Molecular Vision 2006; 12:1153-1159 <http://www.molvis.org/molvis/v12/a131/> Received 20 April 2006 | Accepted 22 September 2006 | Published 2 October 2006

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Thioredoxin, thioredoxin reductase, and α-crystallin revive inactivated glyceraldehyde 3-phosphate dehydrogenase in human aged and cataract lens extracts

Hong Yan,^1,2 Marjorie F. Lou,³ M. Rohan Fernando,³ John J. Harding²
 
(Hong Yan and John J. Harding and their represented Departments contributed equally to this publication)
 
 

¹Nuffield Laboratory of Ophthalmology, University of Oxford, Walton Street, Oxford, UK; ²Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an, China; ³Department of Veterinary and Biomedical Sciences, University of Nebraska-Lincoln, Lincoln, NE

Correspondence to: Hong Yan, Department of Ophthalmology, Tangdu Hospital, Fourth Military Medical University, Xi'an 710038, China; Phone: 0086 29 84777445; FAX: 0086 29 84777445; email: yhongb@fmmu.edu.cn

Abstract

Purpose: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without α-crystallin can revive inactivated glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in both the cortex and nucleus of human aged clear and cataract lenses.

Methods: The lens cortex (including capsule-epithelium) and the nucleus were separated from human aged clear and cataract lenses (grade II and grade IV) with similar average age. The activity of GAPDH in the water-soluble fraction after incubation with or without Trx or/and TrxR for 60 min at 30 °C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens α-crystallin was investigated.

Results: GAPDH activity was lower in the nucleus of clear lenses than in the cortex, and considerably diminished in the cataractous lenses, particularly in the nucleus of cataract lenses grade IV. Trx and TrxR were able to revive the activity of GAPDH markedly in both the cortex and nucleus of the clear and cataract lenses. The percentage increase of activity in the cortex of the clear lenses was less than that of the nucleus in the presence of Trx and TrxR, whereas it was opposite in the cataract lenses. The revival of activity in both the cortex and nucleus from the cataract lenses grade II was higher than that of the grade IV. Moreover, Trx alone, but not TrxR, efficiently enhanced GAPDH activity. The combination of Trx and TrxR had greater effect than that of either alone. In addition, α_L-crystallin enhanced the activity in the cortex of cataract grade II with Trx and TrxR present. However, it failed to provide a statistically significant increase of activity in the nucleus.

Conclusions: This is the first evidence to show that mammalian Trx and TrxR are able to revive inactivated GAPDH in human aged clear and cataract lenses, and α-crystallin helped this effect. The inactivation of GAPDH during aging and cataract development must be caused in part by disulphide formation and in part by unfolding, and can be recovered by reducing agents and a molecular chaperone.

Introduction

Protein-thiol mixed disulphide formation or protein S-thiolation, a non-specific posttranslational modification of proteins, increases with age and cataract development [1-3]. Numerous studies showed an extensively diminished size of the glutathione (GSH) pool with some protein thiols being S-thiolated by oxidized non-protein thiols to form protein-thiol mixed disulphides, either as protein-S-S-glutathione (PSSG) or protein-S-S-cysteine (PSSC) or protein-S-S-γ-glutamylcysteine in aging lenses or lenses under oxidative stress [2,4]. This protein-thiol mixed disulphide formation could cause inactivation of enzymes in the lenses which contributes to development of cataract.

Thioredoxin (Trx) is a 12 kDa protein molecule with a redox active disulphide-dithiol within the conserved active site sequence (-Cys-Gly-Pro-Cys-). These active site cysteines form a disulphide, which is reduced by the homologous Trx reductase. Trx is a multifunctional protein with roles in antioxidation, growth promotion, neuroprotection, inflammatory modulation, anti-apoptosis, and immune function [4,5]. The presence of the thioredoxin genes of both Trx1 and Trx2 in lens was first reported in the Emory mouse lens [6]. Later the distribution of Trx1 gene expression and its activity was reported in human lens [7,8]. The upregulation of the Trx gene by H₂O₂ stress in human lens epithelial cells suggests that the Trx system may protect the lens against oxidative stress [8]. Trx operates via reduction of Trx-S₂ to Trx-(SH)₂ by NADPH.H⁺, catalyzed by Trx reductase. Due to its dithiol:disulphide exchange activity, Trx regulates the reduction-oxidation state of protein thiols [6,9], as part of the Trx-TrxR system (Figure 1) [5].

Thioredoxin reductase (TrxR), a homodimeric protein containing 1 selenocysteine and 1 FAD per subunit of 55 kDa, catalyses the NADPH-dependent reduction of thioredoxin disulfide and of numerous other oxidized cell constituents [10]. It is necessary therefore to restore Trx to its active state (Figure 1). The mammalian TrxR is important for cell proliferation, antioxidant function, and redox signaling [11], and exists in the human lens in both the cytosol and membrane fractions of the capsule-epithelium, as well as the fibers [4,7]. TrxR in human lens epithelial cells is highly sensitive to inactivation by molecular O₂ [12], and easily upregulated in the lens exposed to oxidative stress [13]. Thus, the Trx system (Trx and TrxR) has been regarded as a main regulator of the intracellular redox environment, governing the redox regulation of several cellular processes. These two enzymes may work synergistically to dethiolate protein-thiol mixed disulphides, reduce protein disulphides, and regulate the redox homeostasis in lens proteins and enzymes.

α-Crystallin, a small heat shock protein and molecular chaperone, prevents the aggregation of partially denatured proteins under various stress conditions and promotes their return to native conformations when favourable conditions pertain. The poorly conserved NH₂-terminal domain influences oligomer construction and chaperone activity, whereas the flexible COOH-terminal extension stabilizes quaternary structure and enhances protein/substrate complex solubility [14]. Diminished chaperone-like activity of α-crystallin due to posttranslational modifications may thus result in the accumulation of aggregated/inactivated proteins [15]. Recent studies have shown that α-crystallin assisted the renaturation of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) [16], reactivation of glucose 6-phosphate dehydrogenase (G6PDH) upon refolding [17], and protected against loss of antigenicity of esterase by glycation and a steroid [18].

Glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a key glycolytic enzyme, oxidizes and subsequently phosphorylates glyceraldehyde 3-phosphate in the glycolytic pathway. Its activity decreased in clear human lenses with aging and in cataract lenses [19,20]. An age dependent increase in the number of acidic species of GAPDH was observed in clear human lenses varying in age from fetal to 73 years [21], and it exhibited progressive heterogeneity with ageing and a shift towards an acidic charge while becoming less stable to increased temperatures [22]. In vitro, GAPDH is susceptible to oxidation [23,24] and other chemical modifications [25]. Furthermore, the activity of H₂O₂-treated GAPDH can be recovered by the Trx and TrxR from E. coli [23], by the recombinant human lens Trx-1 [4] and by the recombinant human lens thioltransferase or by DTT [26,27], suggesting that the reduction of the disulphide bonds caused by oxidative stress in GAPDH may contribute to the recovery of activity.

We have shown previously with whole human lenses of grade II cataract that inactivated GAPDH could be revived by chemical reducing agents [28]. The purpose of this study was to attempt to take further step to revive some activity of GAPDH by the mammalian Trx system and α-crystallin in lens extracts from both the cortex and nucleus from aged clear and the cataract lenses.

Methods

Materials

Unless stated otherwise, all chemicals and enzymes were bought from Sigma-Aldrich Company, Ltd. (Dorset, UK.). Recombinant rat thioredoxin reductase (TrxR) was provided by IMCO CORP. LTD (Stockholm, Sweden). Recombinant human thioredoxin (6.5 mg/ml) was purified as described in Yegorova et al. [8].

Human whole cataract lenses were obtained from the Institute of Ophthalmology, University of Nijmegen, Netherlands with informed consent, and donated by Dr R.M. Broekhuyse. The lenses were extracted via intracapsular cataract extraction procedures and graded after extraction on the basis of nuclear color as described by Pirie [29], then kept at -70 °C in the Netherlands and at -20 °C in the Nuffield Laboratory of Ophthalmology until used. The cataract lenses used in this project were all nuclear and of grade II (n=47) and grade IV (n=31; grade IV cataracts have a strong brown nucleus). The average age of patients at the time of surgery was 64±12 years (40-85 years) for grade II lenses and 69±9 years (48-86 years) for grade IV lenses. The cataract lenses used in our study were all cataractous and graded after extraction on the basis of nuclear color as described by Pirie [29]. They were all cataract lenses removed from patients. Furthermore, the color of the cataract lenses graded by the Pirie system has been correlated to a wide variety of biochemical and physiological changes, and nuclear color and opacity have been correlated. Therefore, the degree of color in cataract lenses may correspond to the lens protein modifications.

The 74 whole clear post-mortem human lenses were obtained from the Bristol Eye Bank with informed consent and kept at -20 °C until used. The average donor age at time of death was 67±11 ranging between 50 and 93 years.

Preparation of human lenses

The cataractous and aged clear lenses were thawed at room temperature. The capsule and epithelium were dissected out together with the cortex and was considered as the cortex portion. Then the nucleus was separated from the cortex with the help of needles. Each portion was pooled and gently homogenized in a hand-held homogeniser over ice with 7 times the volume of double purified water (containing 13 mM EDTA) of its wet weight, to extract the water-soluble fraction of the lens proteins containing enzymes from the cortex and nucleus pools.

The suspension was centrifuged at 22,000x g for 40 min at 4 °C. The supernatant was then freeze-dried. The dried material was dissolved in double purified water to a concentration of 15 mg/ml. These samples were stored at -20 °C before enzyme determination and used for all the experiments described. The time from dissection to freeze-drying was kept as short as possible to avoid artifactual oxidation.

The method used for separation of the cortex and nucleus gives a nuclear weight corresponding to the lens weight at birth, and therefore the nuclear fractions contain cells laid down before birth.

Isolation of bovine lens α_L-crystallin

Bovine α_L-crystallin was separated from lenses by size exclusion gel chromatography on Sephacryl S300HR according to the methods described by Slingsby and Bateman [30]. The purity of the crystallins was determined by SDS-PAGE according to Laemmli's discontinuous buffer system using a Mini-PROTEAN II electrophoresis unit (Bio-Rad, Hemel Hempstead, UK.).

Thioredoxin assay

The Trx assay was based on the method described by Holmgren and Bjornstedt [9]. The activity of Trx was determined by the ability of Trx to reduce insulin with NADPH in the presence of an excess of TrxR. The assay mixture contained 50 mM potassium phosphate, pH 7.0, 1 mM EDTA, 80 μM insulin (0.5 mg/ml), 0.2 mM NADPH and 10 μl (14.8 mU) of recombinant rat TrxR at 25 °C. Trx was added to the assay mixture and the final volume of the assay mixture was 1 ml. The reaction rate was followed from the oxidation of NADPH at 340 nm. Activity was calculated as micromoles of NADPH oxidized per minute from the relation with A340.

GAPDH activity assay

GAPDH activity was measured spectrophotometrically at 30 °C by monitoring the decrease in absorbance of NADH for 1 min at 340 nm [31]. The reaction mixture (3.0 ml) consisted of 2.258 ml of 0.1 M triethanolamine buffer, pH 7.6, 200 μl of 50 mg/ml solution of glycerate-3-P, 100 μl of 20 mg/ml solution of ATP, 100 μl of 10 mg/ml solution of EDTA, 50 μl of 0.1 M magnesium sulphate, 50 μl of 10 mg/ml NADH, 12 μl of 3-phosphoglyceric phosphokinase (EC 2.7.2.3) from Baker's yeast suspension (containing 7 units), and finally, 230 μl of the incubation sample, containing 3 mg lens protein, see below. The enzyme activity was calculated from the linear decrease in absorbance between time 0 and 1 min. By definition, one international unit of GAPDH reduces 1.0 μmol of 3-phosphoglycerate to D-glyceraldehyde 3-phosphate in a coupled system with 3-phosphoglyceric phosphokinase at pH 7.6, in 1 min at 30 °C.

GAPDH activity in human aged clear and cataract lenses

The cataractous and clear lens supernatants (200 μl aliquots containing 3 mg lens protein) were incubated with 25 μl of Trx (12.5 μM) and 5 μl TrxR (7.4 mU) for 1 h at 30 °C, respectively. The GAPDH activity was measured in an order by which one sample from each group (controls, with and without agents) was measured in succession, to ensure that all the samples were treated similarly. All samples underwent the same incubation conditions and testing procedures at the same time.

An additional experiment was conducted to determine whether α-crystallin, previously shown to affect the revival of glutathione reductase activity in human cataract lenses [32], would enhance the activity of GAPDH revived by the combination of Trx and TrxR. A separate experiment was performed using 50 μl of bovine α_L-crystallin solution (2 mg/ml) and a new batch of Trx (25 μl, 6.25 uM) and TrxR (5 μl, 7.4 mU).

Statistical analysis

Unless stated otherwise, all experiments were repeated at least three separate times and differences assessed using the Bonferroni correction for multiple comparisons.

Results

Activity of GAPDH from human aged clear and cataract lenses

The activity of GAPDH in both the cortex and the nucleus from aged clear and the cataract lens extracts (grade II and grade IV) are shown in Figure 2. The activity of GAPDH was considerably greater in the cortex than in the nucleus in the aged clear lens extracts (p<0.001), which is in agreement with previous observations [2]. A remarkably decreased activity was observed in the cataract lenses. However, there was no statistically significant difference between the cortex and the nucleus of lenses of grade II and grade IV. The statistically significant difference in activity was observed between the nucleus of clear lens extracts and cataract lens grade II (p=0.016) and the nucleus of grade IV (p=0.001; Figure 2B,D,F).

Revival of GAPDH by Trx and TrxR in the aged clear lenses

Trx and TrxR without lens did not show GAPDH activity. Different amounts of Trx and TrxR were used to determine the minimum amount of agent to give effective revival of the GAPDH activity (data not shown). In all the remaining experiments 25 μl Trx (12.5 μM) and 5 μl TrxR (7.4 mU) were used.

After a preincubation period of 60 min, the activity of GAPDH in the cortex of the aged clear lenses reached 30.5±0.39 (IU/μg lens protein; p<0.001), 26.4±0.52 (p<0.001), and 4.18±0.14 (p=1.000) compared with 3.88±0.41 in the presence of the combination of Trx/TrxR, Trx, and TrxR, respectively (Figure 3). Although TrxR gave no significant enhancement of activity, Trx alone or in combination with TrxR enhanced GAPDH activity by 580% to 686% (Table 1). A similar pattern was observed in the nucleus of the aged clear lens extract (Figure 3). Statistically significant difference was noted with the combination of Trx and TrxR (p<0.001), Trx alone (p<0.001), but not with TrxR alone (p=1.000). Trx/TrxR gave an approximately 1,180% increase in activity compared to an approximately 427% increase with Trx alone (Table 1). The revival of activity in the nucleus was greater than that of the cortex in the presence of the Trx/TrxR, but not in the presence of Trx alone.

Revival of GAPDH by Trx and TrxR in the cataract lenses

The activity of GAPDH in both the cortical and the nuclear lens extracts of the cataract lenses of grade II and grade IV was revived by Trx/TrxR, Trx, or TrxR (Figure 4). The activity in the cortex of the cataract lenses grade II reached 7.69±0.2 (IU/μg lens protein; p<0.001), 3.6±0.25 (p<0.001), and 0.35±0.02 (p=1.000) compared with 0.18±0.05 (no addition) in the presence of Trx/TrxR, Trx, or TrxR, respectively. This is a much greater percentage enhancement of activity than found for clear lens but starting from the diminished activity found in cataracts. In the nucleus the corresponding activities were 1.88±0.12 (p<0.001), 0.57±0.09 (p=0.001), and 0.21±0.05 (p=1.000) compared with 0.13±0.04 in the lens alone (Figure 4). A similar pattern was observed in the cataract lenses grade IV with some increase in activity by Trx alone but much more with the Trx/TrxR (Figure 5).

The combination of Trx and TrxR dramatically recovered GAPDH activity in the cortex of cataract grade II lens extracts by approximately 4,172% (p<0.001) over the control level, and 1,346% (p=0.001) in the nucleus (Figure 4, Table 2). Trx alone increased activity very significantly but not as much as the combination. The enhancement was seen in the cataract lens extracts of grade IV but was smaller (3,164% in the cortex and 700% in the nucleus; Figure 5, Table 2). All of these results are highly statistically significant. Moreover, the greater increase in activity was obtained in the cortex than in the nucleus, indicating that more disulphide bonds are present in the cortex and exist in a way enabling recovery by these dethiolating agents (Figure 4 and Figure 5, Table 2). The revival of activity was less in the presence of Trx alone, suggesting that the Trx/TrxR system, as NADPH-Trx-TrxR, works together as a team for the disulfide reduction. There was no evidence of a statistically significant revival of activity in the cataract lens extracts by TrxR alone (Figure 3 and Figure 4).

Revival of GAPDH by Trx/TrxR and α_L-crystallin

α_L-Crystallin alone gave no statistically significant increase in GAPDH activity in extracts from either cortex (p=0.644) or nucleus (p=0.100) although some increased activity appeared to be present in the cortex in the presence of α_L-crystallin (Figure 6). A significant enhancement of activity in the cortex of the cataract lens extracts grade II (p=0.001) was observed in the presence of Trx and TrxR with α_L-crystallin, where an approximate 167% of revival was achieved (Figure 6), more than found with Trx and TrxR. The extra increase (approximaterly 73%) in activity was gained with α_L-crystallin (p=0.171). An approximately 106% enhanced activity was observed with the combination of these three proteins in the nucleus of the cataract lenses grade II (p=0.010; Figure 6).

Discussion

The present data are the first evidence that mammalian Trx and TrxR can successfully revive the activity of inactivated GAPDH from human aged and cataract lenses after being inactive for many years. α_L-Crystallin, as a molecular chaperone, enhanced the effect on revival of activity from the cortex, but not from the nucleus of cataract lenses, indicating that GAPDH may be less damaged in the cortex of cataract lenses so it can be refolded with the help of a molecular chaperone.

The GAPDH activity, like other enzymes in the lens, is more active in the epithelium and the cortex of clear lenses. Activity is lower in the cataractous lenses. However, there were no remarkable differences in GAPDH activity between the cortex and the nucleus of cataract lenses (grade II and IV) except for the comparison of the cortex in grade II, and the nucleus in grade IV, suggesting that the browning of the lens nucleus may contribute to the extensively diminished enzyme activity. Earlier studies reported that GAPDH activity was lowered in at least some human cataracts [19,33]. Our data showed that the GAPDH activity was significantly decreased in pooled lenses from cataract groups compared to clear lenses of similar average age. The activity was lower in the nucleus of the aged clear lenses compared with the cortex. The lowest activity was found in the nucleus of grade IV cataract lenses, which have suffered extensive posttranslational modification.

The Trx system (Trx, TrxR, and NADPH) is ubiquitous from Archaea to human [34]. Trx, with a dithiol/disulfide active site (CGPC) is the major cellular protein disulphide reductase. It specifically catalyzes the reduction of inter- or intra-protein/protein disulphides and sulphenic acid formation of cysteine moieties of proteins, which affect the activities of the proteins [4,34]. GAPDH has an oxidation-sensitive SH group at its active site. Revival of activity in cataract lenses by reducing agents suggests that a protein thiolation event may have occurred at the active site, thus inhibiting the catalytic activity of GAPDH [4,28]. Our results show that the dethiolation by Trx recovered the inactive GAPDH in human aged and cataract lenses supporting the notion that inactivation of the enzyme during cataract formation could be caused by disulphide bond formation. The Trx system is more physiological than DTT, which has previously provided the best revival [28].

TrxR was originally purified from bacterial sources and named after the reaction it catalyzed, namely the reduction of the 12 kDa disulphide protein Trx to its dithiol-containing reduced form (Figure 1). Reduced Trx, in turn, provides reducing equivalents for a number of processes. As a general reducing enzyme with little substrate specificity, mammalian TrxR and Trx contribute to redox homeostasis and are involved in prevention, intervention, and repair of damage caused by H₂O₂-based oxidative stress [10]. Thus, TrxR may function effectively with Trx. The present data demonstrated that TrxR alone was not able to revive GAPDH activity in the clear and the cataract lenses except for the cortex from cataract lenses grade II. However, the combination of Trx and TrxR works synergistically.

Interestingly, the lowest percentage of revival activity by the Trx and TrxR system was observed in the nucleus of cataract lenses, but it was opposite in the aged clear lenses (Figure 3-Figure 5). Together with failure of enhancement by α-crystallin (Figure 6), it indicates that extensive modification and unfolding may occur in the nucleus of cataract lenses, the oldest part of the lens where the proteins have been present since before birth. The maximum revival of GAPDH achieves activities in the cataract cortices greater than in the unstimulated clear lenses but not as high in the revived clear lens. It appears that much GAPDH activity is lost even from clear lenses with age but that there is a sufficient reserve of activity to maintain transparency. GAPDH is the most abundant lens enzyme. Activity in the cataract lens cortex is lower but would be recoverable by Trx plus TrxR to viable levels.

Our results showed that bovine lens α-crystallin can enhance approximately 73% of the GAPDH activity in the presence of Trx and TrxR, suggesting that unfolding of GAPDH may be in part responsible for its inactivation in the cataract lenses and α-crystallin is able to assist in refolding GAPDH and recovery of its activity after disulphide reduction. The lower revival of activity by Trx and TrxR in the separate experiment may result from the lower amount of Trx used and perhaps a decreased effect of Trx presented. Although most studies on small heat shock proteins relate to protection against denaturation, it has been shown that α-crystallin can assist the renaturation of GAPDH and G6PDH [16,17], and of glutathione reductase in human cataract lens extracts [32]. Extracts from aged lens nuclei lack low molecular mass α-crystallin but still contain high molecular weight α-crystallin and exhibit chaperone activity albeit diminished compared to lens cortex [35].

Together with our previous studies, the present results provide further evidence to support the conclusion that disulphide bond formation may contribute to cataract formation. An approach to repair the initial oxidative damage to lens proteins could help to delay the opacification of lens.

Acknowledgements

We are grateful to the Wellcome Trust for financial support for Dr. Hong Yan (International Research Development Award, number 070667). The authors thank Mr D. Ji for human lens preparation assistance, Dr. D. Rachdan for bovine lens α-crystallin, and Dr. R.M. Broekhuyse, University of Nijmegen, for the cataract lenses.

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