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The possible physiological function of thioltransferase in cells^{1 ,2}

KUIYI XING^*,3 and MARJORIE F. LOU^*,,,4

^* Department of Veterinary and Biomedical Sciences and
Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska, USA; and
Department of Ophthalmology, University of Nebraska Medical Center, Omaha, Nebraska, USA

⁴ Correspondence: 134 VBS, Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE 68583-0905, USA. E-mail: mlou1{at}unl.edu

SPECIFIC AIMS

Oxidative induced protein glutathione conjugation, an intermediate during human senile cataract formation, can be cleaved by thioltransferase. We manipulated cellular thioltransferase (TTase) activity by either high-expressing TTase in TTase-transfected cells or inhibiting endogenous cellular TTase with cadmium in order to investigate the role of TTase in protecting cells from permanent oxidative damage.

PRINCIPAL FINDINGS

1. High expression of TTase in TTase-transfected human lens epithelial (HLE) B3 cells
TTase cDNA, amplified from human lens epithelial cells by using RT-PCR and modified by adding Kozak sequence to the 5' region, was cloned into pCR3.1-uni vector and transfected into HLE B3 cells using lipofectamine. The transient transfected cells displayed increased TTase activity depending on the amount of plasmid DNA used for transfection. However, the increase of TTase activity in stable transfected cells was only twofold regardless of the amount of plasmid DNA. The high expression was further confirmed by Northern blot and Western blot.

2. Enhanced protection against oxidative stress by the TTase-transfected HLE B3 cells
The stable TTase-transfected HLE B3 cells displayed the same viability as normal HLE B3 cells when exposed to a bolus H₂O₂ at concentrations from 0 to 0.2 mM (Fig. 1 A).

View larger version (27K): [in this window] [in a new window]   Figure 1. Enhanced protection against oxidative stress by TTase overexpression. A) Cell viability. Normal (open bars) and stable TTase-transfected HLE B3 cells (filled bars) were exposed to a bolus H2O2 of different concentrations in serum-free MEM for 3 h. Cells were counted with trypan blue and the cell viability was expressed as the percentage of trypan blue unstained cells in total cells. B–D) A bolus of H2O2 (0.1 mM) was used to treat 1.6 million stable transfected cells for a total of 3 h. Culture medium was removed at 0, 5, 10, 15, 30, 60, 90, 120, 150, and 180 min for H2O2 assay. Cells were harvested and homogenized to assay G3PD and GPx activities. Data are expressed as mean ± SD, with n = 3. B) H2O2 concentration in the medium. C) G-3PD activity. D) GPx activity. Open circles: Vector-transfected HLE B3 cells; filled triangles: TTase-transfected HLE B3 cells.

The stable TTase-transfected B3 cells were exposed to a bolus of 0.1 mM H₂O₂ and showed the same ability to detoxify H₂O₂ in the medium as the control cells (transfected with the pCR3.1-uni vector only) (Fig. 1B ).

Overexpression of TTase did not influence the activities of G-3PD and GPx in the B3 cells but enhanced the cells’ ability to protect these enzymes against oxidative stress. As shown in Fig. 1C , in the control cells exposed to H₂O₂, G-3PD was inactivated to below 40% of the initial activity but was restored up to 70% of its normal level at the end of 3 h. TTase-transfected cells under the same condition showed <50% of G-3PD inactivation after 30 min of H₂O₂ exposure and recovered to almost 80–90% of its original activity at the end of the 3 h experiment.

As shown in Fig. 1D , better protection was also observed for GPx in these TTase-transfected cells. The highest inactivation of GPx in the TTase-transfected cells caused by 30 min H₂O₂ treatment was only 30% in comparison to the usually observed 80% in the H₂O₂-treated control cells for 60 min. Recovery of GPx activity in the TTase-transfected cells was nearly complete at 120 min, while the control cells could reach to only 40% of the normal level at the end of the experiment.

3. Reduced formation of PSSG in the TTase-transfected cells under H₂O₂ treatment
Under a bolus of 0.1 mM H₂O₂ treatment, cellular protein-S-S-glutathione mixed disulfide (PSSG) in the control cells showed a transient increase with maximal level appeared at 30 min post-treatment. TTase transfection did not affect the basal level of PSSG, quantified as glutathione sulfonic acid. However, the elevated PSSG level in the H₂O₂ pretreated TTase-transfected cells was much less than that in control cells under the same conditions. The control cells usually generated 60% more PSSG upon oxidation but the transfected cells generated only 30% over the basal level.

4. Inhibiting of TTase activity by cadmium
Purified recombinant human lens thioltransferase (RHLT) was first used to study the inactivation of TTase by cadmium. The reduced RHLT was inactivated by cadmium in a concentration-dependent manner. Only half the original activity remained when RHLT was incubated with 50 µM cadmium.

Cellular TTase activity in HLE B3 cells was inhibited by incubating the cells with cadmium in serum-free medium for 30 min. Cadmium (25 µM) inhibited cellular TTase activity by 55% and only 20% of the original TTase activity remained after being treated with 100 µM cadmium.

5. Effect of cadmium on the recovery of oxidatively damaged enzymes and cell viability
Pretreating the HLE B3 cells with 100 µM cadmium for 30 min did not affect the cellular specific activity of G-3PD. However, treatment of cadmium weakened the recovery of H₂O₂-induced inactivation of G-3PD. As shown in Fig. 2 A, the cellular G-3PD in the HLE B3 cells without cadmium treatment displayed a transient inactivation under a bolus of 0.1 mM H₂O₂ exposure, with the lowest activity at 37% of the original at 30 min and complete recovery by the end of the treatment (3 h). When cadmium pretreated B3 cells were exposed to 0.1 mM H₂O₂ in the presence of cadmium (100 µM), only 15% of the original activity remained at 30 min and the highest recovered activity of G-3PD was only 50% of the original.

View larger version (16K): [in this window] [in a new window]   Figure 2. Effect of cadmium on G-3PD regeneration and cell viability in H2O2-treated HLE B3 cells. A) G-3PD regeneration. HLE B3 cells pretreated with 100 µM Cd2+ were exposed to 0.1 mM H2O2 plus Cd2+ for another 2 h (filled triangles) vs. control cells treated with H2O2 alone (open circles). G-3PD-specific activities were analyzed and expressed as mean ± SD, with n = 3. B) Cell viability. HLE B3 cells pretreated with Cd2+ of various concentrations for 30 min were treated with MEM alone (filled circles) or H2O2 plus Cd2+ of the same concentration used for pretreatment (filled triangles) for another 3 h. Cells were counted with trypan blue.

To test the effect of cadmium on cell viability, HLE B3 cells were pretreated with cadmium (0, 25, 100, or 400 µM) for 30 min, then incubated in either normal MEM medium or 0.1 mM H₂O₂ plus cadmium of the same concentration as before. Cells were counted and the living cells were distinguished from dead ones by trypan blue exclusion. As shown in Fig. 2B , cadmium treatment had no significant effect on cell viability. H₂O₂ alone showed little effect on cell death. The incubation of H₂O₂ together with cadmium enhanced cell death in proportion to the concentration of cadmium used. Only 40% of cells were viable after being treated for 3 h with cadmium (400 µM) plus H₂O₂ (0.1 mM).

CONCLUSIONS AND SIGNIFICANCE

Through gene transfection and the use of TTase inhibitor, we manipulated the cellular TTase activity in order to demonstrate its physiological role in combating oxidative stress. TTase showed high expression in transient-transfected HLE B3 cells, but the stable transfectant resulted in only a twofold increase in TTase activity. Although mammalian TTase cDNA has been overexpressed in Escherichia coli, only an twofold increase of TTase activity was observed in eukaryotic cells by transfection. The small increase of TTase in eukaryotic cells by transfection suggests that high levels of TTase might be cytotoxic, which agrees with the findings that TTase may be involved in many important cellular functions via regulating the thiol/disulfide homeostasis. These functions may include glucose metabolism, signal transduction, and gene regulation. The increase of TTase activity in transfected HLE B3 cells was not proportional to the elevation of mRNA, suggesting that not all mRNA for TTase was translated into proteins and that translation of TTase may be under certain regulation.

As a twofold increase of TTase activity could significantly enhance the activation of transcription factors, a similar increase of TTase activity in HLE B3 cells provided better protection from oxidative damage. As expected, transfection of TTase has no direct effect on the ability of cells to detoxify H₂O₂ in the medium, which was consumed primarily by catalase and GPx. However, TTase transfection displayed good protection on oxidatively inactivated enzymes, G-3PD and GPx, which were both inactivated less and reactivated sooner and more completely. This confirmed our hypothesis that oxidation-induced thiolation leads to enzyme inactivation and that the increase of TTase activity through transfection would accelerate the dethiolation process to restore more damaged enzymes within a shorter period. This was further confirmed by the observation that less PSSG was detected in the TTase-transfected cells.

The data provided here is the first evidence that TTase has a physiological function to repair oxidatively damaged enzymes. G-3PD is a critical enzyme for ATP generation that is particularly important to the lens, since this ocular organ has a minimum number of mitochondria; thus, a minimum amount of ATP can be generated via oxidative phosphorylation. G-3PD-controlled glycolytical pathway is the main supply of ATP for the lens. Similarly, GPx is a key oxidation defense enzyme for the lens. Maintenance of G-3PD and GPx activities thus is vital to proper physiological function of the lens. Certainly, there may be other repair systems to be discovered in the lens, but current findings on the function of TTase in the lens can provide some insight into the inherent repair/protection mechanism of the lens. This repair function of TTase was further demonstrated by cadmium-induced TTase inhibition, resulting in impaired spontaneous reactivation of the oxidatively damaged G-3PD.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-1164fje; doi: 10.1096/fj.02-1164fje

² Presented in part at U.S.-JAPAN Cooperative Research Conference at Kona, HI, November 3-7, 2001.

³ This work was contributed by K.X. as partial fulfillment of Ph.D. dissertation.

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