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,,,
,1
* Department of Veterinary and Biomedical Sciences,
Department of Biochemistry, and
Center for Redox Biology, University of Nebraska-Lincoln, Lincoln, Nebraska, USA; and
Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska, USA
1Correspondence: Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA. E-mail: mlou1{at}unl.edu
ABSTRACT
Thioltransferase (or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. Mitochondrial Grx2 is a recent discovery, but its function is largely unknown. In this study we investigate Grx2 function by examining its potential peroxidase activity using lens epithelial cells (LEC). cDNA for human and mouse Grx2 was cloned into pET21d(+) vector and used to produce respective recombinant Grx2 for kinetic studies. cDNA for human Grx2 was transfected into human LEC and used for in vivo studies. Both human and mouse Grx2 showed glutathione (GSH)-dependent and thioredoxin reductase (TR)-dependent peroxidase activity. The catalytic efficiency of human and mouse Grx2 was lower than that of glutathione peroxidases (2.5 and 0.8x104 s–1M–1, respectively), but comparable with TR-dependent peroxiredoxins (16.5 and 2.7x104 s–1M–1, respectively). TR-dependent peroxidase activity increased 2-fold in the transfected cells and was completely abolished by addition of anti-Grx2 antibody (Ab). Flow cytometry (FACS) analysis and confocal microscopy revealed that cells preloaded with pure Grx2 detoxified peroxides more efficiently. Grx2 over-expression protected cells against H2O2-mediated disruption of mitochondrial transmembrane potential. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play a role in protecting the mitochondria from oxidative damage.—Fernando, M. R., Lechner, J. M., Löfgren, S., Gladyshev, V. N., Lou, M. F. Mitochondrial thioltransferase (glutaredoxin 2) has GSH-dependent and thioredoxin reductase-dependent peroxidase activities in vitro and in lens epithelial cells.
Key Words: peroxiredoxins • oxidative stress • antioxidant enzymes • peroxide removal
MITOCHONDRIA IN MAMMALIAN CELLS are the main site of aerobic energy metabolism. Most of the oxygen utilized by the cell is consumed by the oxidative phosphorylation that operates within the mitochondria. Less than 1% of the total oxygen is converted to reactive oxygen species (ROS), including superoxide anions (1
2
3)
. Superoxide anions thus generated are converted to hydrogen peroxide (H2O2) by the mitochondrial enzyme manganese superoxide dismutase (SOD) (MnSOD). Even though H2O2 is a relatively stable oxidant, it can be converted to the highly reactive hydroxyl radical by a metal ion via the Fenton reaction (3)
. Hydrogen peroxide and free radicals generated in this way within the mitochondria may damage the reactive moieties of biologically important macromolecules such as proteins, lipids, and DNA (4
, 5)
. This damage may eventually challenge the integrity of the organelle. ROS have been implicated in mediating mitochondrial permeability transition, which is an initial event in the process of cell death induced by Ca2+ and inorganic phosphate. This mitochondrial permeability transition can be prevented by antioxidants (6)
. Mitochondria have several oxidation defense systems, including enzymes such as glutathione peroxidase (Gpx), MnSOD, thioredoxin-2 (Trx2), and thioredoxin reductase (TR3). The mitochondria is also defended by antioxidants such as glutathione (GSH), vitamin E, and ascorbate.
Mammalian mitochondrial thioltransferase (glutaredoxin 2 or Grx2) is a recently (7
, 8)
identified 18 kDa protein belonging to the thiol/disulfide exchange oxidoreductase family. This new protein is 34% identical to the previously known cytosolic thioltransferase (also known as Grx1) and exhibits GSH-dependent hydroxyethyl disulfide reducing activity in vitro (7
, 8)
. Grx2 can reduce S-glutathionylated proteins with high affinity, accepting electrons from either GSH or thioredoxin reductase (9)
. It has also been shown that silencing of Grx2 by RNA-mediated short interfering method increases the sensitivity of HeLa cells toward the anticancer drugs doxorubicin and phenylarsine oxide (10)
. Recent findings show that Grx2 plays a role in attenuating apoptosis by preventing cytochrome c release in Grx2 overexpressed HeLa cells (11)
. Human Grx2 has been characterized as an iron-sulfur protein, which may play an important role as redox sensor in mitochondria (12)
. Using mouse and human recombinant Grx2 and overexpressed human Grx2 in mammalian cells, the current study provides evidence that mammalian Grx2 possesses a novel GSH- and TR-dependent peroxidase activity.
MATERIALS AND METHODS
Thioredoxin reductase was purchased from IMCO (IMCO Corp. Ltd, Sweden). 2',7'-dichlorodihydrofluorecin diacetate (DCFH-DA), and the oxidation-insensitive analog of DCFH-DA (carboxy-DCFH-DA) were from Molecular Probes Inc. (Eugene, OR, USA). BioPORTER protein transfection reagent was from Therapy Systems, Inc. (San Diego, CA, USA). Antiglutathione peroxidase monoclonal antibody (mAb) raised against human glutathione peroxidase was purchased from Stressgen (Victoria, BC, Canada). Antiperoxiredoxin 3 Ab raised against human peroxiredoxin 3 was from Upstate (Lake Placid, NY, USA). Anticytochrome c mouse Ab was purchased from BioVision (BioVision, Mountain View, CA, USA). All other chemicals and reagents were standard commercial products of analytical grade.
Cell culture
A human lens epithelial cell line, (HLE-B3), immortalized by infecting with adenovirus 12-SV40, was generously provided by Dr. Usha Andley (Washington University, St. Louis, MO, USA). The cells were grown in MEM medium supplemented with 20% FBS and 50 µg/ml gentamicin in 100 x 20 mm culture dishes in humid atmosphere with 5% CO2 at 37°C. Cells reached confluence with 6 x 106 cells per dish in 4 d.
Purification of recombinant human and mouse Grx2
Human and mouse cDNA for Grx2 was cloned into pET21d(+) vector, expressed in E. coli, and purified using His-bind column as described earlier (7)
.
Peroxidase activity
Both GSH-dependent and TR-dependent peroxidase activities were measured spectrophotometrically (13)
. GSH-dependent peroxidase activity was measured using a reaction mixture containing 50 mM Tris-HCl (pH 8.0), 2 mM NaN3, 0.1 mM EDTA, 0.25 U/ml glutathione reductase, 0.2 mM NADPH, and 50 µM GSH. The reaction was started by addition of 250 µM H2O2, 250 µM tert-butyl hydroperoxide, or 250 µM cumine hydroperoxide, and the decrease of absorbance at 340 nm was monitored (13)
. TR-dependent peroxidase activity was measured in a mixture containing 50 mM HEPES-NaOH (pH 7.3), 5 µM thioredoxin reductase, and 0.2 mM NADPH. The reaction was initiated by the addition of H2O2, tert-butyl hydroperoxide, or cumine hydroperoxide at a final concentration of 0.25 mM, and the decrease in absorbance at 340 nm was monitored (13)
.
Ab against mouse Grx2
Recombinant mouse Grx2 was purified, emulsified with adjuvant, and used subcutaneously (s.c.) to immunize a rabbit. The first booster injection was given 4 wk later, followed by three more booster injections. Antiserum was collected 2 wk after the last booster injection and the IgG fraction was isolated using a protein A Sepharose column (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
Isolation of mitochondria
HLE-B3 cells (2.8x107) were trypsinized and centrifuged. The resultant cell pellet was suspended in 0.25 mM sucrose in 10 mM Tris/HCl (pH 7.4) and homogenized using a glass homogenizer. All steps were performed at 4°C or on ice. The mitochondrial fraction was isolated by the differential centrifugation method described by Lai and Clark (14)
. To verify the purity and intactness of the mitochondrial preparations, specific marker constituents were determined. These markers are compartment-specific, existing in elevated or exclusive amounts in separate cellular locations. Lactate dehydrogenase (cytosolic marker) and succinate cytochrome c reductase (mitochondrial marker) activities were determined as described previously (15
, 16)
. Protein was assayed using bicinchoninic acid (BCA) colorimetry (bicinchoninic acid Protein Assay kit, Pierce, Rockford, IL, USA).
Western blot analysis of mitochondrial lysate
The mitochondrial pellet was lysed using M-PER lysis buffer (Pierce). Proteins were separated by 15% SDS-PAGE and electrotransferred onto Immobilon P membranes (Millipore, Billerica, MA, USA). These membranes were probed with rabbit anti-Grx2 Ab diluted in TBST buffer (10 mM Tris-HCl, pH 7.5; 100 mM NaCl; and 0.1% Tween 20) and then treated with goat anti-rabbit IgG horseradish peroxidase (Santa Cruz Biotechnology). Immunodetection was performed with chemiluminescent reagents (SuperSignal, Pierce). The immunoblot was then analyzed with an imaging system (Fluor-S MAX MultImager, Bio-Rad, Richmond, CA, USA). To use cytochrome c as a loading control, we reprobed the blots with anticytochrome C Ab and rabbit anti-mouse IgG conjugated with horseradish peroxidase (Santa Cruz Biotechnology) and analyzed with chemiluminescent reagents. Before reprobing, blots were washed with a stripping buffer containing 0.63 M Tris-Hcl, pH 6.8; 10% SDS; and 14.3 M 2-mercaptoethanol for 30 min.
Overexpression of Grx2 in HLE-B3 cells
Sense cDNA for human Grx2 was introduced into the multicloning site of geneticin (G418 sulfate)-resistant mammalian expression vector pcDNA3.1(+) to construct sense plasmids. The plasmids were then transfected into HLE-B3 cells using Lipofectamine Plus reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s protocol. Cells were incubated with transfection medium for 1 d and then passaged into new dishes with fresh culture medium. The cells were grown for two days and then changed to fresh medium containing 1 mg/ml geneticin for selection. Thereafter, the cells were fed every four days. After 4 wk of selection, the cells were passaged into new dishes containing fresh medium with 400 µg/ml geneticin and grown to confluency before use. Transfection efficiency for HLE-B3 cells was very low, 
10% as determined by counting the number of cells before transfection and after geneticin selection.
Flow cytometric quantification of DCF fluorescence in H2O2-treated control, ß-galactosidase-loaded, Grx2-loaded, and catalase-loaded HLE-B3 cells
The fluorescent dye DCFH-DA crosses the cell membrane and undergoes deacetylation by intracellular esterases producing the nonfluorescent compound DCFH, which is trapped inside the cells. Oxidation of DCFH by ROS produces the highly fluorescent DCF. Externally added H2O2 easily diffuses into the cells and oxidizes DCFH to give DCF fluorescence. Hence, DCF fluorescence intensity inside cells is proportionate to the intracellular H2O2 level. H2O2 levels in cells can then be quantified by flow cytometric determination of the cellular DCF fluorescence (17)
. Recombinant human Grx2 (mature protein without mitochondrial signal), ß-galactosidase, and catalase were loaded into cells using a protein transfection reagent following the manufacturer’s protocol (BioPORTER, Gene Therapy Systems, Inc, San Diego, CA, USA). BioPORTER protein transfection reagent is a lipid-mediated protein delivery system that delivers protein in a functionally active form into cytoplasm of cells. The delivery efficiency of BioPORTER was determined to be 78% by using flow cytometry and a FITC-tagged Ab provided with the kit. Control cells, ß-galactosidase-loaded cells, Grx2-loaded cells, and catalase-loaded cells were removed by trypsination, washed with Ca2+ and Mg2+-free PBS (PBS, Sigma-Aldrich), and then resuspended in PBS containing 50 µM DCFH-DA. After 5 min the cells were again washed with PBS before the basal DCF fluorescence level was determined by flow cytometry (FACScan flow cytometer; Becton Dickinson, Franklin Lakes, NJ, USA). After baseline was acquired, 50 µM H2O2 was added to all cell groups and DCF fluorescence levels were determined every 1 min. Excitation was set at 488 nm and the green emission was measured at 525 nm in 10,000 gated cells using linear amplification. The arithmetic mean fluorescence channel was derived by CellQuest® software (Becton Dickinson).
Confocal microscopic analysis of H2O2 reduction in normal and protein-loaded cells
HLE-B3 cells were grown to 60–70% confluency in 60 x 15 mm dishes. GSH was depleted by 24 h treatment with 100 µM of the GSH biosynthesis inhibitor L-buthionine-[S,R]-sulfoximine (BSO). GSH-depleted and nondepleted cells were loaded with human Grx2, ß-galactosidase, or catalase using the BioPORTER protein transfection reagent described above. Catalase was used as a positive control and ß-galactosidase as a negative control. The control and protein-loaded cells were treated with 50 µM DCFH-DA fluorescence dye. After 5 min dye incubation, the cells were washed and subjected to confocal analysis as described previously (18)
using Bio-Rad MRC1024ES confocal laser scanning microscope. After acquisition of the baseline fluorescence, the cells were treated with 50 µM H2O2 for 5 min, washed with serum-free medium, and again analyzed by confocal microscopy.
Inhibition of peroxidase activity in recombinant Grx2 and HLE-B3 mitochondrial lysates by anti-Grx2 Ab
Grx2 was incubated with either nonspecific human IgG or anti-Grx2 IgG then TR-dependent and GSH-dependent peroxidase activities were determined as described above. For the TR-dependent peroxidase activity inhibition 100 µg of human recombinant Grx2 was mixed with either 40 µg of nonspecific human IgG or 40 µg of anti-Grx2 IgG and incubated for 2 h at 4°C, with gentle head-to-tail shaking before peroxidase activity measurement. The method for the GSH-dependent peroxidase activity inhibition by anti-Grx2 IgG was similar to TR-dependent activity inhibition except that 400 µg of Grx2 and 80 µg of either nonspecific IgG or anti-Grx2 IgG were used. The effect of anti-Grx2 Ab on TR-dependent peroxidase activity in Grx2 overexpressed HLE-B3 mitochondrial lysate was also investigated. The mitochondrial pellet was lysed in a buffer containing 50 mM Tris/HCl, pH 7.5; 150 mM NaCl; 5 mM EDTA; 0.5% Igepal (Sigma-Aldrich), and protease inhibitor cocktail set III (Calbiochem, La Jolla, CA, USA) and then incubated on ice for 15 min. Equal lysate volumes were mixed with either anti-Grx2 IgG (10 µg/100 µg lysate protein) or human nonspecific IgG (10 µg/100 µg lysate protein). The TR-dependent peroxidase activity was determined after 2 h incubation at 4°C with gentle head-to-tail shaking.
Effect of H2O2 on mitochondrial transmembrane potential (MTP) of control and Grx2 over-expressed HLE-B3 cells
Control and Grx2 over-expressing HLE-B3 cells were cultured in MEM with 20% FBS in 60 mm dishes. When cells were 60–70% confluent, the serum containing medium was removed and cells were washed two times with PBS. Cells were then incubated either with serum-free MEM alone or with serum-free MEM containing 0.3 mM H2O2 for 1 h. After this incubation period, the media was replaced with fresh MEM with 20% FBS and cells were incubated for another 16 h before making mitochondrial transmembrane potential measurements. Mitochondrial transmembrane potential of cells was determined by using JC-1 dye also known as MitoCapture (BioVision), which is a cationic dye that fluoresces differently in cells with intact mitochondria than in cells with altered mitochondrial transmembrane potential. In cells with intact mitochondria, this dye accumulates and aggregates inside the mitochondria, giving a red fluorescence. Whereas in cells with damaged mitochondria, or altered mitochondrial transmembrane potential, the dye cannot aggregate and remains in the cytoplasm in its monomeric form, giving a green fluorescence.
Both control and Grx2 over-expressing cells were washed thoroughly with PBS after exposure to H2O2 and then incubated with prewarmed MitoCapture solution for 15 min at 37°C, followed by immediate observation under an Olympus FV500 confocal laser scanning microscopic system on an inverted fluorescence microscope. Optical images were collected under same magnification for both control and Grx2 over-expressed cells. Confocal settings used were 488 nm laser line excitation and simultaneous dual channel display mode (522/595 nm emission). Mitochondrial transmembrane potential alteration in H2O2-treated cells were quantified and compared with control cells by measuring green fluorescence level using flow cytometry (FACScan flow cytometer; Becton Dickinson). Excitation was set at 488 nm, and the green emission was measured at 525 nm in 10,000 gated cells using linear amplification. The arithmetic mean fluorescence channel was derived by CellQuest® software (Becton Dickinson).
Statistics
All tests were two-sided, and P < 0.05 was considered significant; 1-way ANOVA and 2-way ANOVA with replication were used to analyze the peroxidase data. Subsequent to significant ANOVAs, pair-wise comparisons between the control group and treatment groups were performed by using Dunnett’s modified t test. The groups’ mean levels of ROS production in the FACS assay were analyzed by 1-way ANOVA, and subsequent Dunnett’s testing. Statistical analysis was done by a nonparametric test called Mann-Whitney U test.
RESULTS AND DISCUSSION
Peroxidase activity of mammalian Grx2
Yeast glutaredoxins Grx 1 and Grx 2 can catalytically reduce hydroperoxides in the presence of GSH and GR (19)
. The cytosolic thioltransferase (Grx1) can enhance the GSH-dependent peroxidase activity of plant type II peroxiredoxin (13)
. To investigate whether the mammalian Grx2 also possesses hydroperoxide-reducing activity and the ability to enhance the activity of peroxidases, we purified human and mouse recombinant Grx2 (Fig. 1
A, SDS-PAGE). The purified mouse and human recombinant Grx2 showed considerable peroxidase activity in the presence of GSH, glutathione reductase, and NADPH (Table 1
; Fig. 1B
). Omitting Grx2 from the assay mixture resulted in a significant (P<0.01) 70% drop in the peroxidase activity, indicating that GSH/GR accounts for the remaining 30% activity (Fig. 1C
). Neutralization of Grx2 by specific anti-Grx2 IgG also resulted in a similar significant (P<0.01) decrease in peroxidase activity (Fig. 1C
). Further, the addition of nonspecific human IgG had no effect on the peroxidase activity of Grx2 (Fig. 1C
). This indicates that Grx2 solely contributes to the peroxidase activity and that this activity is not due to contamination of other peroxidase enzymes in the Grx2 preparations. Since Johansson et al. (9)
reported that Grx2 was a substrate for mammalian TR, we also studied the TR-dependent peroxidase activity of Grx2, using H2O2 as the substrate. Our results show that both human and mouse Grx2 had TR-dependent peroxidase activity in the presence of TR and NADPH (Table 1
, Fig. 1D
). When Grx2 was removed from the reaction mixture by anti-Grx2 IgG, the Grx2-contributed peroxidase activity was completely abolished, with a significant (P<0.01) 80% decrease of the total activity (Fig. 1E
). Nonspecific human IgG had no effect on the peroxidase activity of Grx2 (Fig. 1E
), indicating that the TR-dependent peroxidase activity was due to Grx2. The TR-dependent peroxidase activity of human recombinant Grx2 was 6.6-fold greater than that of GSH-dependent peroxidase activity of Grx2 (Table 1)
. Mouse recombinant enzyme also had TR- and GSH-dependent peroxidase activities; however, the peroxidase activity was far less than that of the human recombinant enzyme (Table 1)
. To study whether this observed low-peroxidase activity of mouse Grx2 was due to the inactivation of the mouse enzyme during the preparation, we did a kinetic analysis of the dethiolation and dehydroascorbate reductase activities of Grx2. This study reveled that these activities were comparable in both human and mouse Grx2 (data not shown), indicating that the observed low-peroxidase activity in mouse Grx2 was not due to the inactivation of the mouse enzyme during preparation. It is unclear at this juncture whether the low peroxidase activity of mouse Grx2 is due to some inherent property of the enzyme or due to some modification during preparation.
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Reduction of H2O2, tert-butyl hydroperoxide and cumine hydroperoxide by Grx2
Since Grx2 could reduce H2O2 using either GSH or TR, we studied the ability of human recombinant Grx2 to reduce tert-butyl hydroperoxide and cumine hydroperoxide in vitro. Figure 2
shows the comparison of GSH-dependent reduction of H2O2, tert-butyl hydroperoxide, and cumine hydroperoxide by human Grx2 in vitro. The human enzyme could reduce both hydroperoxides in a concentration-dependent manner (Fig. 2)
. The mean peroxidase activities in the cumine hydroperoxide and the tert-butyl hydroperoxide groups were both significantly (P<0.01) higher than in the H2O2 control group.
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Effect of Grx2 overexpression on the peroxidase activity in HLE-B3 mitochondrial fraction
The purity and integrity of the cytosolic and mitochondrial fractions were determined by the estimation of succinate cytochrome c reductase and lactate dehydrogenase (LDH) activities. In normal control HLE-B3 cells and Grx2 overexpressed cells, the mitochondrial fraction contained 87 and 96% of the total succinate cytochrome c reductase activity, respectively. The mitochondrial fraction in control HLE B3 cells was contaminated with 3% of the total LDH activity, while in the Grx2 overexpressed cells the contamination was 2%. Therefore, these data show that our mitochondrial preparation had a low degree of cytosolic contamination.
The overexpression of Grx2 was successful as shown by Grx2 Western blotting using mitochondrial lysate (Fig. 3
A). The TR-dependent peroxidase activity was significantly (P<0.01) higher in mitochondria lysate from Grx2 overexpressed cells than in control cells (Fig. 3B
). Incubation with anti-Grx2 IgG resulted in a significant (P<0.01) reduction in the peroxidase activity, indicating that the increased peroxidase activity in Grx2 overexpressed cells is due to the increased expression of Grx2 (Fig. 3B
). Manipulation of certain genes may lead to induction of other genes. Therefore, we investigated glutathione peroxidase (Gpx) and peroxiredoxin-3 protein expression in control and Grx2 overexpressed cells, because these two enzymes are well known for their peroxidase activity in mitochondria. The Western blots revealed very little difference in expression of the two enzymes in control cells vs. Grx2 overexpressed cells (Fig. 3C, D
), indicating that Grx2 overexpression did not enhance gene expression of either glutathione peroxidase or peroxiredoxin-3. Thus the increased peroxidase activity in mitochondrial lysate from Grx2 overexpressed cells is directly associated with the increased Grx2 expression.
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H2O2 decomposing ability in cells loaded with Grx2 and catalase
The fluorescence of the ROS marker DCF in H2O2-treated HLE-B3 cells was measured with and without preloading of Grx2 or catalase. The FACS-determined baseline DCF fluorescence in all groups indicated a low level of endogenous ROS before the addition of H2O2. The DCF fluorescence gradually increased in all three groups after addition of H2O2 (Fig. 4
). The mean DCF fluorescence in the control group was significantly higher than both the catalase (P<0.01) and the Grx2 (P<0.01) groups. Since catalase is a H2O2 detoxifying enzyme, we attribute the effect of Grx2 loading on decreased ROS levels to its peroxidase activity. Figure 5
shows the confocal microscopic analysis of DCF fluorescence in cells loaded with ß-galactosidase, Grx2, and catalase. Control cells were analyzed before as well as after H2O2 treatment. To ensure that the increased DCF fluorescence was related to oxidation of DCFH by H2O2 and not increased uptake, ester cleavage, or efflux, the oxidation-insensitive analog of DCFH-DA (carboxy-DCFH-DA) was used in control experiments. No difference was found in DCF fluorescence intensity among the four groups (Fig. 5
, 1st row), indicating that when changes were seen using the oxidation-sensitive DCFH-DA they were truly representative of altered DCFH oxidation. Using oxidation-sensitive DCFH-DA dye, the DCF fluorescence intensity was higher in control and ß-galactosidase-loaded cells as compared to Grx2 or catalase-loaded cells (Fig. 5
, 2nd row), indicating that cells loaded with either Grx2 or catalase are removing endogenous ROS more efficiently than control cells and cells with ß-galactosidase. Exposing the DCFH-DA pretreated cells to H2O2 increased the DCF fluorescence (Fig. 5
, 3rd row) as compared to dye-loaded cells not exposed to H2O2 (Fig. 5
, 2rd row). The DCF fluorescence was higher in H2O2-exposed control and ß-galactosidase-loaded cells as compared to the Grx2 or catalase-loaded cells (Fig. 5
, 3rd row), demonstrating that these cells remove exogenous H2O2 more efficiently as compared to the control cells. GSH depletion by preincubation with BSO resulted in increased DCF fluorescence (Fig. 5
, 4th vs. 2nd row), indicating that a depleted GSH pool hindered ROS removal. Again, in this group, the DCF fluorescence intensity was higher in control or ß-galactosidase-loaded cells as compared to Grx2 or catalase-loaded cells (Fig. 5
, 4th row).
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In another experiment, when GSH depleted cells were exposed to H2O2, the DCF fluorescence intensity was significantly higher in control and ß-galactosidase-loaded cells as compared to Grx2 or catalase-loaded cells (Fig. 5
, 5th row), demonstrating that added catalase or Grx2 also reduced H2O2 in cells despite the low GSH levels. Catalase does not depend on GSH for its H2O2 detoxifying activity, but Grx2 has both GSH- and TR-dependent H2O2 detoxifying activity. It is likely that the cells used the latter electron donor system under this condition. This explains the observed low DCF fluorescence in Grx2 or catalase-loaded cells. Even though catalase does not depend on GSH for its peroxidase activity, catalase-loaded and GSH-depleted HLE-B3 cells appear to show higher levels of DCFH fluorescence as compared to catalase-loaded control cells (Fig. 5
, raw 2 vs. raw 4). This may be due to the inability of glutathione peroxidases to remove H2O2 in the absence of required levels of GSH. We speculate that peroxide removal in cells is carried out by several systems both enzymatic and nonenzymatic. GSH also can remove peroxide nonenzymatically. Therefore, the low levels of GSH and low glutathione peroxidase activity may hinder the efficient removal of H2O2 in GSH-depleted and catalase-loaded cells as compared to catalase-loaded control cells (Fig. 5
, raw 2 vs. raw 4).
Effect of Grx2 over-expression on mitochondrial transmembrane potential (MTP) of HLE-B3 cells
Alteration in MTP is an early event of apoptotic process (20)
. Hydrogen peroxide can alter MTP by damaging mitochondrial membrane (21)
. We studied the effect of Grx2 over-expression on H2O2-induced mitochondrial membrane damage in control (vector only) and Grx2 over-expressed HLE-B3 cells using a cationic dye, MitoCapture (BioVision), as described in Materials and Methods. According toFig. 6
A, cells in untreated control group and Grx2 over-expressed group showed bright red fluorescence, indicating that MTP was not altered in those cells. Whereas green fluorescence had replaced red fluorescence in cells pretreated with H2O2 (Fig. 6A
), indicating that MTP disruption occurred. However, Grx2 over-expressed cells pretreated with H2O2 gave red fluorescence, and very little green fluorescence was observed in these cells, indicating that MTP was not disrupted as compared to H2O2-treated control cells. We have quantified the green fluorescence in untreated and treated control and Grx2 over-expressed cells using FACS analysis. Figure 6B
shows the mean green fluorescence intensity in H2O2-treated, untreated control, and the H2O2-treated Grx2 over-expressed cells. In H2O2-treated control cells, the intensity of green fluorescence increased nearly 4.5-fold over the untreated control cells, whereas only 2-fold increase was observed in H2O2-treated cells enriched with Grx2. The observed protective effect of Grx2 against H2O2-mediated MTP disruption may be due to its peroxidase activity.
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We have purified human and mouse recombinant Grx2 and have shown for the first time that Grx2 has both GSH-dependent and TR-dependent peroxidase activity in vitro. We manipulated Grx2 expression in HLE-B3 cells through Grx2 cDNA transfection and produced Grx2 overexpressed cells, which showed an increased peroxidase activity in the mitochondrial fraction. Manipulation of Grx2 expression had no effect on mitochondrial glutathione peroxidase or peroxiredoxin 3 expressions. Treating mitochondrial lysate from Grx2 overexpressed cells with anti-Grx2 IgG completely abolished the increase in peroxidase activity, indicating that the augmented peroxidase activity is a direct result of increased Grx2 expression. Loading HLE-B3 cells with Grx2 improved their ability to remove H2O2 as judged from DCF fluorescence in Grx2-loaded and unloaded HLE-B3 cells. Our in vitro experiments carried out using purified recombinant Grx2 showed that catalytic efficiency for TR-dependent peroxidase activity of Grx2 was similar to those seen for other peroxiredoxins (104–105 s–1M–1) (22)
. The catalytic efficiency for GSH-dependent peroxidase activity of Grx2 was considerably lower than those of Gpx (108 s–1M–1) (22)
.
It has been reported that human Grx2 is an iron-sulfur center-containing dimer and this dimer is enzymatically inactive (12)
. Monomerization of the Grx2 dimer is prevented by cellular GSH. However, oxidized glutathione and one-electron oxidants can activate the monomerization of Grx2, thereby releasing the active enzyme (12)
. Therefore, under high oxidative stress conditions, active monomeric Grx2 is formed inside the mitochondria.
Note that Grx2 can remove peroxides by accepting electrons either from GSH system or thioredoxin reductase. This dual electron accepting capability may be very important to cells, especially those under high oxidative stress conditions where cellular GSH level is low. Under such conditions Grx2 may be effective in peroxide removal as it has the ability to accept electrons from thioredoxin reductase, whose activity and expression are induced quickly under oxidative stress conditions (23)
. We have shown that Grx2 over-expression in HLE-B3 cells protected cells from H2O2 mediated alterations of mitochondrial transmembrane potential. This observation is in agreement with the findings of Enoksson et al. that overexpression of Grx2 was shown to inhibit oxidation-induced apoptosis in HeLa cells (11)
, and it is likely that Grx2 mediates this function through its peroxidase activity. Our findings suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may protect the mitochondria from oxidative damage.
ACKNOWLEDGMENTS
This work was presented in part at the annual meeting for the Association for Research in Vision and Ophthalmology at Fort Lauderdale, FL, May 2005. This research was supported through a grant from the National Institutes of Health, EY RO1–10590 (MFL). S. L. is a Swedish Research Council and a Swedish Society of Medicine postdoctoral fellowship awardee. We gratefully acknowledge the expertise and assistance given by Dr. Y. Zhou (University of Nebraska, Lincoln) in confocal microscopy and Dr. C. A. Kuszynski for FACS analysis (University of Nebraska Medical Center).
Received for publication February 14, 2006. Accepted for publication August 18, 2006.
REFERENCES
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), 0.25 mM cumine hydroperoxide (
), and 0.25 mM tert-butyl hydroperoxide (
), as substrates. Error bars indicate SD, n = 5. Asterisk indicates significant difference from the H2O2 control group (P<0.01), determined by Dunnett’s test after significant ANOVA.
