Thioltransferase is Present in the Lens Epithelial Cells as a Highly Oxidative Stress-resistant Enzyme

doi:10.1006/exer.1997.0464

Experimental Eye Research

Volume 66, Issue 4, April 1998, Pages 477-485

https://doi.org/10.1006/exer.1997.0464 Get rights and content

Abstract

The redox homeostasis is controlled by several enzyme systems. Sulfhydryl groups in lens proteins are very sensitive to oxidative stress and can easily conjugate with nonprotein thiols (S-thiolation) to form protein-thiol mixed disulfides. We have observed an elevation of protein S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC) in cataractous lenses from humans and from animal models subjected to oxidative stress. We also observed that these protein-thiol mixed disulfides could be spontaneously dissociated and lowered to basal levels if the lens which was pre-exposed to H₂O₂was subsequently cultured in H₂O₂-free medium. This suggests that the lens has a system to repair oxidative damage through dethiolation thereby restoring its redox homeostasis. In other tissues, an enzyme, thioltransferase (TTase), has been shown to be responsible for thiol/disulfide regulation. We recently demonstrated the presence of this enzyme in the lens and in cultured lens epithelial cells. Here, we investigated the response of TTase to H₂O₂stress and its possible repair function in cultured lens epithelial cells. Rabbit lens epithelial cell line N/N 1003A was raised to confluence, trypsinized and plated at 0.8 million cells per 60 mm culture dish. The cells were incubated overnight in Eagle's minimum essential medium (MEM) with 1% rabbit serum and then in serum-free MEM for 30 min before a bolus of 0.5 mmH₂O₂was added. At intervals of 5, 15, 30 min and up to 3 hr, the cells were harvested and used for enzyme assays for TTase, glutathione reductase (GR), glutathione peroxidase (GPx) and glyceraldehyde-3-phosphate dehydrogenase (G-3PD). Free GSH, total SH and PSSG and PSSC were also determined. Hydrogen peroxide in the medium was measured at each time point. Cells incubated without H₂O₂were used as controls. The results showed that the H₂O₂concentration was reduced to 50% within 30 min and was undetectable at 2 hr. Cellular GSH dropped to 40% within 5 min and stayed at this level before it began to increase at 90 min and completely recovered by 2 hr. The total SH groups were similar to free GSH. PSSG and PSSC increased 6.5 and 2 times respectively before 30 min and then decreased when GSH started to recover. G-3PD was most sensitive to H₂O₂and lost 95% activity within 5 min. The activity was regained quickly when H₂O₂diminished in the medium. A similar but less severe pattern was observed in both GPx (60% loss at 60 min) and GR (30% loss at 90 min). In contrast, TTase activity remained constant during the entire 3 hr. Only when a higher dose of H₂O₂(0.8–1.0 mm)was used, did TTase activity show a brief loss (<30% at 60 min) and a swift recovery. Cells exposed to H₂O₂exhibited a normal morphology with no evidence of DNA fragmentation. The lens epithelial cells showed a remarkable ability to repair the early damages induced by H₂O₂. The unusual oxidative stress-resistant property displayed by TTase, coupled with its known function suggest that it plays an important role in the repair of oxidative damage.

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Lens homeostasis and transparency are dependent on the function and intercellular communication of its epithelia. While the lens epithelium is uniquely equipped with functional repair systems to withstand reactive oxygen species (ROS)-mediated oxidative insult, ROS are not necessarily detrimental to lens cells. Lens aging, and the onset of pathogenesis leading to cataract share an underlying theme; a progressive breakdown of oxidative stress repair systems driving a pro-oxidant shift in the intracellular environment, with cumulative ROS-induced damage to lens cell biomolecules leading to cellular dysfunction and pathology. Here we provide an overview of our current understanding of the sources and essential functions of lens ROS, antioxidative defenses, and changes in the major regulatory systems that serve to maintain the finely tuned balance of oxidative signaling vs. oxidative stress in lens cells. Age-related breakdown of these redox homeostasis systems in the lens leads to the onset of cataractogenesis. We propose eight candidate hallmarks that represent common denominators of aging and cataractogenesis in the mammalian lens: oxidative stress, altered cell signaling, loss of proteostasis, mitochondrial dysfunction, dysregulated ion homeostasis, cell senescence, genomic instability and intrinsic apoptotic cell death.
Susceptibility of Atlantic salmon lenses to hydrogen peroxide oxidation ex vivo after being fed diets with vegetable oil and methylmercury
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In the present study, VO control lenses had a significantly higher expression of MT-B compared to FO control lenses, while the histidine enrichment appeared to have a slightly modulating effect, and the FO control lenses had the lowest expression levels. Lou et al. (1998) showed that the enzyme thioltransferase (TTase), that is present in lens epithelial cells, can dethiolate, and repair oxidative damage caused by previous exposure to H2O2, and restore the redox homeostasis in the lens. Glutaredoxin (thioltransferase) was significantly higher expressed in FO lenses cultured in histidine enriched medium, despite similar histidine, NAH and GSH concentration between the examined groups.
The development of cataract in farmed Atlantic salmon (Salmo salar L.) has been related to changes in feed composition resulting in sub-optimal lens nutrition. The present study was performed to investigate the ability of Atlantic salmon lenses to withstand oxidative stress ex vivo, with focus on the nutritional lipid history and exposure to methylmercury (MeHg) as a relevant dietary contaminant. Since dietary histidine has been shown to have a mitigating effect on the prevalence of cataract in farmed salmon, the antioxidative abilities of histidine and NAH, a major imidazole in the salmon lens, was also investigated ex vivo. Lenses from Atlantic salmon prefed diets based on either fish oil (FO) or vegetable oil (VO) as lipid source, with or without addition of 5 mg MeHg kg⁻¹ feed, were cultured for 96 h in normal medium (control), medium added 5 mM H₂O₂ or in histidine enriched medium. Lipid class composition of the lenses was not affected by the dietary lipids; while VO fed fish had a decrease in lens n − 3/n − 6 fatty acid ratio due to minor but significant increase in the concentration of 18:2 n − 6 and 20:4 n − 6, and decrease in 20:5 n − 3 fatty acids compared to FO fed fish. The lenses accumulated mercury in response to dietary levels, but neither the oxidative status nor any physiological responses were affected. The cultured lenses responded to H₂O₂ exposure with loss of transparency, accumulation of auto-fluorescent compounds, volume increase and reduced glutathione concentration similarly and irrespective of the dietary history. Lenses extracted histidine from the media, and synthesised NAH during the culture period. The innate antioxidative defence system appeared to be influenced both by the dietary lipid history and histidine enrichment on a transcriptional level. Catalase and SPARC were expressed higher in lenses from FO fed fish, and glutaredoxin showed elevated expression levels in FO lenses cultured in histidine enriched medium, suggesting that histidine is related to the innate antioxidant defence in salmon lenses. Further, the concentration of NAH was significantly reduced in oxidatively stressed lenses. Based on the results from this study it is suggested that NAH has a novel role as antioxidant in the Atlantic salmon lens.
Revival of inactive glyceraldehyde 3-phosphate dehydrogenase in human cataract lenses by reduction
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In this study, endogenous as well as synthetic reducing systems were shown to reduce the disulphide bonds formed in glyceraldehyde 3-phosphate dehydrogenase, an important glycolytic enzyme previously reported to have lost its activity in human cataract lenses, resulting in reviving the activity of this enzyme.
Disulphide bond formation is a non-specific posttranslational modification of proteins, which leads to a loss of function of the affected protein. When an enzyme is targeted, this harmful effect can be easily detected by monitoring the change of activity. Endogenous reducing systems are responsible for breaking these bonds and returning the protein (enzyme) to its natural state, when these mechanisms fail to do so, the loss of enzyme activity will be permanent.
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Citation Excerpt :
The above described unique property of thioltransferase in resisting oxidative stress has effectively displayed in vivo. It was demonstrated both in rabbit lens epithelial N/N 1003A cells (Lou et al., 1998) and in human lens epithelial (HLE) B3 cells (Xing and Lou, 2002), when a bolus of H2O2 (0.5 mM for rabbit cells and 0.1 mM for HLE cells) was added to cultured cells in medium without serum and followed for 3 h. This created an environment for the cells to be oxidatively stressed in the first 90 min followed by a recovery period in the following 90 min (H2O2-free) when H2O2 in the medium was completely detoxified by the cells.
The high content of glutathione (GSH) in the lens is believed to protect thiols in structural proteins and enzymes for proper biological functions. The lens has both biosynthetic and regenerating systems for GSH to maintain its large pool size. However, ageing lenses or lenses under oxidative stress show an extensively diminished size of GSH pool with some protein thiols being S-thiolated by oxidized non-protein thiols to form protein–thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC) or protein-S-S-γ-glutamylcysteine. It was shown in an H₂O₂-induced cataract model that PSSG formation precedes a cascade of events before cataract formation, starting with protein disulfide crosslinks, protein solubility loss and high molecular weight aggregation. Furthermore, this early oxidative damage in protein thiols can be spontaneously reversed in H₂O₂ pretreated lenses if the oxidant is removed in time. This dethiolation process appears to have mediated through a redox-regulating enzyme, thioltransferase (TTase), which is ubiquitously present in microbial, plant and animal tissues, including the lens. The GSH-dependent, low molecular weight (11.8 kDa) cytosolic enzyme plays an important role in oxidative defense and can modulate key metabolic enzymes in the glycolytic pathway. The enzyme repairs oxidatively damaged proteins/enzymes through its unique catalytic site with a vicinal cysteine moiety, which can specifically dethiolate protein-S-S-glutathione and restore protein free SH groups for proper enzymatic or protein functions. Most importantly, it has been demonstrated that thioltransferase has a remarkable resistance to oxidation (H₂O₂) in cultured human and rabbit lens epithelial cells under oxidative stress conditions when other oxidation defense systems of GSH peroxidase and GSH reductase are severely inactivated. A second repair enzyme, thioredoxin (TRx), which is NADPH-dependent, is widely found in many lower and higher life forms of life. It can dethiolate protein disulfides and thus is an extremely important regulator for redox homeostasis in the cells. Thioredoxin has been recently found in the lens and has been shown to participate in the repair process of oxidatively damaged lens proteins/enzymes. These two enzymes may work synergistically to regulate and repair thiols in lens proteins and enzymes, keeping a balanced redox potential to maintain the function of the lens.
Propyl gallate is a superoxide dismutase mimic and protects cultured lens epithelial cells from H<inf>2</inf>O<inf>2</inf> insult
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n-Propyl gallate (nPG) is a food preservative that is generally regarded as safe by the US FDA. It suppresses oxidation in biological systems. The mechanism by which nPG acts in biological systems is uncertain. We investigated whether nPG protected cultured lens epithelial cells from H₂O₂-induced damage. Cells were treated with H₂O₂ or with nPG and then H₂O₂. H₂O₂ inhibited growth, caused membrane blebbing, decreased lactate production, increased the level of GSSG, decreased the levels of GSH, ATP and NAD⁺, and G3PDH activity, stimulated the hexose monophosphate shunt and induced single-strand breaks in DNA. nPG prevented the H₂O₂-induced growth inhibition, membrane blebbing, drop in NAD⁺ and single-strand breaks in DNA.
The mechanism by which nPG acts at the chemical level was investigated using electron paramagnetic resonance (EPR), direct spectrophotometric kinetic measurements, and cyclic voltammetry. When nPG at low concentrations (nM to μM) was mixed with a large excess of O₂⁻, the superoxide signal was destroyed as indicated by UV visible spectroscopy and EPR. Kinetic analysis indicated that nPG dismutated O₂⁻ in repetitive additions of superoxide with little loss of activity. The rate constant for the overall reaction of nPG with O₂⁻ was ca. 10⁶ M⁻¹ s⁻¹. nPG had a very low specific binding constant for Fe²⁺ as determined by cyclic voltammetry. The evidence indicates that nPG dismutates the superoxide ion in a catalytic manner.
Effect of H<inf>2</inf>O<inf>2</inf> on human lens epithelial cells and the possible mechanism for oxidative damage repair by thioltransferase
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Human lens epithelial (HLE) B3 cells were used to study the oxidative damage and cellular repair with respect to the redox homeostasis, the oxidative defense enzymes and the glucose metabolic pathway. The effect of oxidative stress on cell growth was initially analyzed by culturing the cells with a bolus amount (0.02–0.1 mM ) of hydrogen peroxide (H₂O₂) in minimal essential medium (MEM) containing 20% fetal bovine serum (FBS) for 1 week. Concentration of H₂O₂greater than 0.03 mM showed progressive inhibition of cell growth. However, the cells were also shown to tolerate H₂O₂concentrations up to 0.5 m M by detoxifying the exogenous oxidant within 3 hr without any detectable DNA damage. Therefore, this short-term H₂O₂exposure model was chosen to study the effect of oxidative stress on the cellular redox homeostasis. HLE B3 cells were first grown to confluence in MEM with 20% FBS. Approximately 1.6 million cells were gradually weaned off serum by subculturing in 2% FBS overnight, followed by serum-free medium for 30 min before subjecting to a bolus of 0.1 m M H₂O₂for up to 180 min. These cells were used for biochemical analysis, which included H₂O₂detoxification (H₂O₂in the medium), glutathione (GSH) level and lactate production. Activity measurements were conducted on the oxidation defense enzymes: glutathione-S-transferase (GST), glutathione reductase (GR) and glutathione peroxidase (GPx); the dethiolating enzyme, thioltransferase (TTase); and a key glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (G-3PD). While the B3 cells were shown to tolerate and detoxify 0.1 m M H₂O₂within 60 min, the GSH pool was transiently depleted in the first 60 min before fully recovered. GPx suffered more than 80% loss in activity and was unable to recover fully. GST showed slight inactivation but neither GR nor TTase was affected. G-3PD was inactivated to < 50% within 15 min of oxidative stress and was reactivated gradually to 80% of normal at the end of 180 min, concurrent with the transient loss of lactate production in the same cells. The reactivation of G-3PD was both temperature- and GSH-dependent, occurring only at physiological temperature and failing to reactivate when the intracellular GSH pool was depleted by BCNU (GR inhibitor) pretreatment. The inactivated cellular G-3PD in the cell extract could be partially reactivated by DTT (6 m M) or by recombinant human lens thioltransferase (RHLT) but not by GSH (1 mM ), GR or GST. HLE cells cultured in the presence of L-³⁵S-cystine and cycloheximide displayed an extra radiolabelled protein band on the autoradiograph in the H₂O₂treated cells. The labelled band was positively reacted with G-3PD antibody and could be removed by RHLT, indicating that S-thiolation of G-3PD occurred. The H₂O₂pre-exposed cells also transiently accumulated proteins modified by thiolation, including protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). It can be concluded that HLE could endure up to 0.1 m M of H₂O₂oxidative stress since the cell could be protected by its effective repair systems, including dethiolating the inactivated key SH-sensitive enzymes. TTase may play a role in this. One of the mechanisms may be through preserving glucose metabolism and supplying ATP needed for maintaining cell viability.

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^☆: Preliminary results were presented at the Annual Meeting of the Association for Research in Vision and Ophthalmology, Fort Lauderdale, FL, U.S.A., 1996.

^☆☆: Albert, D. M.Jakobiec, F. A.

^f2: For reprint requests: Marjorie F. Lou, 134 VBS, Department of Veterinary and Biomedical Sciences, University of Nebraska–Lincoln, Lincoln, NE 68583-0905, U.S.A.

^f3: Current address: Manhattan Eye, Ear and Throat Hospital, New York City, NY, U.S.A.

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Regular articleThioltransferase is Present in the Lens Epithelial Cells as a Highly Oxidative Stress-resistant Enzyme☆,☆☆

Abstract

FEBS Lett.

Arch. Biochem. Biophys.

Exp. Eye Res.

Arch. Biochem. Biophys.

Exp. Eye Res.

Exp. Eye Res.

Meth. Enzymol.

Exp. Eye Res.

J. Immunol. Methods

Exp. Eye Res.

Exp. Eye Res.

Differentiation

Exp. Eye Res.

Exp. Eye Res.

Anal. Biochem.

Exp. Eye Res.

Free Radical Biol. Med.

Am. J. Ophthalmol.

Exp. Eye Res.

Biochemical mechanisms of age-related cataract

Principles and practice of ophthalmology,

Protein modification in cataract: possible oxidative mechanisms

Regular article
Thioltransferase is Present in the Lens Epithelial Cells as a Highly Oxidative Stress-resistant Enzyme☆,☆☆