Differences in α-Crystallin isomerization reveal the activity of protein isoaspartyl methyltransferase (PIMT) in the nucleus and cortex of human lenses.

Although it is well-known that protein turnover essentially stops in mature lens fiber cells, mapping out the ensuing protein degradation and its effects on lens function over time remains challenging. In particular, isomerization is a common, spontaneous post-translational modification that occurs over long timescales and generates products invisible to most analytical methods. Nevertheless, isomerization can significantly impact protein structure, function, and solubility, which are all necessary to maintain clarity and proper refractive index within the lens. Herein, we examine the degree of isomerization occurring in crystallin proteins in the human eye lens as a function of both age and location within the lens. A novel mass spectrometric technique leveraging radical chemistry enables detailed characterization of proteins extracted from the cortex and nucleus of the lens. It is observed that the degree of isomerization increases significantly between the cortex and nucleus and between water-soluble and water-insoluble fractions. Interestingly, the abundance of L-isoAsp is low in the water-soluble cortex despite being the dominant product generated by isomerization of Asp in vitro, suggesting that Protein L-isoaspartyl methyltransferase (PIMT) is active in the cortex and suppresses the accumulation of L-isoAsp. The abundance of L-isoAsp increases dramatically in the nucleus, revealing that PIMT activity decreases over time in the center of the lens. In addition, the growth of L-isoAsp in the nuclear fraction suggests protein isomerization continues within the nucleus, despite the fact that most of the protein within the nucleus has become insoluble. Additionally, it is demonstrated that sequential Asp residues lead to isomerization hotspots in human crystallin proteins and that the isomerization profiles for αA and αB crystallin are notably different. Although αA is more prone to isomerization, αB loses solubility more rapidly upon modification. These differences are likely related to the distribution of Asp residues within αA and αB, which are in turn connected to refractive index. The high Asp content of αA is a hazard in terms of isomerization and aging, but it serves to enhance the refractive index of αA relative to αB, and may explain why αA is only found in the eye.

The LEGSKO mouse: a mouse model of age-related nuclear cataract based on genetic suppression of lens glutathione synthesis.

Age-related nuclear cataracts are associated with progressive post-synthetic modifications of crystallins from various physical chemical and metabolic insults, of which oxidative stress is a major factor. The latter is normally suppressed by high concentrations of glutathione (GSH), which however are very low in the nucleus of the old lens. Here we generated a mouse model of oxidant stress by knocking out glutathione synthesis in the mouse in the hope of recapitulating some of the changes observed in human age-related nuclear cataract (ARNC). A floxed Gclc mouse was generated and crossed with a transgenic mouse expressing Cre in the lens to generate the LEGSKO mouse in which de novo GSH synthesis was completely abolished in the lens. Lens GSH levels were reduced up to 60% in homozygous LEGSKO mice, and a decreasing GSH gradient was noticed from cortical to nuclear region at 4 months of age. Oxidation of crystallin methionine and sulfhydryls into sulfoxides was dramatically increased, but methylglyoxal hydroimidazolones levels that are GSH/glyoxalase dependent were surprisingly normal. Homozygous LEGSKO mice developed nuclear opacities starting at 4 months that progressed into severe nuclear cataract by 9 months. We conclude that the LEGSKO mouse lens mimics several features of human ARNC and is thus expected to be a useful model for the development of anti-cataract agents.

Matrix metalloproteinase-9 activity in human lens epithelial cells of cortical, posterior subcapsular, and nuclear cataracts.

PURPOSE: To evaluate the level of matrix metalloproteinase-9 (MMP-9) activity in lens epithelial cells (LECs) derived from different types of cataract in patients having phacoemulsification. SETTING: Iladevi Cataract & IOL Research Centre, Memnagar, Ahmedabad, India. METHODS: This observational study of 275 patients having phacoemulsification was performed to evaluate the level of MMP-9 activity in LECs. All anterior lens capsules harboring the LECs and derived from the surgical curvilinear capsulorhexis were obtained during phacoemulsification. The anterior lens capsule samples were processed to analyze MMP-9 activity using a succinylated gelatin assay. The samples were grouped based on age and on pure cataract type. RESULTS: The level of MMP-9 activity in LECs was highest in eyes with cortical cataract. A significant difference in the level of MMP-9 activity was found in different types of cataract (P<.001). The highest level of MMP-9 activity was in patients older than 60 years. The MMP-9 activity increased gradually with age irrespective of cataract type (P<.001). CONCLUSIONS: A significant difference was observed in the level of MMP-9 activity between different types of cataract. The activity of MMP-9 was highest in eyes with cortical cataract. The level of MMP-9 activity increased with age in the LECs of patients with age-related cataract.

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

PURPOSE: To investigate whether mammalian thioredoxin (Trx) and thioredoxin reductase (TrxR), with or without alpha-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 degrees C was measured spectrophotometrically. In addition, the effect of a combination of Trx/TrxR and bovine lens alpha-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, alpha(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 alpha-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.

Proteasome activity in human lens nuclei and correlation with age, gender and severity of cataract.

PURPOSE: The aim of this study was to measure proteasome activity in human lens nuclei resulting from cataract surgery and in different regions of donor lenses. METHODS: The chymotrypsin-like, the trypsin-like and the peptidylglutamyl-peptide hydrolysing activities of the proteasome were studied using synthetic flourogenic substrates. RESULTS: Proteasome activity did not show any correlation with age of the patients or with gender. Increased opacification of the lens nucleus, as estimated prior to surgery using a 4-grade scale, was significantly correlated with decreased activity of all peptidase activities in the insoluble fraction. In the donor lenses, all peptidase activities were highest in the epithelium and decreased rapidly towards the nucleus. CONCLUSIONS: The present study demonstrates that proteasome activity is preserved in the nucleus of lenses from elderly individuals, although a decrease can be seen with cataract formation. This finding may be of importance for elucidating the mechanism behind the formation of nuclear cataract.

Glutathione peroxidase-1 deficiency leads to increased nuclear light scattering, membrane damage, and cataract formation in gene-knockout mice.

PURPOSE: Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals. METHODS: In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times. RESULTS: Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months. CONCLUSIONS: The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.

Xanthurenic acid derivative formation in the lens is responsible for senile cataract in humans.

BACKGROUND: The tryptophan degradation pathway leads to NAD production via 3-hydroxykynurenine. Kynurenine aminotransferase (KAT) transforms 3-hydroxykynurenine into xanthurenic acid. In this study, we measured the activity of KAT in human lenses and studied the consequences of xanthurenic acid formation METHODS: KAT activity was determined by the method of Tobes. Fluorescence spectroscopy and SDS-PAGE were used for the protein studies. Thin-layer chromatography and infrared and fast atom bombardment spectrometry were used for substance characterization. RESULTS: The KAT activity was detected in senile cataratous lenses, but was absent in the young lenses. Xanthurenic acid at physiological pH exists in equilibrium with its tautomeric form reported by us as oxo-xanthurenic acid (OXA), which is oxidized to di-oxoxanthurenic acid (DOXA), a naphthoquinone-like substance. The incubation of DOXA with crystallins in a solution of physiological pH led to crystallin crosslinking and formation of conjugates with glutathione. CONCLUSIONS: Xanthurenic acid is formed in human lenses. Its tautomerization and oxidation leads to a naphthoquinone-like substance, DOXA. DOXA provoked formation of conjugates with glutathione and crosslinking of crystallins. Thus, KAT activity seems to be the initial event in senile cataract formation in humans.

Ferricyanide reductase activity in cataractous human lens.

We assayed ferricyanide reductase activity (one of NADH-dependent diaphorase activities) in the soluble and insoluble fractions of cataractous human lenses. Activity of this reductase in both the soluble and insoluble fractions tended to decrease in order of cortex > nucleus periphery > nucleus center, and it was suggested that a decrease of the reductase activity is closely correlated with lens protein aggregation, and to some extent associated with the development of nuclear sclerosis (coloration) and cortical cataract. Furthermore, insoluble fraction had very high specific activity per mg insoluble protein in cortex, and the activity decreased sharply with an increase in the level of insoluble protein. The reductase activity in the insoluble fraction may be also related to the metabolic activity of plasma membranes.

Distribution of lens sodium-potassium-adenosine triphosphatase.

PURPOSE: The specific activity of sodium-potassium-adenosine triphosphatase (Na-K-ATPase) in lens fiber cells is lower than the specific activity in lens epithelium. To test whether there is a reduction in the expression of Na-K-ATPase molecules in lens fibers, a Western blot technique was used. METHODS: Membrane material was isolated from different regions of the rabbit lens. Na-K-ATPase (adenosine triphosphate hydrolysis) activity was measured in each membrane sample and Western blots were performed using an antibody to rabbit kidney Na-K-ATPase. RESULTS: By immunoblotting, Na-K-ATPase polypeptide was detected in all lens cells. In contrast, adenosine triphosphate hydrolysis by the Na-K-ATPase (Na-K-ATPase activity) was not detectable or was detectable only at very low levels in fiber membranes from the lens nucleus and in cortex. CONCLUSION: These findings suggest that plasma membrane adenosine triphosphatase enzyme responsible for sodium-potassium transport is expressed in newly formed lens fibers and the transport molecules are retained as the fibers age and are compressed toward the center of the lens. However, with fiber aging there is a loss of functional ability of the Na-K-ATPase to hydrolyze adenosine triphosphate.

Distribution and activity of glutathione-S-transferase in normal human lenses and in cataractous human epithelia.

The distribution of glutathione-S-transferase (GST) activity was determined in frozen normal human lenses. The highest activity of GST was found in the peripheral and equatorial regions, whereas the lowest activity was found in the nucleus. Western blot showed that both mu and pi isoenzymes of GST were present in human lenses. This result is similar to that found in rat lenses. In addition, GST activity was analyzed in 50 lens epithelia which were obtained during cataract surgery. Twenty-seven lens epithelia showed no activity. Statistically significant association was found between cortical and mixed cortical--nuclear cataract and loss of GST activity. No association was found between pure nuclear cataract and loss of epithelial GST activity.

Regional and subcellular distribution of ascorbate free radical reductase activity in the human lens.

This paper reports that the human lens is endowed with a characteristic regional and subcellular distribution of ascorbate free radical (AFR) reductase (EC 1.6.5.4) activity, and it is associated with the soluble fraction of the lens protein. AFR reductase activity in either immature senile cataractous or transparent lens was higher in the cortex, where the level of insoluble protein is lower, than in the nucleus. The high reductase activity and low insoluble protein content of the cortex were comparable between the assayed cataractous and transparent lenses. In the nucleus, in contrast, the reductase activity tended to decrease with an increase in the level of insoluble protein and with the development of nuclear coloration (sclerosis). As for the subcellular distribution of AFR reductase activity, 70-90% of the enzyme activity in the lens was located in the cytosol fraction. Based on the above results, it was proposed that cytosolic free radicals may be involved in oxidation, coloration and aggregation of lens protein in senile cataractogenesis and in lens aging.

Age-related changes in calpain II and calpastatin in rat lens.

The purpose of these experiments was to determine how the activity and regulation of calpain in rat lens changed during aging. Calpain II enzyme activity and immunoreactivity decreased with both chronological and anatomical age. Two pieces of data suggested that loss of soluble calpain II was a result of both autolysis and insolubilization during aging: (i) proteolytic fragments of calpain were detected in lenses with molecular weights similar to fragments produced during incubation of purified calpain II with calcium; (ii) the water-insoluble fraction of lens cortex contained increasing amounts of calpain antigen during aging both the 75-kDa calpain subunit and a unique high-molecular-weight immunoreactive protein. The regulation of calpain II also appeared to change with age. The activity of calpain II in vivo may be regulated by the relative concentrations of calpain II and its endogenous inhibitor calpastatin. Calpain II concentrations decreased in the rat lens with age, whereas levels of the endogenous inhibitor calpastatin were maintained. Assays of calpain II and calpastatin indicated that upon aging there was insufficient activity of calpain II to overcome the inhibition of calpastatin in the nucleus. These findings were confirmed by incubation of crude lens homogenates of 2-week- and 7-month-old rat lens regions with calcium. It is hypothesized that binding of calpain II to membrane may be important for calpain II activation, especially in older lens regions, because it may allow escape from the inhibitory action of calpastatin.

The distribution of aldose reductase and aldehyde reductase II in different regions of bovine lens.

The distribution of aldose reductase and aldehyde reductase II in the epithelium, cortex and nuclear regions of the bovine lens has been studied. The levels of the two enzymes in different regions of the bovine lens were determined after partial purification by DEAE-cellulose (DE-52) column chromatography. Aldose reductase was present in all the three regions of the lens, whereas aldehyde reductase II was present mainly in the epithelium and cortex. The activity of the enzymes, expressed per mg protein, was 10-15 fold higher in lens epithelium as compared to cortex and when expressed per g tissue wet weight, was approximately 2 fold higher. Substrate specificity of aldose reductase purified from all three regions of the lens was comparable, but the susceptibility to inhibition by various aldose reductase inhibitors was significantly different. As compared to the enzyme of cortex and nucleus, the epithelial aldose reductase was less (30-40%) susceptible to inhibition by aldose reductase inhibitors such as sorbinil, tolrestat, statil and tetramethylene glutaric acid. The substrate specificity and characteristics of inhibition of aldehyde reductase II purified from epithelium and cortex were similar.

Localization of denatured enzyme molecules in rat lenses.

Immunohistochemical localization of altered enzyme molecules was detected by the use of antibodies to denatured enzymes (ADE) conjugated with fluorescein. Denatured aldolase, glucose 6-phosphate dehydrogenase and superoxide dismutase are mostly located in the subcortical region and in the nucleus of the rat lens. In the nuclear fibres the enzyme is located near the membrane of the fibres. This study provides additional evidence that altered enzyme molecules accumulate in the lens, and indicates their exact localization. ADE antibody can distinguish between inactive enzyme molecules and active ones, using immunohistochemical techniques.

Acid hydrolases in the bovine lens epithelium.

Acid hydrolases (acid phophatase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, alpha-L-fucosidase, and beta-D-glucuronidase) in the bovine lens epithelium were studied biochemically. p-Nitrophenyl derivatives were used as substrate. All enzymatic activity was found to be much higher in the epithelium than in the cortex and nucleus. The properties of acid phosphatase, N-acetyl-beta-D-glucosaminidase, and alpha-D-mannosidase were also studied, yielding Km values of 0.28, 0.95, and 0.53 mM, respectively. The optimal pH of these enzymes was acidic. Among the subcellular fractions, both acid phosphatase and N-acetyl-beta-D-glucosaminidase had the highest enzymatic activities in the 20,000 g precipitate fraction, while alpha-D-mannosidase showed no difference in activity among the subcellular fractions, suggesting that alpha-D-mannosidase in the bovine lens epithelium is nonlysosomal.

G6PD molecules devoid of catalytic activity are present in the nucleus of the rat lens.

The specific activity of glucoso-6-phosphate dehydrogenase (G6PD) in the rat lens declines as a function of animal age. G6PD activity could be found only in the cortex of the lens. Previous work showed that antibodies against the 'native' form of G6PD could not recognize any cross-reacting material (CRM) in the lens. It was speculated that this could be due either to disappearance of G6PD from the nucleus by degradation or by alterations of the molecules which resulted in the loss of their antigenic determinants. In order to investigate this question, an antibody was prepared against denatured G6PD. This antibody did not interact with 'native' (active) forms of the enzyme; it was shown, however, to recognize antigenically cross-reactive but catalytically inactive intact G6PD molecules in the nucleus of the lens. This finding suggests that altered enzyme molecules are at least partially denatured and accumulate in the lens without being further cleaved proteolytically.

Age-related changes in carbonic anhydrase of toad lens.

Carbonic anhydrase in the toad lens is immunologically identical to the (high activity) enzyme form present in the erythrocyte. As with the erythrocyte carbonic anhydrase, electrofocusing of the freshly-extracted lens enzyme separated three variants with isoelectric points of 6.1, 5.7 and 5.4, all of which exhibited inhibition properties characteristic of the high activity type of carbonic anhydrase. No evidence of the low activity form of the enzyme was found. In contrast to the erythrocyte isoelectric variants, which are stable in vitro, the three variants of lens carbonic anhydrase exhibited progressive anodization in the course of purification and during storage, and could not be isolated in the original form. Acidification of lens carbonic anhydrase appears to be part of the in vivo aging process of the enzyme protein: the still-active enzyme from the oldest lens region--the nucleus--exhibited a considerably lower isoelectric point than the enzyme extracted from the younger, soft fibers of the lens. Although the specific activity of the carbonic anhydrase from the fibers of lens nucleus was considerably lower than the activity of the enzyme from the younger fibers, the observed modification (acidification) of the aged enzyme did not appear to affect substantially its binding properties towards acetazolamide nor the heat lability of the active protein.

The purification and properties of human lens glutathione reductase.

A very simple and rapid method for the purification of human lens glutathione reductase has been developed. The method involves only two steps--affinity chromatography on 2',5'-ADP-Sepharose 4B and gel filtration on Sephacryl S-200. With whole lenses, the purification achieved is over 18000-fold and 80% of the activity in the tissue homogenate is recovered as an enzyme with a specific activity of 218 IU/mg-1. Glutathione reductase was purified from the nucleus and cortex of type I cataractous human lenses and the properties of the two enzymes were compared. No differences could be detected between these enzymes in their specific activities, molecular weights, pH-activity profiles, heat labilities, reactivity towards various substrates and kinetic parameters (Vmax and Km) for glutathione, NADPH2 and NADH2. Therefore, it was concluded that specific alterations in the properties of nuclear glutathione reductase were not responsible for the decreased ability of the lens nucleus to protect itself against oxidative insults. Several different glutathione reductase preparations were examined for their ability to cleave mixed disulphides of lens proteins and glutathione. Only crude tissue extracts (wheat germ and whole lens) were able to cleave the mixed disulphides: no activity was observed with purified lens or yeast glutathione reductases. Therefore, it was concluded that glutathione reductase does not cleave mixed disulphides.

Age-related and distributional changes in the trypsin inhibitor activity of bovine lens.

Age-related changes in the bovine lens trypsin inhibitor activity were measured by assaying water soluble extracts of 10 concentric slices from the periphery to the center of the lens. Inhibitor assays were carried out at pH 7.0 and 7.9 using prenatal, calf and mature lenses. The inhibitor at pH 7.0 remained constant throughout the lens but the pH 7.9 activity decreased sharply in the lens nucleus. This was particularly true for the prenatal and calf lenses. Agarose A-15 m gel filtration of the water soluble inhibitor activity showed a decrease in inhibitor in the alpha-crystallin region and a corresponding increase in inhibitor activity in the HMW protein peak. Inhibitor assays were carried out on the water insoluble fractions following solubilization in 6.0 M-urea. Little or no inhibitor activity was seen in the outer cortical fractions but the inner cortex and nucleus contained high levels of inhibitor activity in the water insoluble fraction with specific activities 7- to 10-fold higher than the comparable crude lens extracts. These data suggest that the lens inhibitor activity at pH 7.0 and 7.9 aggregate into a HMW complex and with time preferentially enter the water insoluble fraction. The distribution of a purified 5.5 K inhibitor protein between the water soluble and the water insoluble fraction was measured. In the periphery all of this inhibitor was in the water soluble fraction, but toward the center of the lens this inhibitor began shifting to the water insoluble fraction. Slices taken from the lens nuclear region showed that all the inhibitor was in the water insoluble fraction as detected by both activity measurements and SDS-PAGE.

Superoxide dismutase, catalase and glutathione peroxidase in the human cataractous lens.

The activities of the protective enzymes, superoxide dismutase, catalase and glutathione peroxidase have been measured in the cortical and nuclear sections of 76 human cataractous lenses as well as in calf, rabbit and rat lenses. No changes was observed in the activity of catalase with the progressive development of cataract. However, a precipitous decrease (70%) in both superoxide dismutase and glutathione peroxidase in the nuclear region of the lens was found at the onset of nuclear cataract. Further decreases accompanied the progression of the cataract and similar, but less marked, decreases were observed in the cortical region of the lens. It is suggested that the inactivation of these enzymes may result in an elevation of the H2O2 and O2.- levels in the lens and that this may be responsible for the oxidative modification of lens proteins observed in nuclear cataracts.