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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
SPECIFIC AIMS
Thioltransferase (glutaredoxin or Grx) belongs to the oxidoreductase family and is known to regulate redox homeostasis in cells. The mitochondrial thioltransferase (glutaredoxin 2 or Grx2) is a recent discovery, but its function is largely unknown. In this study we investigate the functions of Grx2 by examining its potential peroxidase activity using human lens epithelial cells (HLE-B3).
PRINCIPAL FINDINGS
1. Mammalian Grx2 has GSH-dependent and thioredoxin reductase-dependent peroxidase activity in vitro
Human and mouse cDNA for Grx2 were cloned into pET21d(+) vector, expressed in E. coli, and recombinant proteins purified using His-bind column. The purification of the human and mouse Grx2 was successful as judged by SDS-PAGE (Fig. 1
A). In vitro experiments showed that mouse and human recombinant Grx2 has significant peroxidase activity in the presence of glutathione (GSH), glutathione reductase, and NADPH (Fig. 1B
). Omitting Grx2 from the assay mixture resulted in a significant (P<0.01) 70% drop in the peroxidase activity, indicating that GSH/glutathione reductase accounts for the remaining 30% activity (Fig. 1C
). Neutralizing Grx2 by specific anti-Grx2 IgG also resulted in significant (P<0.01) and complete abolition of the peroxidase activity contributed by Grx2 (Fig. 1C
). Further addition of nonspecific human IgG had no effect on the peroxidase activity of Grx2 (Fig. 1C
). This indicates that Grx2 solely contributes such peroxidase activity and this activity is not due to potential contamination of other peroxidase enzymes in our Grx2 preparations. Since it has been reported that Grx2 is a substrate for mammalian thioredoxin reductase (TR), we also studied the TR-dependent peroxidase activity of Grx2, using H2O2 as the substrate. Our results show that human Grx2 had TR-dependent peroxidase activity in the presence of TR and NADPH (Fig. 1D
). When human Grx2 was removed from the reaction mixture by anti-Grx2 IgG, the Grx2-contributed peroxidase activity was completely abolished and the total activity was significantly (P<0.01) decreased by 
80% (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. The mouse recombinant enzyme also had showed TR- and GSH-dependent peroxidase activities; however, the peroxidase activity was far less than that of the human recombinant enzyme.
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2. Grx2 overexpression in HLE-B3 cells increases the peroxidase activity of HLE-B3 mitochondrial fraction
Human Grx2 cDNA was cloned into pcDNA3.1(+) vector and transfected into HLE-B3 cells using lipofectamine and stably transfected cells were selected using geneticin treatment. The Western blot analysis of mitochondrial lysates showed an increase in Grx2 level in Grx2 overexpressed cells. 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 of the control HLE B3 cells was contaminated with 3% of the total LDH activity, while the Grx2 overexpressed mitochondrial fraction showed a 2% contamination. These low levels of LDH show that our mitochondrial preparation had a low degree of cytosolic contamination. The TR-dependent peroxidase activity was significantly (P<0.01) higher in mitochondrial lysate from Grx2 overexpressed cells than in control cells. 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 was due to the increased expression of Grx2. Manipulation of certain genes may lead to induction of other genes. Therefore, we investigated expression of glutathione peroxidase (Gpx) and peroxiredoxin 3 (Tpx) in control and Grx2 overexpressed cells because these two enzymes are well-known for their peroxidase activity in mitochondria. The densitometric analysis of Western blots revealed negligible difference in the expression of Gpx and Tpx in control cells vs. Grx2 overexpressed cells, indicating that Grx2 overexpression did not enhance gene expression of these two enzymes. Thus the increased peroxidase activity in mitochondrial lysate from Grx2 overexpressed cells is directly associated with the increased Grx2 expression.
3. 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. Human Grx2 could reduce both hydroperoxides in a concentration-dependent manner. The mean peroxidase activities in the cumine hydroperoxide and tert-butyl hydroperoxide groups were both significantly (P<0.01) higher than in the H2O2 group.
4. Loading HLE-B3 cells with either Grx2 or catalase increases the H2O2 decomposing ability of cells
We loaded HLE-B3 cells either with catalase or purified recombinant Grx2 using a protein transfection reagent (BioPORTER, Gene Therapy Systems, Inc, San Diego, CA, USA). The fluorescence of the reactive oxygen species (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. The mean DCF fluorescence in the control group was significantly (P<0.01) higher than both the catalase and the Grx2 groups. Since catalase is a H2O2 detoxifying enzyme, we attribute the effect of Grx2 loading on decreased ROS levels to its peroxidase activity.
5. Confocal microscopic analysis of DCF fluorescence in either ßbeta;-galactosidase (negative control), Grx2, or catalase-loaded HLE-B3 cells before and after H2O2 treatment
HLE-B3 cells were loaded either with ßbeta;-galactosidase (negative control), Grx2, or catalase (as a positive control). The loaded cells and unloaded control cells were treated with DCFH-DA dye for 5 min. After removal of excess dye, the DCF fluorescence was measured by confocal microscopy before and after 5 min of 50 µM H2O2 treatment. Without H2O2 treatment, the DCF fluorescence intensity was higher in control and ßbeta;-galactosidase-loaded cells than the Grx2 or catalase-loaded cells, indicating that with excess Grx2 or catalase, the cells are removing endogenous ROS more efficiently. H2O2 addition increased the intensity of DCF fluorescence, which was higher in control (no loading) and ßbeta;-galactosidase-loaded cells in comparison to cells preloaded with either Grx2 or catalase. This indicates that cells enriched with Grx2 or catalase can more efficiently detoxify exogenous and endogenous H2O2.
In another experiment, cells were first depleted of GSH by L-buthionine-[S,R]-sulfoximine (BSO) treatment before repetition of the above experiment. The GSH depletion resulted in increased intensity of DCF fluorescence, indicating that a depleted GSH pool hinders ROS removal. Again, in this group, the control or ßbeta;-galactosidase loaded cells had higher intensity of DCF fluorescence than that of Grx2 or catalase loaded cells. When GSH-depleted cells were exposed to H2O2, the DCF fluorescence intensity was significantly higher in control and ßbeta;-galactosidase-loaded cells as compared to Grx2 or catalase loaded cells, demonstrating that added catalase or Grx2 can still lower H2O2 levels in cells despite the low GSH level. Catalase does not depend on GSH for its H2O2 detoxifying activity, but Grx2 has both GSH- and TR-dependent H2O2 detoxifying activities. It is likely that the cells use the latter electron donor system under this condition. This explains the observed low DCF fluorescence in Grx2 or catalase-loaded cells.
CONCLUSIONS AND SIGNIFICANCE
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 the mitochondrial lysate taken from Grx2 overexpressed cells with anti-Grx2 IgG completely abolished the increase in peroxidase activity, indicating that this augmented 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.
It is remarkable 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 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 high oxidative stress conditions. Overexpression of Grx2 has been shown to inhibit oxidation induced apoptosis in HeLa cells. Grx2 may mediate this function through its peroxidase activity. These results suggest that Grx2 has a novel function as a peroxidase, accepting electrons both from GSH and TR. This unique property may play an important role in protecting the mitochondria from oxidative damage.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-5919fje
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