From the
We examined the intracellular distribution of 8-oxo-dGTPase
(8-oxo-7,8-dihydrodeoxyguanosine triphosphatase) encoded by the MTH1 gene, a human mutator homologue. The activity of
8-oxo-dGTPase mainly located in cytosolic and mitochondrial soluble
fractions of Jurkat cells, a human T-cell leukemia line. Electron
microscopic immunocytochemistry, using a specific antibody against MTH1
protein, showed localization of MTH1 protein in the mitochondrial
matrix. Activity in the mitochondria accounted for about 4% of the
total activity. The specific activity in the mitochondrial soluble
fraction (8093 units/mg protein) was as high as that in the cytosolic
fraction (8111 unit/mg protein). The 8-oxo-dGTPase activities in
cytosolic and mitochondrial soluble fractions co-eluted with MTH1
protein by anion-exchange chromatography, and the molecular mass of the
mitochondrial MTH1 protein was much the same as that of the cytosolic
MTH1 protein (about 18 kDa). HeLa cells expressing MTH1 cDNA showed an
increased cytoplasmic signal together with a weak signal in the nucleus
in in situ immunostaining of MTH1 protein, and the
overexpressed MTH1 protein was recovered from both cytosolic and
mitochondrial fractions. Thus, the 8-oxo-dGTPase encoded by MTH1 gene is localized in mitochondria and cytosol.
Oxygen radicals generated through the process of
oxidation-reduction reactions in living cells attack many reactive
moieties of DNA. When DNA is subjected to such oxidative lesions,
strand breaks of DNA occur as does modification of bases, and cellular
dysfunction, mutagenesis, and carcinogenesis
follow(1, 2, 3) . Among the oxidative lesions of
DNA, 8-oxoguanine
Organisms are equipped
with elaborate mechanisms to counteract such mutagenic effects of
8-oxoguanine. In Escherichia coli, two DNA glycosylases
encoded by mutM and mutY genes function to repair
8-oxoguanine. MutM protein removes 8-oxoguanine paired with cytosine (6) and MutY protein removes adenine paired with 8-oxoguanine in
DNA(7) . The oxidized form of guanine is also formed in the
nucleotide pool of the cell and can be eliminated by the mutT gene product. MutT protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP,
thereby preventing misincorporation of 8-oxo-dGMP into DNA (5, 8). In a mutT-deficient strain, the rate of spontaneous occurrence of
A:T to C:G transversion increases hundreds to thousandsfold compared to
the wild type(9, 10, 11) . While the spontaneous
mutation rate in mutM or mutY-deficient strain is
10-50 times higher than that in wild type
strain(7, 11) , a rate of mutation in the double mutants
of mutM and mutY is equivalent to that of the mutT mutant(11) .
Mammalian cells contain enzyme
activities similar to those E. coli enzymes. 8-Oxo-dGTPase has
been purified from human Jurkat cells, a T cell leukemia cell
line(12) . The cDNA was isolated, and the genomic sequence was
determined(13, 14) . The human 8-oxo-dGTPase shows a
considerable degree of amino acid sequence homology with the E.
coli MutT protein(13) , and expression of the human cDNA in mutT-deficient E. coli cells efficiently reduced the
increased frequency of A:T to C:G transversion to the level seen in
wild type strain(14) . Hence, the enzyme is considered to be a
human counterpart of MutT protein, and the gene was named MTH1 (for mutT homologue 1)(14) .
In eukaryotic cells, a pool
of dNTP for nuclear DNA replication is present mainly in the
cytosol(15) . For mitochondrial DNA synthesis, mitochondria
preserve a pool of dNTP, consisting of more than 10% of the total
intracellular dNTP. The amount of the mitochondrial DNA is roughly 1%
of the total DNA in the cell(15, 16) . Judging from the
different behavior of nucleotides in mitochondrial and cytosolic pools
(15-17), nucleotides in the former seem to be synthesized in the
mitochondria and are not derived from the cytosolic pool. The activity
of ribonucleotide reductase that catalyzes synthesis of
deoxyribonucleoside 5`-diphosphate from ribonucleoside 5`-diphosphate
is present in both the cytpolasm and mitochondria(17) .
The
mitochondrial respiratory chain located on inner membranes is a major
site for the initiation of lipid peroxidation (18, 19) which can lead to oxidation of the guanine base
to 8-oxoguanine(20) . In addition, the mitochondrial respiratory
chain produces superoxide (21) which can be converted to
hydroxyl radical via hydrogen peroxide. The hydroxyl radical is the
main species of active oxygens that attack the guanine
base(22) . Thus, DNA and dNTP in the mitochondrial pool may be
exposed to a greater oxidative stress. The repair of oxidized
mitochondrial DNA and elimination of 8-oxo-dGTP from the mitochondrial
dNTP pool may be crucial to maintain integrity of mitochondrial DNA. A
system for repair of oxidatively damaged DNA in the mitochondria has
been described(23) . However, it is uncertain whether the
mitochondria possess mechanism(s) for eliminating 8-oxo-dGTP from dNTP
pool. We examined the intracellular distribution of 8-oxo-dGTPase in
human cells with special reference to role of the enzyme 8-oxo-dGTPase
in the mitochondria. Several lines of evidence obtained in this study
show that the mitochondrial matrix possesses 8-oxo-dGTPase and suggest
that the 8-oxo-dGTPase present in mitochondria and cytosol fractions is
the product of the same gene.
For
preparation of the nuclear fraction, cells were suspended in hypotonic
buffer (10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 15
µg/ml leupeptin, 5 µg/ml APMSF, and 50 ng/ml pepstatin)
containing 5 mM CaCl
Mitoplasts
were prepared as follows. Freshly prepared mitochondria (3 mg) in 0.3
ml of TES were diluted 10-fold with buffer containing 5 mM Tris-HCl, pH 7.4, and 1 mM EDTA and centrifuged at 14,000
Polyclonal rabbit antibodies against human MTH1 protein were
prepared using TrpE-MTH1 fusion protein, as described by Nakabeppu and
Nathans (25). E. coli BL21(DE3) carrying pET:TrpE-MTH1 was
cultured at 37 °C in 250 ml of broth containing 50 µg/ml of
ampicillin to OD
For electron
microscopic immunocytochemistry, the washed fraction of crude
mitochondria was used because the hypertonic procedure had resulted in
mitochondria with a highly condensed configuration. Isolated
mitochondria were pelleted, fixed with 8% paraformaldehyde, and
embedded in LR white resin at 50 °C. Thin sections were cut with a
diamond knife, treated with 1% bovine serum albumin for 10 min, and
incubated with anti-hMTH1 antibody at a concentration of 25 µg/ml
for 2 h at room temperature. After washing with phosphate-buffered
saline, the sections were incubated with anti-rabbit IgG-gold
(OD
Light microscopic immunocytochemistry of HeLa MR cells was done as
described by Ishibashi et al.(29, 32) after
the cells had been fixed with 0.2% glutaraldehyde for 1 h at 4 °C
and permeabilized with 50% methanol and 50% acetone for 2 min at room
temperature.
The total activities of 8-oxo-dGTPase in
cytosol and mitochondria fractions were calculated on the basis of
recovery of lactate dehydrogenase and succinate-cytochrome c reductase activities in those fractions. The combined activities
of 8-oxo-dGTPase in the cytosolic and mitochondrial fractions almost
correspond with the total activities (). The 8-oxo-dGTPase
activity in the mitochondria was about 4% of that in the entire cell.
In the case of promyelocytic leukemia HL60 cells, mitochondrial
8-oxo-dGTPase occupied 9.6% of the total activities of the cells.
Each
subcellular fraction was analyzed by immunoblotting using the
anti-hMTH1 (Fig. 1B). A single band corresponding to the
18-kDa polypeptide was detected in the cytosol and in the mitochondria.
The intensity of the signal/mg of protein in each fraction essentially
paralleled to the activity of 8-oxo-dGTPase (see Fig. 1B and ). This would suggest that a single molecular
species of MTH1 protein is responsible for 8-oxo-dGTPase activities
present both in the cytosolic and mitochondrial fractions.
To see whether mitochondrial 8-oxo-dGTPase is localized in the
intermembrane space or in the matrix, we prepared mitoplasts which do
not have components of intermembrane space. The specific activities of
fumarase and 8-oxo-dGTPase in the mitoplasts were 76 and 65% of those
in the intact mitochondrial fraction, respectively, while the specific
activity of adenylate kinase, a marker enzyme of the intermembrane
space, was only 6% of that in the mitochondrial fraction (I). This low, specific activity of adenylate kinase in
the mitoplasts is not due to inactivation of the enzyme during the
preparation of mitoplasts since about 70% of the total activities was
recovered in the post-mitoplast supernatant. These results would
suggest that 8-oxo-dGTPase is mainly localized in the mitochondrial
matrix.
The localization of 8-oxo-dGTPase in the matrix was further
confirmed by electron microscopic immunocytochemistry of mitochondria
stained with anti-hMTH1 (Fig. 2). To rule out the possibility
that the cytosolic 8-oxo-dGTPase might give a false positive signal on
the staining of mitochondrial 8-oxo-dGTPase, we used isolated
mitochondria for the immunostaining experiments. The mitochondria
showed a condensed configuration usually observed in mitochondria
isolated by the method used here(37) . Proteins reactive to the
anti-hMTH1 were predominant on the matrix (electron dense area), not on
the intermembrane space (Fig. 2). When non-immune IgG was used,
signals were nil (results not shown).
In the cytosolic fraction, we detected activity
converting 8-oxo-dGTP to 8-oxo-dGDP, which eluted more slowly than did
the actual 8-oxo-dGTPase peak (Fig. 3, lowerpanel). This activity represents a nonspecific nucleotide
triphosphatase that was not found in the mitochondrial soluble fraction (Fig. 3, upper panel). This evidence further supports
the notion that 8-oxo-dGTPase in the mitochondrial fraction is not due
to the contamination with the cytosolic fraction.
The spontaneous oxidation of dGTP forms 8-oxo-dGTP, which can
be inserted opposite dA and dC residues of template DNA with almost
equal efficiency, and the MutT protein of E. coli specifically
degrades 8-oxo-dGTP to the monophosphate(5) . Since defects in
the mutT gene increase the occurrence of A:T to C:G
transversions 100-10,000-fold over the wild type
level(9, 10, 11) , elimination of the oxidized
form of guanine nucleotide from the nucleotide pool is important for
the high fidelity of DNA replication. An enzyme similar to the MutT
protein was detected in human cells (12) and the cDNA was
cloned(13) . As expression of human cDNA in E. coli
mutT
We
have shown in the present study that the MTH1 protein is mostly present
in the cellular cytosolic fraction. In subcellular fractionation,
little MTH1 protein was recovered in the nuclear fraction. In situ immunostaining of MTH1-overexpressing HeLa cells revealed a
typical cytoplasmic staining with slightly weaker nuclear staining.
MTH1 protein present in nuclei may be lost into the cytosolic fraction
during isolation of nuclei since the MTH1 protein does not have a
typical nuclear localization signal (13) and size is small
enough to freely traverse nuclear pores(38) . Even taking this
into account, it is reasonable to conclude that a large part of the
8-oxo-dGTPase is localized in the cytosol. Larger pools of dNTP are
present in cytosol than in nucleus, the former being supply of
materials for the replication of chromosomal
DNA(15, 16) .
We have shown that a considerably high
level of the 8-oxo-dGTPase protein is present in the mitochondrial
matrix. Mitochondria synthesize their own dNTPs (17) and form an
independent pool of dNTP from pools in the cytosol and the
nucleus(15, 16) . The mitochondrial dNTP pool is
probably exposed to a stronger oxidative stress than are the cytosolic
and nuclear pools because the respiratory chain producing a large
amount of active oxygen species is present. DNA polymerase
Mitochondria are central for aerobic energy production in eukaryotic
cells and, therefore, the proper function of mitochondria is critical
for cell survival. Many cases of mitochondrial neuromyopathy caused by
dysfunction of mitochondrial respiration have been noted(40) .
Since the integrity of mitochondrial DNA is essential for the normal
functions of mitochondria(41) , it is important to protect
mitochondrial DNA from attack by oxygen radicals inevitably produced by
the mitochondrial own activity. Accumulation of mutations in
mitochondrial DNA has been correlated with the decline of the oxidative
phosphorylation with aging and with the impairment of the respiratory
chain in degenerative diseases(1, 42) . It has also been
reported that content of 8-oxoguanine in mitochondrial DNA increases
with aging(1, 43) . Therefore, some portion of mutations
leading to dysfunction of the mitochondrial respiratory chain may be
caused by 8-oxoguanine. Involvement of 8-oxoguanine in deletion of
mitochondrial DNA which is often observed in the Parkinson disease has
also been discussed(43) . The content of 8-oxoguanine in DNA may
be determined by a dynamic equilibrium between generation of
8-oxoguanine in DNA and excision repair of base from DNA. It is an
intriguing question whether impairment of the 8-oxo-dGTPase or other
repair enzymes is one of the causes for mitochondrial dysfunction in
aging as well as in some degenerative diseases.
We have shown that
an identical molecular form of MTH1 protein is present both in cytosol
and mitochondria. There are several examples that isozymes exist in the
cytosol and in the mitochondria(44) . Enzymes which locate in
the two sites are classified into three categories: (i) enzymes coded
by two separate genes, such as alcohol dehydrogenase; (ii) enzymes
derived from alternatively spliced mRNAs, e.g. 2-isopropylmalate synthase; and (iii) enzymes differentially
translated from single mRNA, for example, fumarase. Prefumarase
carrying a signal sequence is imported into mitochondria, processed,
and eventually takes on the same size as cytosolic fumarase (45). The
last mechanism might relate to 8-oxo-dGTPase but there is no possible
alternative initiation sites at the 5` region of cDNA with a signal
sequence which would be processed(13) . There is the possibility
that the intracellular localization of MTH1 protein is determined by
post-translational modification of the protein.
Subcellular fractions of Jurkat cells were prepared as
described under ``Experimental Procedures.'' Activities of
lactate dehydrogenase, succinate-cytochrome c reductase, and
8-oxo-dGTPase in subcellular fractions are expressed as percent of
those in cytosol, mitochondria, and cytosol, respectively. Each value
is a mean ± S.D. (n
The total activities of 8-oxo-dGTPase in cytosol
and mitochondria fractions were calculated on the basis of recovery of
the lactate dehydrogenase and succinate-cytochrome c reductase
activities present in the fractions. The activities are expressed as
percent of those in the homogenate. Each value in Jurkat cells is a
mean ± S.D. of three independent experiments. Values in HL60
cells are from a single experiment.
Submitochondrial fractions were prepared from mitochondria
of Jurkat cells. Activities are expressed as percent of those in
mitochondria. Values are means of two independent experiments.
hMTH1 protein in cytosol and
mitochondrial fractions of each strain was quantified with
immunoblotting by anti-hMTH1. Each value in parenthesis indicates
percent of that in the cytosol.
We extend special thanks to Dr. H. Sumimoto for
helpful discussions and M. Ohara for useful comments on the manuscript.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
(
)(8-oxo-7,8-dihydroguanine),
an oxidized form of guanine, is a major causative lesion for
mutagenesis by oxygen radicals, since during DNA replication it can
pair with adenine as well as cytosine, with almost equal
efficiency(4, 5) . Thus, 8-oxoguanine would cause A:T to
C:G and G:C to T:A transversion mutations.
Materials
8-Oxo-dGTP was synthesized from dGTP
as described previously(5) . [-
P]GTP
(>15TBq/mmol) and
I-protein A (1.1 GBq/mg) were
purchased from Amersham International plc (Buckinghamshire, United
Kingdom). Other reagents were of analytical grade.
Fractionation of Cultured Cells
Jurkat cells, a
human T cell leukemia line, were grown in RPMI 1640 medium containing
10% fetal calf serum. Cells in midlog growing phase were harvested,
washed in an isotonic sucrose buffer (TES) composed of 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.25 M sucrose, 15
µg/ml leupeptin, 5 µg/ml (p-amidinophenyl)methanesulfonyl fluoride hydrochloride
(APMSF) and 50 ng/ml pepstatin, and suspended in the same buffer (5
10
cells/ml). The following procedures were
performed at 4 °C. The cells were homogenized in a Potter-Elvehjem
homogenizer, the homogenate was centrifuged at 600
g for 10 min, and the supernatant (post-nuclear supernatant) was
centrifuged again at 600
g for 10 min to minimize
contamination of the post-nuclear supernatant with nuclei and intact
cells. The post-nuclear supernatant was centrifuged at 7,000
g for 10 min. The pellet (crude mitochondrial fraction) was
used for further purification of mitochondria. The 7,000
g supernatant was centrifuged at 320,000
g for 1 h
and the resulting supernatant was used as the cytosolic fraction. The
320,000
g pellet was washed three times with TES and
served as the microsomal fraction. The crude mitochondrial fraction was
layered on discontinuous sucrose gradient made by successive layering
4.5 ml of 1.5 M and 1.0 M sucrose from the bottom and
then centrifuged at 80,000
g for 1 h. The phase
between the layers of 1.5 and 1.0 M sucrose was collected and
washed three times with TES (mitochondrial fraction).
which stabilizes nuclear
membranes and was then kept on ice for 20 min. After homogenization of
the cells, the tonicity of the homogenate was made isotonic by adding
2.2 M sucrose, then the homogenate was centrifuged at 600
g for 10 min. The supernatant was centrifuged at
320,000
g for 1 h. The supernatant showed essentially
the same levels of activities of lactate dehydrogenase and
8-oxo-dGTPase as seen in the cytosolic fraction prepared without
CaCl
(results not shown). The 600
g pellet
was recentrifuged at 600
g for 10 min. The resulting
pellet was homogenized in TES containing 5 mM CaCl
, layered on discontinuous sucrose gradient made
by successive layering of 4.5 ml of 2.3 and 1.5 M sucrose
containing 1 mM MgCl
and centrifuged at 80,000
g for 1 h. The pellet at the bottom was collected and
washed three times with TES with 0.25 M sucrose containing 1
mM MgCl
(nuclear fraction).
Subfractionation of Mitochondria
To separate
mitochondria into membrane and soluble fractions, 4 mg of the
mitochondrial fraction in 1 ml of TES was sonicated at output 3 for
three 1-min cycles, using a Branson sonifier and then centrifuged at
320,000 g for 1 h at 4 °C. The supernatant was
used as the mitochondrial soluble fraction. The pellet was homogenized
in TES and served as the mitochondrial membrane fraction.
g for 20 min at 4 °C. The pellet was resuspended
in 3 ml of the same hypotonic buffer and left to stand on ice for 20
min. The suspension was centrifuged at 14,000
g for 20
min at 4 °C and washed three times with 3 ml of the hypotonic
buffer (mitoplasts). The supernatant at each step was also stored until
measurement of enzyme activities.
8-Oxo-dGTPase Assay
8-Oxo-dGTPase activity was
assayed by measuring the hydrolysis of 8-oxo-dGTP to
8-oxo-dGMP(5) . The reaction mixture (12.5 µl) contained 20
mM Tris-HCl, pH 8.0, 4 mM MgCl, 40 mM NaCl, 20 µM 8-oxo-dGTP, 80 µg/ml bovine serum
albumin, 8 mM dithiothreitol, 10% glycerol, and the enzyme
fraction to be examined. The reaction was run at 30 °C for 20 min
and stopped by spotting 2 µl of the reaction mixture onto a
polyethyleneimine-cellulose plate (Merck, Darmstadt, Germany). The
product was separated from the substrate by thin layer chromatography
with 1 M LiCl for 1 h, and the radioactivity was measured
using a Fujix 2000 Bio-image analyzer (Fuji Photo Film Co., Ltd.,
Tokyo, Japan). One unit of activity was defined as amount of enzyme
that produced 1 pmol of 8-oxo-dGMP/min.
Antibody Preparation
A peptide corresponding to
Lys to Val
(M78) was synthesized and purified
by HPLC. The polypeptide was coupled with bovine serum albumin and
hemocyanin by 0.1% glutaraldehyde(24) . To obtain polyclonal
antibodies against the peptide, each of the coupled peptides (200
µg) was emulsified with adjuvant, Titer Max (Vaxel, Inc., Norcross,
GA) and injected into a Japanese white rabbit. Four weeks later, the
first booster injection (100 µg) was given, followed by three
booster injections at 2-week intervals. Sera were obtained 2 weeks
after the last booster injection, and antibodies against the peptides
were purified on immunoaffinity columns in which the peptide was
covalently linked to activated CH-Sepharose 4B (Pharmacia
LKB)(24) . The purified antibody was designated anti-M78.
= 0.6 and then
isopropyl-
-D-thiogalactoside was added at a final
concentration of 1 mM(26) . After cultivation for 6 h,
the cells were harvested by centrifugation, washed with Tris-buffered
saline, and disrupted by sonication on ice in 50 mM Tris-HCl,
pH 7.5, 0.5 mM EDTA, and 0.3 M NaCl.The insoluble
protein fraction contained about 10 mg of TrpE-MTH1 fusion protein and
was electrophoresed on SDS-12.5% polyacrylamide gels. A region of the
gel containing the fusion protein was excised (approximately 200
µg), emulsified with adjuvant (Titer Max, Vaxel, Inc., Norcross,
GA), and injected into a Japanese white rabbit. Four weeks later, the
first booster injection (approximately 100 µg) was given, followed
by booster injections at 2-week intervals for 1 year. Sera were
obtained after the second injection of booster, and antibodies were
purified with the aid of TrpE-MTH1 fusion protein-Sepharose and TrpE
protein-Sepharose affinity columns(25, 27) . This
antibody preparation was designated as anti-hMTH1.
Immunoblotting Analysis of Subcellular
Fractions
Proteins were separated by 15% sodium dodecyl
sulfate-polyacrylamide gel electrophoresis (PAGE)(28) , and
immunoblotting analysis was performed as described
elsewhere(27, 29) . Briefly, a blot was blocked in
Tris-buffered saline (10 mM Tris-HCl, pH 7.6, and 150 mM NaCl) containing 0.05% Tween 20 and 5% bovine serum albumin at 52
°C for 1 h, probed with 10 µg/ml anti-hMTH1 antibody in the
blocking solution at 4 °C overnight, washed with Tris-buffered
saline containing 0.05% Tween 20, and reacted with I-protein A in the blocking solution at 4 °C for 1 h.
The antibody-reactive protein was visualized, and the radioactivity was
measured using a Fujix 2000 Bio-image analyzer. In the case of Fig. 1A and Fig. 3, the protein reactive to the
antibody was probed with biotinylated anti-rabbit IgG and the
avidin-biotin complex (Vector Laboratory, Burlingame, CA) and detected
using the chemiluminescence reagent (DuPont NEN).
Figure 1:
Immunoblotting of
subcellular fractions with the anti-hMTH1. A, proteins of the
total homogenates (14.6 µg which corresponds to 2.5 µg of the
cytosol) were separated on 15% SDS-PAGE and immunoblotted with 10
µg/ml of non-immune IgG or the anti-hMTH1 as described under
``Experimental Procedures.'' B, proteins were
separated on 15% SDS-PAGE and analyzed. MTH1 in each fraction was
quantified and is expressed as radioactivity/mg of protein (arbitrary
unit). Applied protein contents per lane are
shown.
Figure 3:
Analyses by anion-exchange chromatography.
The mitochondrial soluble (0.85 mg) and cytosolic (2 mg) fractions were
analyzed by MonoQ anion-exchange chromatography. The activity
hydrolyzing 8-oxo-dGTP to 8-oxo-dGDP () or to 8-oxo-dGMP (
)
was assayed using 2 µl from 1 ml of each fraction. The proteins
reactive to the anti-hMTH1 in each fraction (10 and 2.5 µl/lanes in upper and lower panels, respectively) were also
analyzed. Upper panel, mitochondrial soluble fraction; lower panel: cytosolic fraction.
Immunocytochemistry
Colloidal gold with a diameter
of 8 nm was prepared by reducing tetrachloroauric acid with tannic acid
and sodium citrate(30) . Goat antibody to rabbit IgG was
conjugated to the colloidal gold at pH 9.0 according to De Mey et
al.(31) . The resulting IgGgold complex was
centrifuged and resuspended in 20 mM Tris-HCl, pH 8.2,
containing 1% bovine serum albumin and 50% glycerol.
= 0.08) for 1 h. Immunolabeled sections were
then stained with uranyl acetate and lead citrate and examined under a
Hitachi HU12 electron microscope at 100 kV. Control experiments were
done using non-immunized rabbit IgG instead of anti-hMTH1 antibody.
MonoQ Anion-exchange Chromatography
The cytosolic
fraction (2 mg) and the mitochondrial soluble fraction (0.85 mg) were
analyzed by MonoQ anion-exchange chromatography, as
described(13) . The activity of 8-oxo-dGTP hydrolysis in each
fraction was assayed.
Plasmids
A mammalian expression vector pcDEB
which carries the SR
promoter and the hph gene for
selection with hygromycin B has been described(27) . An entire
cDNA for human MTH1 protein (13) was inserted downstream the
SR
promoter to generate pcDEB
-MTH1. A coding region (NarI-BamHI fragment of the cDNA) corresponding to
amino acid residues 3-156 of MTH1 protein was fused to the TrpE
coding region at the PstI-BamHI site of pYN3103-TrpE
vector(25) . The NcoI-BamHI fragment of the
plasmid, yielding TrpE-MTH1 fusion protein, was subcloned into NcoI- and BamHI-digested T7 promoter expression
vector pET8c(26) , resulting in pET:TrpE-MTH1.
Culture of HeLa MR Cells
HeLa MR cells (32) were maintained in Dulbecco's modified Eagle's
medium, supplemented with 100 µg/ml of streptomycin, 100 units/ml
of penicillin, and 5% each of fetal bovine serum and horse serum. Cells
carrying pcDEB or pcDEB
-MTH1 were maintained in medium
containing 150 µg/ml of hygromycin B.
DNA Transfection
HeLa MR cells were transfected
with pcDEB or pcDEB
-MTH1, using the procedure of Chen and
Okayama (33). Transformants were selected in medium containing 300
µg/ml of hygromycin B. Established transformant with pcDEB
was
designated MRV11, and three transformants with pcDEB
-MTH1 were
designated MR11, MR51, and MR81.
Other Methods
The activity of lactate
dehydrogenase was measured by the method of Bergmeyer et
al.(34) . One unit of the activity was defined as the
absorbance change of 0.001/min. The activities of succinate-cytochrome c reductase(35) , adenylate kinase(35) , and
fumarase (36) were measured as described. One unit of the
activities for adenylate kinase and fumarase was defined as the
absorbance change of 0.01/min. Protein concentration was determined
using a Bio-Rad DC protein assay kit with bovine serum albumin as a
standard, according to the manufacturer's instruction.
Intracellular Distribution of
8-Oxo-dGTPase
Jurkat cells were separated into cytosolic,
mitochondrial, microsomal, and nuclear fractions characterized by the
existence of specific marker enzymes; most lactate dehydrogenase was
present in the cytosolic fraction while almost all of
succinate-cytochrome c reductase was in the mitochondria (). The specific activity of 8-oxo-dGTPase in the
mitochondrial fraction was about 17% of that in the cytosolic fraction.
Since the specific activity of lactate dehydrogenase, a marker enzyme
of cytosol, in the mitochondrial fraction was less than 1% of that in
the cytosolic fraction, it is unlikely that the presence of
8-oxo-dGTPase in the mitochondrial fraction was due to contamination
with the cytosolic fraction. On the other hand, 8-oxo-dGTPase
activities in the microsomal and nuclear fractions may be explained by
contamination with the cytosol, since the values were essentially the
same as those of lactate dehydrogenase in the microsomal and nuclear
fractions ().
Immunological Detection of MTH1 Protein
To
determine whether the activity of 8-oxo-dGTPase measured with
subcellular fractions actually represents the amount of MTH1 protein
itself, we prepared an antibody against TrpE-MTH1 fusion protein and
used them for immunological analyses. When the total homogenate was
immunoblotted with the anti-hMTH1 (Fig. 1A), the
antibody reacted with a single protein with a molecular mass of 18 kDa,
a mass corresponding to the molecular mass deduced from the MTH1 cDNA.
When non-immune IgG was used, signals were hardly visible.
Localization of 8-Oxo-dGTPase in the Mitochondrial
Matrix
The sublocalization of 8-oxo-dGTPase (MTH1 protein) in
the mitochondria was next examined. When mitochondria were
subfractionated into membrane and soluble fractions, more than 80% of
the activity was recovered in the soluble fraction. The specific
activity of 8-oxo-dGTPase in the mitochondrial soluble fraction was
five times higher than that of the intact mitochondrial fraction, the
level being as high as that of the cytosolic fraction (Tables I and
III). This value was higher than a factor of enrichment of fumarase, a
marker enzyme for the mitochondrial matrix. This would imply that
8-oxo-dGTPase is present in soluble form in the mitochondria while
fumarase may have a weak association with mitochondrial membranes.
Figure 2:
Electron microscopic immunocytochemical
localization of MTH1 in mitochondria. Isolated mitochondria from Jurkat
cells were stained with 20 µg/ml of the anti-hMTH1. The length of a
bar indicates 0.1 µm.
Chromatographic Resolution of 8-Oxo-dGTPase
Protein
We analyzed 8-oxo-dGTPase activities in cytosolic and
mitochondrial fractions by anion-exchange chromatography, using a MonoQ
column, the objective being to further confirm that the MTH1 protein is
actually responsible for activity seen in those fractions. The
8-oxo-dGTPase activity eluted as a single peak and essentially the same
elution patterns were obtained with the enzyme from cytosolic and
mitochondrial fractions (Fig. 3). The activity of 8-oxo-dGTPase
co-eluted with proteins reactive to the anti-hMTH1 both in the
cytosolic and mitochondrial soluble fractions, thereby indicating that
8-oxo-dGTPase present in the cytosolic and mitochondrial fractions is
the same or similar molecular species of protein, coded by the MTH1 gene.
Overexpression of MTH1 Protein in HeLa Cells
It
was of interest to determine whether the MTH1 protein would locate in
cytosolic and mitochondrial fractions when the protein in a cell is
overproduced. We transfected MTH1 cDNA into HeLa cells and selected
several transfectants overproducing the MTH1 protein. We established
three clones of MR11, MR51, and MR81 which produced about 10-300
times larger amounts of MTH1 protein ( Fig. 4and ).
In MRV11, a clone transfected with the vector alone, the signal for the
immunoreactive protein represents the level of endogenous MTH1 protein.
When the mitochondrial fraction was prepared from each of these cells,
the activity of lactate dehydrogenase in these materials contained less
than 1% of the activity found in the cytosolic fraction (), thereby indicating that these mitochondrial fractions
were not contaminated with cytosolic materials. The amount of MTH1
protein in the mitochondrial fraction of various strains varied
considerably. Compared to values in the cytosolic fraction, the range
was from 4 to 10% ( Fig. 4and ). Thus, the relative
distribution of 8-oxo-dGTPase was much the same even with
overproduction of the protein. Such being the case, the sequence
encoded by the MTH1 cDNA probably has the full information leading to
localization of the protein in mitochondria as well as in cytoplasm.
Figure 4:
Immunoblotting analyses of MTH1
overexpressed in HeLa cells. HeLa cells were transfected with
pcDEB-MTH1 to overproduce MTH1 protein. The cytosolic and
mitochondrial fractions were prepared from HeLa cells and analyzed as
for Fig. 2. Applied proteins/lane were 1.25 and 10 µg for the
cytosolic and mitochondrial fractions, respectively. C,
cytosol; M, mitochondria.
Light Microscopic
Immunocytochemistry
Intracellular distribution of MTH1 protein
was further examined by light microscopic immunocytochemistry.
Immunostaining signals were rare in case of normal cells, thus we
analyzed HeLa MR51 cells which overproduce MTH1 protein. When the cells
were stained with the anti-hMTH1 antibody, there was a typical
cytoplasmic staining and a slightly weaker nuclear staining (Fig. 5B). A similar staining was observed when
anti-peptide antibody for MTH1, anti-M78, was used (Fig. 5D). Such a signal was hardly visible in HeLa
MRV11 cells which carry the vector alone (Fig. 5, A and C).
Figure 5:
Light microscopic immunocytochemistry of
HeLa cells. HeLa MRV11 (A and C) and MR51 (B and D) cells were immunostained with 0.33 µg/ml of
the anti-hMTH1 (A and B) and the anti-M78 (C and D).
mutant cells suppresses almost completely
the occurrence of specific A:T to C:G mutation(14) , the human
8-oxo-dGTPase probably has the same antimutagenic capacity as the MutT
protein. The human gene was named MTH1 (for mutT homologue 1) and locates on chromosome 7p22(14) .
, the
replicative polymerase for mitochondrial DNA, readily misincorporates
8-oxo-dGMP opposite adenine(39) . Thus, the 8-oxo-dGTPase in the
mitochondrial matrix may play an important role in maintaining genetic
integrity of the mitochondria DNA. In mitochondria, naked DNA is
surrounded by membranes containing proteins for the respiratory chain
which catalyze lipid peroxidation reaction yielding active
oxygens(18, 19, 21) . Such a situation is close
to that of bacterial DNA, in which mutT deficiency causes a
high frequency of A:T to C:G transversion(9, 10) .
Table: Enzyme activities in subcellular fractions of
Jurkat cells
3). Values without S.D. are
means of two independent experiments.
Table: Distribution of 8-oxo-dGTPase in cytosol
and mitochondria
Table: Enzyme activities in submitochondrial
fractions
Table: Mitochondrial localization of MTH1
overexpressed in HeLa cells
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.