The Possible Physiological Function of Thioltransferase in the Cells

Abstract

Abstract: : Purpose : To study the possible physiological function of thioltransferase (TTase) in combating oxidative damage in the lens epithelial cells. Methods : The human lens thioltransferase gene (hTTase) was obtained from the total RNA of human lens epithelial cells (HLE B3 cells) using RT-PCR and cloned into pCR3.1-uni vector. The plasmid was transfected into HLE B3 cells using lipofectamine method. The cells transfected with either TTase-containing plasmid or vector only were then compared for their resistance to oxidative stress in the presence of a bolus of H₂O₂ (0.1 mM) for up to 180 min, including the G-3PD, GPx activities and the level of PSSG generation. Cells depleted of TTase activity upon cadmium treatment were examined of the resistance to oxidative stress under the same conditions. Results : TTase activity assay, western blot and northern blot analyses confirmed that hTTase gene was successfully transfected into the HLE B3 cells and was over-expressed. The TTase activity in the transient-transfected cells could be increased as high as 10 folds over control. However, stable-transfected cells (geneticin selected) showed about 2-fold increase in both TTase activity and its protein level (western blot), although 6-fold increase in mRNA expression was detected by northern blot. The TTase-transfected B3 cells detoxified H₂O₂ as efficiently as the control cells but displayed a faster and more complete recovery of oxidatively-inactivated G-3PD and GPx activities. Furthermore, these cells suppressed protein thiolation (PSSG formation) under the same oxidative stressed conditions. The purified recombinant human lens TTase (RHLT) was inactivated by cadmium in vitro. Same inhibition of cellular TTase activity could be achieved by incubating the cells with cadmium. The spontaneous reactivation of G-3PD under bolus H₂O₂ treatment was not accomplished in cadmium-pretreated HLE B3 cells. Conclusion : These data indicate that the physiological function of TTase involves in the reactivation of the oxidatively-inactivated enzymes through dethiolation, thus this redox regulating enzyme can protect the human lens epithelial cells and may be other cell types by preventing them from permanent oxidative damage. Supported by NIH grant EY 10590. None.

Keywords: enzymes/enzyme inhibitors • oxidation/oxidative or free radical damage • protein modifications-post translational