, to
our knowledge, been characterized as rigorously as that of their
mammalian counterparts.
ACKNOWLEDGEMENT
FOOTNOTES
To whom correspondence should be addressed. Tel.:
44-1382-660111; Fax: 44-1382-669993; E-mail:
mclellan@icrf.icnet.uk.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Huang, C.-S.,
Anderson, M. E.,
and Meister, A.
(1993)
J. Biol. Chem.
268,
20578-20583
2.
Huang, C.-S.,
Chang, L.-S.,
Anderson, M. E.,
and Meister, A.
(1993)
J. Biol. Chem.
268,
19675-19680
3.
Misra, I.,
and Griffith, O. W.
(1998)
Protein Expression Purif.
13,
268-276
4.
Tu, Z. H.,
and Anders, M. W.
(1998)
Arch. Biochem. Biophys.
354,
247-254
5.
Watanabe, K.,
Yamano, Y.,
Murata, K.,
and Kimura, A.
(1986)
Nucleic Acids Res.
14,
4393-4400
6.
Ohtake, Y.,
and Yabuuchi, S.
(1991)
Yeast
7,
953-961
7.
Wilson, R., Ainscough, R., Anderson, K., Baynes, C., Berks, M., Burton,
J., Connell, M., Bonfield, J., Copsey, T., Cooper, J., Coulson, A.,
Craxton, M., Dear, S., Du, Z., Durbin, R., Favello, A., Fraser, A.,
Fulton, L., Gardner, A., Green, P., Hawkins, T., Hillier, L., Jier, M.,
Johnston, L., Jones, M., Kershaw, J., Kirsten, J., Laisster, N.,
Latreille, P., Lloyd, C., Mortimore, B., Ocallaghan, M., Parsons, J.,
Percy, C., Rifken, L., Roopra, A., Saunders, D., Shownkeen, R., Sims,
M., Smaldon, N., Smith, A., Smith, M., Sonnhammer, E., Staden, R.,
Sulston, J., Thierrymieg, J., Thomas, K., Vaudin, M., Vaughan, K.,
Waterston, R., Watson, A., Weinstock, L., Wilkinsonsproat, J., and
Wohldman, P.
8.
Lueder, D. V.,
and Phillips, M. A.
(1996)
J. Biol. Chem.
271,
17485-17490
9.
Brekken, D. L.,
and Phillips, M. A.
(1998)
J. Biol. Chem.
273,
26317-26322
10.
Saunders, R. D. C.,
and McLellan, L. I.
(2000)
FEBS Lett.
467,
337-340
11.
Bradford, M. M.
(1976)
Anal. Biochem.
72,
248-254
12.
Seelig, G. F.,
Simondsen, R. P.,
and Meister, A.
(1984)
J. Biol. Chem.
259,
9345-9347
13.
Cleland, W. W.
(1979)
Methods Enzymol.
63,
103-138
14.
Towbin, H.,
Staehelin, T.,
and Gordon, J.
(1979)
Proc. Natl. Acad. Sci. U. S. A.
76,
4350-4354
15.
Galloway, D. C.,
Blake, D. G.,
and McLellan, L. I.
(1999)
Biochim. Biophys. Acta
1446,
47-56
16.
Adams, M. D.,
Celniker, S. E.,
Holt, R. A.,
et al..
(2000)
Science
287,
2185-2195
17.
Rubin, G. M.,
Hong, L.,
Brokstein, P.,
Evans-Holm, M.,
Frise, E.,
Stapleton, M.,
and Harvey, D. A.
(2000)
Science
287,
2222-2224
18.
Griffith, O. W.,
and Meister, A.
(1979)
J. Biol. Chem.
254,
7558-7560
19.
Griffith, O. W.
(1999)
Free Radic. Biol. Med.
27,
922-935
20.
Lebo, R. V.,
and Kredich, N. M.
(1978)
J. Biol. Chem.
253,
2615-2623
21.
Seelig, G. F.,
and Meister, A.
(1982)
J. Biol. Chem.
257,
5092-5096
22.
Seelig, G. F.,
and Meister, A.
(1984)
J. Biol. Chem.
259,
3534-3538
23.
Hayes, J. D.,
and McLellan, L. I.
(1999)
Free Radic. Res.
31,
273-300
24.
Wild, A. C.,
and Mulcahy, R. T.
(2000)
Free Radic. Res.
32,
281-301
25.
Soltaninassab, S. R.,
Sekhar, K. R.,
Meredith, M. J.,
and Freeman, M. L.
(2000)
J. Cell. Physiol.
182,
163-170
26.
Lüersen, K.,
Müller, S.,
Hussein, A.,
Liebau, E.,
and Walter, R. D.
(2000)
Mol. Biochem. Parasitol.
111,
243-251
27.
Lüersen, K.,
Walter, R. D.,
and Müller, S.
(1999)
Mol. Biochem. Parasitol.
98,
131-142
28.
Coblenz, A.,
and Wolf, K.
(1995)
Yeast
11,
1171-1177
29.
May, M.,
and Leaver, C.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10059-10063
30.
May, M. J.,
Vernoux, T.,
Sanchez-Fernandez, R.,
Van Montagu, M.,
and Inze, D.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
12049-12054
31.
Hussein, A. S.,
and Walter, R. D.
(1995)
Mol. Biochem. Parasitol.
72,
57-64
32.
Griffith, O. W.,
and Mulcahy, R. T.
(1999)
Adv. Enzymol. Relat. Areas Mol. Biol.
73,
209-267
33.
Sekura, R.,
and Meister, A.
(1977)
J. Biol. Chem.
252,
2599-2605
34.
Yan, N.,
and Meister, A.
(1990)
J. Biol. Chem.
265,
1588-1593
35.
Davis, J. S.,
Balinsky, J. B.,
Harington, J. S.,
and Shepherd, J. B.
(1973)
Biochem. J.
133,
667-678
36.
Fenton, M. J.
(2000)
Nat. Immunol.
1,
279-281
37.
Kockel, L.,
Homsy, J. G.,
and Bohmann, D.
(2001)
Oncogene
20,
2347-2364
38.
Tsuchiya, K.,
Mulcahy, R. T.,
Reid, L. L.,
Disteche, C. M.,
and Kavanagh, T. J.
(1995)
Genomics
30,
630-632
39.
Spradling, A. C.,
Stern, D.,
Kiss, I.,
Roote, J.,
Laverty, T.,
and Rubin, G. M.
(1995)
Proc. Natl. Acad. Sci. U. S. A.
92,
10824-10830
40.
Spradling, A. C.,
Stern, D.,
Beaton, A.,
Rhem, E. J.,
Laverty, T.,
Mozden, N.,
Misra, S.,
and Rubin, G. M.
(1999)
Genetics
153,
135-177
41.
Shi, Z. Z.,
Osei-Frimpong, J.,
Kala, G.,
Kala, S. V.,
Barrios, R. J.,
Habib, G. M.,
Lukin, D. J.,
Danney, C. M.,
Matzuk, M. M.,
and Lieberman, M. W.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
5101-5106
42.
Dalton, T. P.,
Dieter, M. Z.,
Yang, Y.,
Shertzer, H. G.,
and Nebert, D. W.
(2000)
Biochem. Biophys. Res. Commun.
279,
324-329
43.
Easterby, J. S.
(1996)
Hyper, Version 1.1s
, University of Liverpool, Liverpool, United Kingdom
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