Protein-thiol Mixed Disulfides Research Articles

Glutaredoxin 1 protects lens epithelial cells from epithelial-mesenchymal transition by preventing casein kinase 1α S-glutathionylation during posterior capsular opacification

Oxidative stress drives protein S-glutathionylation, which regulates the structure and function of target proteins and is implicated in the pathogenesis of many diseases. Glutaredoxin 1 (Grx1), a cytoplasmic deglutathionylating enzyme, maintains a reducing environment within the cell under various conditions by reversing S-glutathionylation. Grx1 performs a wide range of antioxidant activities in the lens and prevents protein-thiol mixed disulfide accumulation, reducing protein–protein aggregation, insolubilization, and apoptosis of lens epithelial cells. Oxidative stress is related to epithelial–mesenchymal transition (EMT) during posterior capsular opacification (PCO). However, whether Grx1-regulated protein S-glutathionylation plays an essential role in PCO remains unclear. In this study, we revealed that Grx1 expression was decreased in mice following cataract surgery. Furthermore, the absence of Grx1 elevated oxidative stress and protein S-glutathionylation and aggravated EMT in both in vitro and in vivo models. Concurrently, these results could be reversed by Grx1 overexpression. Notably, liquid chromatography–tandem mass spectrometry results showed that casein kinase 1α (CK1α) was susceptible to S-glutathionylation under oxidative stress, and CK1α S-glutathionylation (CK1α-SSG) was mediated at Cys249. The absence of Grx1 upregulated CK1α-SSG, subsequently decreasing the CK1α-induced phosphorylation of β-catenin at Ser45. The consequential downregulation of degradative β-catenin and upregulation of its nuclear translocation activated the Wnt/β-catenin signaling pathway and aggravated EMT. In conclusion, the downregulated expression of Grx1 in mice following cataract surgery aggravated EMT by upregulating the extent of CK1α-SSG. To the best of our knowledge, our study is the first to report how S-glutathionylation regulates CK1α activity under oxidative stress.

Glutathione and Glutaredoxin in Redox Regulation and Cell Signaling of the Lens.

The ocular lens has a very high content of the antioxidant glutathione (GSH) and the enzymes that can recycle its oxidized form, glutathione disulfide (GSSG), for further use. It can be synthesized in the lens and, in part, transported from the neighboring anterior aqueous humor and posterior vitreous body. GSH is known to protect the thiols of the structural lens crystallin proteins from oxidation by reactive oxygen species (ROS) so the lens can maintain its transparency for proper visual function. Age-related lens opacity or senile cataract is the major visual impairment in the general population, and its cause is closely associated with aging and a constant exposure to environmental oxidative stress, such as ultraviolet light and the metabolic end product, H2O2. The mechanism for senile cataractogenesis has been hypothesized as the results of oxidation-induced protein-thiol mixed disulfide formation, such as protein-S-S-glutathione and protein-S-S-cysteine mixed disulfides, which if not reduced in time, can change the protein conformation to allow cascading modifications of various kinds leading to protein–protein aggregation and insolubilization. The consequence of such changes in lens structural proteins is lens opacity. Besides GSH, the lens has several antioxidation defense enzymes that can repair oxidation damage. One of the specific redox regulating enzymes that has been recently identified is thioltransferase (glutaredoxin 1), which works in concert with GSH, to reduce the oxidative stress as well as to regulate thiol/disulfide redox balance by preventing protein-thiol mixed disulfide accumulation in the lens. This oxidation-resistant and inducible enzyme has multiple physiological functions. In addition to protecting structural proteins and metabolic enzymes, it is able to regulate the redox signaling of the cells during growth factor-stimulated cell proliferation and other cellular functions. This review article focuses on describing the redox regulating functions of GSH and the thioltransferase enzyme in the ocular lens.

Molecular Mechanisms and Potential Clinical Significance ofS-Glutathionylation

Protein S-glutathionylation, the reversible binding of glutathione to protein thiols (PSH), is involved in protein redox regulation, storage of glutathione, and protection of PSH from irreversible oxidation. S-Glutathionylated protein (PSSG) can result from thiol/disulfide exchange between PSH and GSSG or PSSG; direct interaction between partially oxidized PSH and GSH; reactions between PSH and S-nitrosothiols, oxidized forms of GSH, or glutathione thiyl radical. Indeed, thiol/disulfide exchange is an unlikely intracellular mechanism for S-glutathionylation, because of the redox potential of most Cys residues and the GSSG export by most cells as a protective mechanism against oxidative stress. S-Glutathionylation can be reversed, following restoration of a reducing GSH/GSSG ratio, in an enzyme-dependent or -independent manner. Currently, definite evidence of protein S-glutathionylation has been clearly demonstrated in few human diseases. In aging human lenses, protein S-glutathionylation increases; during cataractogenesis, some of lens proteins, including alpha- and beta-crystallins, form both mixed disulfides and disulfide-cross-linked aggregates, which increase with cataract severity. The correlation of lens nuclear color and opalescence intensity with protein S-glutathionylation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses. Recently, specific PSSG have been identified in the inferior parietal lobule in Alzheimer's disease. However, much investigation is needed to clarify the actual involvement of protein S-glutathionylation in many human diseases.

Protein–thiol substitution or protein dethiolation by thiol/disulfide exchange reactions: The albumin model

Dethiolation experiments of thiolated albumin with thionitrobenzoic acid and thiols (glutathione, cysteine, homocysteine) were carried out to understand the role of albumin in plasma distribution of thiols and disulfide species by thiol/disulfide (SH/SS) exchange reactions. During these experiments we observed that thiolated albumin underwent thiol substitution (Alb-SS-X+RSH<-->Alb-SS-R+XSH) or dethiolation (Alb-SS-X+XSH<-->Alb-SH+XSSX), depending on the different pK(a) values of thiols involved in protein-thiol mixed disulfides (Alb-SS-X). It appeared in these reactions that the compound with lower pK(a) in mixed disulfide was a good leaving group and that the pK(a) differences dictated the kind of reaction (substitution or dethiolation). Thionitrobenzoic acid, bound to albumin by mixed disulfide (Alb-TNB), underwent rapid substitution after thiol addition, forming the corresponding Alb-SS-X (peaks at 0.25-1 min). In turn, Alb-SS-X were dethiolated by the excess nonprotein SH groups because of the lower pK(a) value in mixed disulfide with respect to that of other thiols. Dethiolation of Alb-SS-X was accompanied by formation of XSSX and Alb-SH up to equilibrium levels at 35 min, which were different for each thiol. Structures by molecular simulation of thiolated albumin, carried out for understanding the role of sulfur exposure in mixed disulfides in dethiolation process, evidenced that the sulfur exposure is important for the rate but not for determining the kind of reaction (substitution or dethiolation). Our data underline the contribution of SH/SS exchanges to determine levels of various thiols as reduced and oxidized species in human plasma.

Biochemical and Biological Aspects of Protein Thiolation in Cells and Plasma

Protein thiolation is elicited by oxidation by different mechanisms and is involved in a variety of biological processes. Thiols, protein SH (PSH) and non-protein SH groups (NPSH, namely GSH), are in competition in all biological environments in the regulation of oxidant homeostasis because oxidants thiolate proteins, whereas GSH dethiolates them (e.g., GSSG + PSH --> GSSP + GSH). Although poorly investigated, the elimination of disulfides from thiolated proteins to regenerate critical PSH is important. These aspects are poorly known in cells, where glutaredoxin and peroxiredoxin operate as enzymes or potential chaperones to accelerate dethiolation. On the contrary, studies with plasma or albumin have highlighted the importance of protein conformation in dethiolation processes and have clarified the reason why homocysteine (thiol with potential toxicity) is preferentially bound to albumin as protein-thiol mixed disulfide with respect to other NPSH. Here we provide an overview of protein thiolation/dethiolation processes, with an emphasis on recent developments and future perspectives in this field.

The effects of age and hyperhomocysteinemia on the redox forms of plasma thiols

We assayed the redox forms of cysteine (reduced [CSH], oxidized [CSSC], and bound to protein [CS-SP]), cysteinylglycine (CGSH; cysteinylgycine disulfide [CGSSGC] and cysteinylglycine-protein mixed disulfide [CGS-SP]), glutathione (GSH; glutathione disulfide [GSSG] and glutathione-protein mixed disulfide [GS-SP]), homocysteine (Hcy; homocystine [HcyS] and homocystine-protein mixed disulfides [bHcy]), and protein sulfhydryls in the plasma of healthy subjects (divided into 8 groups ranging in age from birth to 70 years) and patients with mild hyperhomocysteinemia associated with cardiovascular disease (heart-transplant patients) or vascular atherosclerosis, with or without renal failure. In healthy individuals, levels of disulfides and protein-mixed disulfides were more abundant than those of thiols, and those of protein-thiol mixed disulfides were higher than disulfides. Concentrations of CSH, GSH, and CGSH in the various groups had profiles characterized by a maximum over time. The concentration of Hcy was unchanged up to the age of 30 years, after which it increased. CSSC concentration increased gradually with age, whereas concentrations of the other disulfides were essentially unchanged. By contrast, the concentrations of all protein-thiol mixed disulfides, especially those with CSH, increased gradually with age. Ranks of distribution of the reduced forms changed with age (at birth, CSH > CGSH > GSH > Hcy; in 1- to 2-year-olds, CSH > GSH > CGSH > Hcy; and in 51- to 70-year-olds, CSH > CGSH = GSH > Hcy), whereas those of disulfides and protein-thiol mixed disulfides were substantially unchanged (in all age groups, CSSC > CGSSGC > GSSG = HcyS and CS-SP > CGS-SP > bHcy > GS-SP). In patients with pathologic conditions, plasma levels of disulfide forms CSSC, HcyS, CS-SP, and bHcy were significantly increased, whereas other redox forms of thiols were unchanged or showed variations opposite (increasing or decreasing) to control values. Maximal increases in disulfides and protein-thiol mixed disulfides were associated with renal failure. Our data suggest that increases in plasma bHcy concentrations in subjects with pathologic conditions were more likely the result of activation of thiol-disulfide exchange reactions between free reduced Hcy and CS-SP than of a direct action of reactive oxygen species.

Thiolation and nitrosation of cysteines in biological fluids and cells.

Thiols (RSH) are potent nucleophilic agents, the rates of which depend on the pKa of the sulfhydryl. Unlike compounds having other nucleophile moieties (-OH or -NH(2)), RSH are involved in reactions, such as conjugations, redox and exchange reactions. Although protein SH groups (PSH) react like non-protein thiols (NPSH), the biochemistry of proteins is much more complex for reasons such as steric hindrance, charge distribution and accessibility of PSH to the solvent (protein conformation). The reaction rates and types of end-products of PSH vary a lot from protein to protein. The biological problem is even more complex because in all compartments and tissues, there may be specific competition between thiols (namely between GSH and PSH), regulated by the properties of antioxidant enzymes. Moreover, PSH are divided biologically into essential and non-essential and their respective influence in the various biological systems is unknown. It follows that during phenomena eliciting a prompt thiol response (oxidative stress), the antioxidant PSH response and reaction mechanisms vary considerably from case to case. For example, in spite of a relatively low pKa that should guarantee good antioxidant capacity, PSH of albumin has much less propensity to form adducts with conjugating agents than NPSH; moreover, the structural characteristics of the protein prevent albumin from forming protein disulfides when exposed to oxidants (whereas protein-thiol mixed disulfides are formed in relative abundance). On the other hand, proteins with a relatively high reactivity, such rat hemoglobin, have much greater antioxidant capacity than GSH, but although human hemoglobin has a pKa similar to GSH, for structural reasons it has less antioxidant capacity than GSH. When essential PSH are involved in S-thiolation and S-nitrosation reactions, a similar change in biological activity is observed. S-thiolated proteins are a recurrent phenomenon in oxidative stress elicited by reactive oxygen species (ROS). This event may be mediated by disulfides, that exchange with PSH, or by the protein intermediate sulfenic acid that reacts with thiols to form protein-mixed disulfides. During nitrosative stress elicited by reactive nitrogen species (RNS), depending on the oxygen concentration of the system, nitrosation reactions of thiols may also be accompanied by protein S-thiolation. In this review we discuss a number of cell processes and biochemical modifications of enzymes that indicate that S-thiolation and S-nitrosation may occur simultaneously in the same protein in the presence of appropriate interactions between ROS and RNS.

Redox regulation in the lens.

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-gamma-glutamylcysteine. It was shown in an H(2)O(2)-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 H(2)O(2) 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 (H(2)O(2)) 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.

UVA Light In vivo Reaches the Nucleus of the Guinea Pig Lens and Produces Deleterious, Oxidative Effects

The possible role of ultraviolet light in the formation of cataract is not well understood. In this study, guinea pigs were exposed to a chronic, low level of UVA light (0.5mWcm−2, 340–410nm wavelength, peak at 365nm) for 4–5 months. It is known that the lens of the guinea pig possesses unusually high levels of the UVA chromophore NADPH. In a preliminary analysis, it was found that isolated guinea pig corneas transmitted 70–90% of 340–400nm light, and that UVA radiation was able to penetrate deep into the nucleus of the guinea pig lens, where it was absorbed. Exposure of guinea pigs to UVA in vivo produced a 60% inactivation of lens epithelial catalase; however, analysis by transmission electron microscopy (TEM) showed no apparent morphological effects on either the lens epithelium or the cortex. A number of UVA-induced effects were found in the nucleus of the guinea pig lens, but were observed either not at all or to a lesser extent in the cortex. The effects included an increase in light scattering (two-fold; slit-lamp examination), distention of intercellular spaces (TEM), an increase in lipid peroxidation (30–35%; infrared spectroscopy), a decrease in GSH level (30%), an increase in protein–thiol mixed disulfide levels (80%), loss of water-soluble protein (20%), an increase in the amount of protein disulfide (two-fold; two-dimensional diagonal electrophoresis), degradation of MIP26 (15%) and loss of cytoskeletal proteins including actin, α- and β- tubulin, vimentin and α-actinin (60–100%). The results indicate that a 4–5 month exposure of guinea pigs to a biologically relevant level of UVA light produces deleterious effects on the central region of the lenses of the animals. UVA radiation, coupled presumably with the photoreactive UVA chromophore NADPH and trace amounts of O2 present in the lens nucleus, produced significant levels of oxidized products in the nuclear region over a five month period. The data demonstrate the potentially harmful nature of UVA light with respect to the lens, and highlight the importance of investigating a possible role for this type of radiation in the formation of human cataract.

Modulation of Lens Glycolytic Pathway by Thioltransferase

The observation that the level of S-thiolated proteins (protein–thiol mixed disulfides) was transiently increased in the lens epithelial cells correlation with the transient inactivation of glyceraldehyde-3-phosphate dehydrogenase (G-3PD), a key glycolytic enzyme, when the cells were treated with a bolus of hydrogen peroxide, prompted our speculation that G-3PD may have been transiently thiolated at the SH sensitive active center. In the meantime, thioltransferase (TTase), a thiol regulating enzyme, whose activity remained constant under the same condition, may be regulating G-3PD and other sulfhydryl-sensitive glycolytic enzymes through thiol–disulfide exchange reactions (11). To prove this hypothesis, several purified glycolytic enzymes from a commercial source, including hexokinase (HK), G-3PD, pyruvate kinase (PK) and fructose 1,6-bisphosphatase (FBPase), an enzyme in gluconeogenesis, were made into protein–thiol mixed disulfide and used for this study. Glycolytic enzymes in cultured rabbit lens epithelial cells pre-exposed to H2O2(0.5m M for 15min) were also studied for this purpose. Recombinant human lens thioltransferase (RHLT), which was isolated and purified previously in this laboratory, reactivated these pure glycolytic enzymes inactivated by forming protein-S-S-gluthathione (PSSG), protein-S-S-cysteine (PSSC) or, protein-S-S-cysteamine after thiolating with oxidized glutathione, cystine or cystamine respectively. RHLT also reactivated these enzymes in the cell extract of cultured rabbit lens epithelial cells after being briefly exposed to 0.5m M H2O2. The S-thiolation and dethiolation of FBPase however, showed an opposite effect to that of glycolytic enzymes. These results suggest that TTase may participate in the repair process of glycolytic enzymes during oxidative stress and restore their activities in situ.

Thiol Regulation in the Lens

The high content of glutathione (GSH) in the lens is believed to protect the 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 (4-6 mM). However, we have observed that, in aging lenses or lenses under oxidative stress, the size of GSH pool is diminished; and some protein thiols are being S-thiolated by oxidized nonprotein thiols to form protein-thiol mixed disulfides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC). We have shown in an H2O2-induced cataract model that PSSG formation precedes a cascade of events starting with protein disulfide crosslinks, protein solubility loss, and eventual lens opacification. Recently, we discovered that this early oxidative damage in protein thiols could be spontaneously reversed in H2O2 pretreated lenses if the oxidant was removed in time. This dethiolation process is likely mediated through a redox regulating enzyme, thioltransferase (TTase), which has been discovered recently in the lens. To understand if the role of oxidative defense and repair is the physiological function of TTase in the lens, we cloned the TTase gene and purified the recombinant human lens TTase. Although TTase required GSH for its activity, TTase was far more efficient in dethiolating lens proteins than GSH alone. It favored PSSG over PSSC and dethiolated gamma-crystallin-S-S-G better than the alpha-crystallin counterparts. Furthermore, TTase showed a remarkable resistance to oxidation (H2O2) in cultured rabbit lens epithelial cells when GSH peroxidase, GSH reductase, and glyceraldehyde-3-phosphate dehydrogenase were severely inactivated. We further showed that activity loss in those SH sensitive enzymes could be attributed to S-thiolation, but reactivation via dethiolation could be attributed to TTase. We conclude that TTase can regulate and repair the thiols in lens proteins and enzymes through its dethiolase activity, thus contributing to the maintenance of the function of the lens.

Does Glutathione-S-Transferase Dethiolate Lens Protein-Thiol Mixed Disulfides?—A Comparative Study With Thioltransferase

Protein S-thiolation is a process in which under oxidative stress, vulnerable sulfhydryl groups of proteins are conjugated to non-protein thiols such as glutathione (GSH) or cysteine resulting in the formation of protein-thiol mixed disulfides, protein-S-S-glutathione (PSSG) and protein-S-S-cysteine (PSSC). This process spontaneously disrupts the redox homeostasis of the cells, which in turn leads to functional disturbances in the respective tissue. In the ocular lens, such modification of proteins may trigger a cascade of events starting with the alteration of protein conformation, protein/enzyme deactivation, protein-S-S-protein aggregation and eventually lens opacification or cataract. Generally, the first line of defense system in the cells protects the lens proteins against such damage. Recent studies in our laboratory have shown that in addition to this defense system, lens cells also possess a well developed system to repair the oxidative damage to the lens proteins. We have identified this repair system as thioltransferase (TTase) and have proved that TTase by its dethiolase activity reverses the protein S-thiolation process which returns the oxidatively damaged lens proteins/enzymes to their original reduced state and restores their physiological functions. We investigated if this repair mechanism was mediated by enzymes other than TTase. We studied glutathione S-transferase (GST) and report here for the first time the cloning, high level expression, and purification of human lens μ and π isoforms of GST. A comparative study of recombinant human lens TTase and GST (μ and π) on their dethiolating abilities using lens crystallin-thiol mixed disulfides showed that the lens TTase is 60–70% more efficient in the dethiolation/repair process than GST. When TTase and GST were tested in conjunction for the dethiolation of thiol mixed disulfides, there was no significant enhancement of dethiolase activity. These findings suggest that TTase by itself is an efficient enzyme in the dethiolation/repair process and hence can be considered a crucial system to counteract oxidative stress in the lens.

Correlation of Nuclear Color and Opalescence with Protein S-thiolation in Human Lenses

Human lens nuclei were collected during routine cataract surgery and used to study the role of oxidation in cataract formation and brunescence. This study focused on the comparison of the intensities of nuclear opacity and pigmentation (brunescence) with the changes in free glutathione (GSH) and the three species of protein-thiol mixed disulfides: protein-S-S-glutathione (PSSG), protein-S S-cysteine (PSSC) and protein-S-S-γ-glutamylcysteine (PSSGC). Eighty-one freshly excised human lens nuclei from a population with a mean age of 77 were used. The nuclear color was graded using the CCRG system, ranging from yellow to dark brown. The nuclear cataract opalescence of these lenses was also graded using the LOCS II system, ranging from LOCS II NO-1 to NO-4. Three normal human lenses (average age of 88 yr) were also included in the study as controls. The nuclear samples were each analyzed for free GSH and protein-thiol mixed disulfides, respectively. It was found that nuclear GSH decreased as the nuclear color increased from yellow to dark brown (from 0.73±0.13 to 0.13±0.03 μmole g wet wt−1) and as the nuclear opalescence increased from NO.1 to NO.4 (from 0.80±0.19 to 0.20±0.01 μmole g wet wt−1). All these values were lower than that of GSH in normal controls (1.43±0.59 μmole g wet wt−1). Levels of both PSSG and PSSC progressively increased, however, as the nuclear color intensified. PSSG increased from 0.29±0.05 to 0.91±0.11 μmole g wet wt−1while PSSC increased from 0.13±0.04 to 0.41± 0.06 μmole g wet wt−1. PSSGC concentration progressively increased with increases in both nuclear pigmentation (from 0.05±0.01 to 0.23±0.05 μmole g wet wt−1) and nuclear opacity (from 0.02±0.00 to 0.20±0.02 μmole g wet wt−1). In comparison, normal controls had lower levels of all three mixed disulfide species: PSSG, 0.22±0.06; PSSC, 0.08±0.02; PSSGC, 0.02±0.06 μmole g wet wt−1, respectively. The correlation of lens nuclear color and opalescence intensity with nuclear protein S-thiolation indicates that protein-thiol mixed disulfides may play an important role in cataractogenesis and development of brunescence in human lenses.

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

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.

Cloning, High Level-expression and Characterization of Human Lens Thioltransferase

Polymerase chain reaction (PCR) primers, directed against the nucleotide sequence of pig liver thioltransferase (PLTT) were used to amplify human lens thioltransferase (HLTT) from a pool of human lens cDNA. The 520 bp PCR fragment obtained was cloned unidirectionally into pCR 3.1-Uni vector and sequenced. The cDNA sequence of the lens thioltransferase had 98% and 87% homology to pig liver and human placental thioltransferases (TTase) respectively. Nhe1 and EcoR1 fragment of the recombinant PCR 3.1-Uni vector was subcloned in pET 23a Expression vector. High level expression of HLTT was accomplished inEscherichia coliand the expressed protein was characterized by immunoblot analysis with anti PLTT andN-terminal amino acid sequence analysis. The recombinant enzyme efficiently dethiolated protein thiol mixed disulfides conjugated to both cystine (PSSC) and glutathione (PSSG) and had a significant dehydroascorbate reductase activity. Human lens thioltransferase thus displayed structural and functional characteristics identical to pig liver and human placental thioltransferases.

Nuclear light scattering, disulfide formation and membrane damage in lenses of older guinea pigs treated with hyperbaric oxygen

Nuclear cataract, a major cause of loss of lens transparency in the aging human, has long been thought to be associated with oxidative damage, particularly at the site of the nuclear plasma membrane. However, few animal models have been available to study the mechanism of the opacity. Hyperbaric oxygen (HBO) has been shown to produce increased nuclear light scattering (NLS) and nuclear cataract in lenses of mice and human patients. In the present study, older guinea pigs (Initially 17-18 months of age) were treated with 2.5 atmospheres of 100% O2 for 2-2.5-hr periods, three times per week, for up to 100 times. Examination by slit-lamp biomicroscopy showed that exposure to HBO led to increased NLS in the lenses of the animals after as few as 19 treatments, compared to lenses of age-matched untreated and hyperbaric air-treated controls. The degree of NLS and enlargement of the lens nucleus continued to increase until 65 O2-treatments, and then remained constant until the end of the study. Exposure to O2 for 2.5 instead of 2 hr accelerated the increase in NLS; however, distinct nuclear cataract was not observed in the animals during the period of investigation. A number of morphological changes in the experimental lens nuclei, as analysed by transmission electron microscopy, were similar to those recently reported for human immature nuclear cataracts (Costello, Oliver and Cobo, 1992). O2-induced damage to membranes probably acted as scattering centers and caused the observed increased NLS. A general state of oxidative stress existed in the lens nucleus of the O2-treated animals, prior to the first appearance of increased NLS, as evidenced by increased levels of protein-thiol mixed disulfides and protein disulfide. The levels of mixed disulfides in the experimental nucleus were remarkably high, nearly equal to the normal level of nuclear GSH. The level of GSH in the normal guinea pig lens decreased with age in the nucleus but not in the cortex; at 30 months of age the nuclear level of GSH was only 4% of the cortical value. HBO-induced changes in the lens nucleus included loss of soluble protein, increase in urea-insoluble protein and slight decreases in levels of GSH and ascorbate; however, there was no accumulation of oxidized glutathione. Intermolecular protein disulfide in the experimental nucleus consisted mainly of gamma-crystallin, but crosslinked alpha-, beta- and zeta-crystallins were also present.(ABSTRACT TRUNCATED AT 400 WORDS)

Further studies on the dynamic changes of glutathione and protein-thiol mixed disulfides in H2O2 induced cataract in rat lenses: distributions and effect of aging.

To further investigate the role of protein-thiol mixed disulfides in cataractogenesis, an in vitro H2O2 cataract model was used with rat lenses to study the effect of aging, and the dynamic changes in the cortex, nucleus and the lens protein fractions. A group of lenses was exposed to H2O2-containing media (0.6 mM) for 1 to 3 days so that cortical cataract was induced gradually. Another group of lenses was first subjected to H2O2 exposure for one day and then recovered in the oxidant-free media for one or two days. These lenses were examined for the distribution of free glutathione and protein-thiol mixed disulfides (protein-glutathione and protein-cysteine) in the cortical and nuclear regions as well as in the water soluble and water insoluble fractions. Similar to the results reported earlier, the glutathione depletion in the whole lens occurred immediately and extensively during the 3-day H2O2 exposure. This loss was evenly distributed in the cortical and nuclear fractions. The level of protein-glutathione increased rapidly and continued throughout the 3 days. Most of the accumulation was found in the cortex and in both lens protein fractions. The protein-cysteine modification responded more slowly and less to oxidative stress. The delayed formation occurred mainly in the nucleus and in both lens protein fractions. In the recovery group, glutathione depletion was less drastic in the cortical and nuclear regions, but the elevated protein-glutathione in both regions and both protein fractions spontaneously decreased to its respective basal level within 1 day. Protein-cysteine on the other hand remained quite high, and in some cases it continued to rise in the absence of oxidation. Aging showed little effect on the response of rat lenses to oxidative stress. Similar patterns in glutathione and protein-thiol mixed disulfides occurred in both age groups (1, 23 months) and in both chronic oxidative stress and recovery conditions.

The culture of rat lenses in high sugar media: effect on mixed disulfide levels.

Lens proteins are long lived proteins with those in the center of the lens predating the birth of the individual. As a result, they are subject to a host of modifications and damage through a variety of mechanisms. Two such modifications have been proposed as primary events which could cause conformational changes potentiating further modifications. These are non-enzymatic glycation and mixed disulfide formation. Human lenses accumulate protein-thiol mixed disulfides of three kinds throughout the lifespan. The presence of one of these, protein-glutathione (PSSG) mixed disulfide has been shown to be intimately involved in protein aggregation. We have utilized ex vivo lens culture and in vitro incubations of purified gamma-crystallin to evaluate the following hypotheses. A) Lenses cultured with a high sugar media will form higher mixed disulfide levels than controls; B) glycation of lens proteins will be dependent on initial mixed disulfide level. Xylose levels in the cultured lens rise rapidly (to 23 mM by 4 h), and the level of glycation after one week is elevated 6-7% over control values. Mixed disulfide levels are also substantially increased but not more than for lenses cultured in control media. gamma-Crystallin modified with 0, 1, or 5 equivalents of GSH was differentially glycated by radioactive fructose. The amount of fructose bound by the protein was found to be inversely related to the extent of mixed disulfide formation. These results indicate that 1) protein modification of one kind may influence further modifications of other types; 2) glycation of lens proteins has no effect on mixed disulfide formation in this system; 3) the sulfhydryl status of lens proteins can affect the potential for protein glycation.

The Effect and Recovery of Long-term H 2O 2 Exposure on Lens Morphology and Biochemistry

Oxidative stress has long been speculated to play an important role in cataractogenesis. In the H 2O 2-induced cataract model, rat lens showed extensive biochemical damage but very mild morphological changes after being exposed to H 2O 2 (0·5 mM) for 24 hr in culture. This damage included reduced glutathione (GSH) depletion, protein-GSH mixed disulfide (PSSG) elevation but not protein-protein disulfide (PSSP) formation. In order to understand the role of protein-thiol mixed disulfide formation in relation to the sequence of events during cataract induction, we conducted a long term H 2O 2 exposure study for up to 96 hr to monitor the dynamic changes in GSH and PSSG levels, the formation of PSSP aggregate, protein solubility, and the progression in lens opacity. Rat lenses were cultured in 0·5 mM H 2O 2 and harvested at intervals of 24, 48, 72 and 96 hr for the examination of morphological and biochemical changes. Contralateral lenses cultured in H 2O 2-free media were used as controls. It was found that the lenses had only patchy opacity at the equator after 24 hr, but became hydrated suddenly at 48 hr (31% heavier than the control), with an opacity which involved the entire outer cortical region. By 72 hr incubation, the nucleus was opacified. Lens GSH progressively decreased with time of H 2O 2 exposure, 40% was lost by 24 hr and over 95% by 48 hr. There was a concomitant elevation of PSSG, 16-fold over the controls by 24 hr and 45-fold by 48 hr followed by a decline to 34-fold after 72 hr. In addition, the level of protein-cysteine mixed disulfide (PSSC) was elevated after 48 hr incubation in H 2O 2. At this time point, PSSP aggregates began to appear both in water soluble (WS) and urea soluble (US) fractions along with a drastic reduction in protein solubility. Western blot analysis of the protein fractions identified β and γ, but not α-crystallin in the disulfide-containing aggregates. The lens clarity and biochemical changes partially recovered if the oxidant was removed within 24 hr, indicating a potential therapeutic role for antioxidants. The complete normalization of PSSG level under this recovery condition signifies that cells may have a natural defense system for controlling PSSG elevation.

Protein-thiol mixed disulfides in human lens

Protein-thiol mixed disulfide formation has been implicated as a possible mechanism for the protein-protein aggregation in cataractogenesis. Previously we have found that two species of thiols are bound to proteins: GSH (PSSG) and cysteine (PSSC). In this study we found these molecules are ubiquitous in animal lenses with the highest levels in human, dog and rat, and lowest in monkey. However, the relative amount of PSSG to PSSC is quite different in each animal species. The ratio of PSSG PSSC was 1 10 in rat lens, 4 1 in human and dog lenses and 2 1 in monkey lens. We also studied the effect of aging on the protein-thiol mixed disulfide levels in human donor lenses between 3 months and 88 years. Lens GSH levels were inversely related to age, similar to earlier reports, but PSSC levels increased linearly with age. PSSG levels showed a triphasic pattern with an initial sharp and linear increase from a low content in infants to a highest level at age 20; fell back about 50% to a new steady state level that was maintained for four more decades; finally, above 60 years, the levels in some lenses were two to three-fold higher while some lenses remained at the same low value. PSSC in human lens appeared to concentrate in the nuclear region and in the water insoluble proteins while PSSG was more evenly distributed. Besides the aging effect on the protein-thiol mixed disulfides, oxidative stress also potentiated protein modification in the human lens. As demonstrated by the hydrogen peroxide induced cataract in organ culture, old human lenses showed biochemical and morphological changes similar to those in published rat lens studies under the same conditions except that human lenses were more resistant to oxidation and in addition to PSSG, the PSSC level was also elevated.