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Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan; and
* Department of Microbiology, Institute of Medical Science, The University of Tokyo, Minato-ku, Tokyo 108-8639, Japan
1Correspondence: Laboratory of Molecular and Biochemical Toxicology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai 980-8578, Japan. E-mail: naganuma{at}mail.pharm.tohoku.ac.jp
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Key Words: gene screening • glucosamine • glucosamine-6-phosphate • resistance • p-chloromercuribenzoate
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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2
3)
. Many reports have contributed MeHg poisoning to
contaminated foods and release into the environment
(4
5
6
7)
. In fact, pollution of the environment with MeHg is
still occurring in some regions in the world, and its influence on the
health of local inhabitants is a great concern (8
9
10)
.
Recently, it was reported that a scientist who was exposed to MeHg
during experiments died of neurological manifestations
(11)
. Despite many studies on the pathogenesis of
MeHg-induced central neuropathy, no useful mechanism of toxicity has
been established. A first step in the investigation is to identify the
molecular targets of MeHg toxicity. We have searched for a gene from an
MeHg-resistant strain Saccharomyces cerevisiae that, when
transfected into MeHg-sensitive host cells, would provide the host with
MeHg-resistance. The hypothesis of drug resistance acquisition by the
elevation of intracellular concentrations of the target molecule
(12
13
14
15)
suggests that the gene encoding the target
molecule of MeHg toxicity may be included in the genes transferring
MeHg resistance. In this study, we found that
L-glutamine:D-fructose-6-phosphate amidotransferase (GFAT), which has
been identified as factor in conferring MeHg-resistance, is the target
molecule of methylmercury in yeast. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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leu2 his3 trp 1 canl-100
ade2 trp1 ura3) was transfected with the yeast genomic DNA library
by the lithium acetate method and ~2 x
104 Leu+ transformants that
expressed the transfected gene were obtained. The transformed cells
were seeded on synthetic minimal medium (SDM; -leucine) agar plates
containing 20 nM MeHg, a concentration that inhibited the growth of
W303B transformed with vector alone. After 3 days, 10 colonies (D1 to
D5 and H1 to H5) remained that were able to grow in the presence of
MeHg. Plasmids were isolated from these clones and retransformed into
the parent strain, and the sensitivity of secondary transfectant to
methylmercury was examined. Yeast transformed with the vector YEp13 was
used for the control.
Identification of the gene confers MeHg resistance
The yeast genomic DNA fragment in the plasmid from clone H5 was
digested with several appropriate restriction enzymes and analyzed by
agarose gel electrophoresis. Plasmid from the H5 clone contained a 9.2
kb genomic DNA fragment with two BamHI sites and one
HindIII site. The plasmid was cleaved with these enzymes
into a BamHI genomic fragment of ~4.6 kb and
HindIII fragment, containing the genomic and plasmid
sequence, of ~7.5 kb. These two fragments were subcloned in the
expression vector pYES2. The parent strain was transformed with these
vectors containing the inserts and examined for MeHg resistance. Colony
formation in the presence of 20 nM MeHg was observed only in the yeasts
transformed with the BamHI fragment. Approximately 300 bp
nucleotide sequences at both ends of the BamHI fragment were
determined, and homologies for these sequences were found in the
Saccharomyces Genome Database. The BamHI fragment
corresponds to the nucleotide sequences from 241442 to 246087 in the
yeast chromosome number 11. This region contains one intact open
reading frame (ORF), GFA1, as well as the truncated ORFs
YKL105C and LAP4.
Establishment of GFA1-transfected yeast clone
GFA1 gene was isolated from the BamHI genomic
fragment containing GFA1 by EcoRI digestion. The
GFA1 gene isolated as 3.7 kb EcoRI fragment was
subcloned in pYES2 and then transfected into yeast (W303B). The clones
that overexpressed GFA1 and transfected with the vector
pYES2 alone were designated W303B/pGFA1 and W303B/pYES2, respectively.
Measurement of MeHg sensitivity of yeast by suspension culture
The yeasts (W303B/pGFA1 and W303B/pYES2) were seeded in SDM
medium and cultured at 30°C overnight. After these culture fluids
were diluted with SDM medium to a cell density of 1 x
106 cells/ml and MeHg was added, the cells were
further cultured with shaking for 24 h at 30°C. The cell
viability was obtained by measuring the absorbance at 620 nm.
Preparation of yeast extract
After the yeast was seeded in SDM medium and cultured at 30°C
overnight, the culture was diluted with SDM medium and further cultured
with shaking for 3 h at 30°C. Glass beads were added to the
cells obtained and vigorously stirred for 20 min at 4°C to destroy
the cells; centrifugation at 1200 g, 4°C for 20 min,
followed and the supernatant was used as yeast extract.
GFAT activity measurement
To 50 µl of the yeast extract, 450 µl of reaction solution
(6 mM D-fructose-6-phosphate, 12 mM L-glutamine, 1.25 mM EDTA, 40 mM
sodium phosphate buffer-pH 7.5) was added and incubated at 374°C for
2 h. Then the solution was heated in boiling water for 3 min to
stop the enzyme reaction, centrifuged at 12,000 g for 10
min, and the supernatant was obtained. Glucosamine-6-phosphate in the
supernatant was measured by the modified Elson-Morgan method
(16)
. The activities of alcohol dehydrogenase (ADH)
(17)
, glutathione reductase (GSHR) (18)
, and
lactate dehydrogenase (LDH) (19)
were determined by the
spectrophotometric method.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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a). GFA1 (20)
encodes
L-glutamine:D-fructose-6-phosphate amidotransferase (EC 2.6.1.16),
which catalyzes the synthesis of glucosamine-6-phosphate from glutamine
and fructose-6-phosphate (16)
. GFAT is conserved in a wide
range of organisms such as Escherichia coli, mice, rats, and
humans, as well as yeasts (21)
. Since
glucosamine-6-phosphate produced by GFAT-catalyzed reaction is the
starting material in all amino sugar biosynthesis pathways, GFAT is
considered essential for growth and functional maintenance of cells.
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To assess the effect of MeHg on GFAT activity, the wild-type yeast
strain W303B was suspended in yeast-peptone-adenine-dextrose (YPAD)
medium and cultured in the presence of MeHg. GFAT activity was measured
in a cell extract preparation. The GFAT activity was inhibited by MeHg
in a dose-dependent manner, reduced to ~10% of the normal value by 3
µM MeHg (Fig. 1b
). Furthermore, the growth rate of the
cultured yeast in the presence of 3 µM of MeHg was decreased to
~25% of the control rate after 24 h of culture. These results
suggest a correlation between cytotoxicity and the inhibition of GFAT
activity by MeHg (Fig. 1b
). These findings suggest that
GFAT, identified as factor in conferring MeHg-resistance, may be the
intracellular target molecule of MeHg toxicity.
To clarify the possibility that GFAT is the target of MeHg toxicity,
the following experiments were performed. First, MeHg was added
directly to an extract of wild-type yeast strain, and its effect on the
activities of GFAT and other SH enzymes such as alcohol dehydrogenase,
glutathione reductase, and lactate dehydrogenase was examined. GFAT
activity was decreased by addition of MeHg at 1 µM or higher, and
90% or more of the activity was inhibited at 4 µM. In contrast,
there were almost no effects of MeHg on the other SH enzymes up to a
concentration of 4 µM (Fig. 2
). The inhibition constant (Ki) of MeHg for each of
the four enzymes was obtained by the Dixon’s plot analysis
(22)
. The inhibition constant of MeHg for GFAT was 4 µM,
whereas those for ADH, GSHR, and LDH were 50.4 µM, 71.0 µM, and
79.6 µM, respectively. These findings indicate that yeast GFAT is
10-fold or more sensitive to MeHg than ADH, GSHR, and LDH. Generally,
mercury compounds such as MeHg have a high affinity for the SH group
and are thought to inhibit SH enzyme activity by binding to the SH
group present in the activity center. Since the amino-terminal Cys-2 in
GFAT is required for binding to glutamine (23)
, GFAT is
likely to be bound by MeHg at Cys-2 in the tertiary structure, which
may account for selective inhibition of GFAT activity by MeHg. Since
GFAT is also present in humans, the effect of MeHg on human GFAT was
investigated using HepG2 cells. MeHg also markedly inhibited the GFAT
activity in HepG2 cells in a dose-dependent manner. The survival rate
of HepG2 cells was also decreased in a MeHg dose-dependent manner. The
LDH activity was measured as an example of SH enzymes other than GFAT.
There was almost no LDH inhibition detected within the concentration
range used in this experiment (data not shown). These findings
suggested that GFAT is selectively inhibited by MeHg not only in yeast,
but also in human cells.
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The organic mercury compound p-chloromercuribenzoic acid
(pCMB) is known to inhibit GFAT activity (24)
.
We investigated the relationship between GFAT activity and cytotoxicity
of pCMB using the yeast suspension culture described above.
Wild-type yeast transformed with the multicopy plasmid pYES2 carrying
GFA1 (W303B/pGFA1) shows a marked MeHg resistance when
compared to the control yeast strain transformed with vector alone
(W303B/pYES2) (Fig. 3
). However, the two yeast strains exhibit almost identical cytotoxicity
to pCMB. Therefore, while the elevation of intracellular
GFAT activity increases resistance to methylmercury, the toxicity of
other GFAT inhibitors persists. These results suggest the possibility
that GFAT is an intracellular target molecule that is specifically
inhibited by MeHg. The W303B/pGFA1 strain contains higher
concentrations of GFAT protein than those in the W303B/pYES2 strain;
therefore, full inhibition of the GFAT activity in W303B/pGFA1 may
require higher concentrations of MeHg than in W303B/pYES2 indicating a
resistance to MeHg. The levels of GFAT activity do not affect
pCMB toxicity, indicating a target molecule of
pCMB in yeast that has a higher affinity for pCMB
than for GFAT.
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As described above, MeHg may have a high binding affinity for GFAT.
Accordingly, the MeHg resistance observed in the W303B/pGFA1 strain may
be due to the overexpression of GFAT protein, which firmly binds MeHg
and reduces the free MeHg concentration. To investigate this
possibility, we examined the effects of elevated intracellular levels
of glucosamine-6-phosphate on MeHg toxicity. Glucosamine-6-phosphate is
not incorporated into cells from the culture medium and its
intracellular levels are dependent on the reaction catalyzed by GFAT.
When added to the culture medium, glucosamine is taken up by the cells
and converted to glucosamine-6-phosphate by hexokinase (25
, 26)
. Yeast cells cultured in medium containing glucosamine
exhibit a markedly reduced sensitivity to MeHg in a dose-dependent
manner (Fig. 4A
). pCMB toxicity, however, was only slightly
affected by the addition of glucosamine (Fig. 4B
). In
addition, no direct binding of glucosamine or glucosamine-6-phosphate
to MeHg was observed after incubation in a test tube, and glucosamine
showed almost no influence on incorporation of MeHg into cells (data
not shown). Based on these findings, the reduction of free MeHg by GFAT
binding as a mechanism for the reduction of MeHg toxicity can be
excluded. The findings of this study, that increased levels of
glucosamine-6-phosphate inhibit MeHg toxicity in yeast (Fig. 4A
), strongly suggest that the inhibition of GFAT activity
(i.e., the inhibition of glucosamine-6-phosphate synthesis) is the
major cause of MeHg toxicity in yeast.
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The reaction catalyzed by GFAT is the initial reaction in the
biosynthesis of each of three amino sugars—N-acetylglucosamine,
N-acetylgalactosamine,and N-acetylneuraminic acid—which
constitute the conjugated polysaccharide of cells such as
glycoproteins, glycolipids, and mucopolysaccharides. In this function,
GFAT is considered to play an important role in the growth and
functional maintenance of cells. Indeed, it has been shown that a
GFA1-deficient yeast cell is glucosamine auxotroph
(20)
. In this study, we have shown that MeHg selectively
inhibits yeast GFAT activity and that MeHg cytotoxicity is suppressed
by supplying the reaction product of this enzyme,
glucosamine-6-phosphate, into the cells. These results indicate for the
first time that GFAT is the target molecule of MeHg in yeast.
Methylmercury inhibition of GFAT activity reduces the amount of
reaction product, glucosamine-6-phosphate, resulting in a reduction in
biosynthesis of hexosamine and amino sugars that is essential for cell
function. These events lead to the development of severe cytotoxicity.
GFAT is an essential enzyme not only in yeast, but also in humans, and
there is high homology of amino acid sequence between yeast GFAT and
human GFAT (27)
. Therefore, a mechanism of expression of
cellular toxicity of MeHg similar to that in yeast may also present in
humans.
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FOOTNOTES |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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