|Molecular Vision 2002;
Received 7 June 2002 | Accepted 14 August 2002 | Published 21 August 2002
Effect of long-term dietary manipulation on the aggregation of rat lens crystallins: Role of α-crystallin chaperone function
G. Bhanuprakash Reddy, P. Yadagiri
Reddy, A. Vijayalakshmi, M. Satish Kumar, P. Suryanarayana, B.
National Institute of Nutrition (ICMR), Hyderabad - 500 007, India
Correspondence to: Dr. G. Bhanuprakash Reddy, Senior Research Officer, Biochemistry Division, National Institute of Nutrition, Hyderabad - 500 007, India; FAX: 91-40-7019074; email: firstname.lastname@example.org
Purpose: To investigate the effect of food, protein, and vitamin restriction on the susceptibility of lens crystallins to aggregation and chaperone activity of α-crystallin.
Methods: Thirty day old Wistar/NIN rats were maintained on regular rodent diet (C), 50% food restriction (FR), 75% protein restriction (PR), and 50% vitamin restriction (VR) diet for 20 weeks. At the end, α-, β-, and γ-crystallins were isolated from the lenses of these animals and subjected to in vitro aggregation induced by oxidation, UV irradiation and heat. Aggregation and chaperone activity was assessed by light scattering methods.
Results: Dietary restriction has been shown to extend the mean and maximum life span and retard age-related diseases, including cataract. In this study, we demonstrate that while β- and γ-crystallins isolated from FR and PR groups were less susceptible to in vitro induced aggregation, β- and γ-crystallins from the VR group were more susceptible, compared to controls. α-Crystallin from any of the groups did not shown a considerable amount of aggregation. On the other hand, the chaperone activity of α-crystallin from FR and PR groups was not significantly different from controls. However, α-crystallin from the VR group demonstrated substantially higher chaperone activity than controls.
Conclusions: These results indicate that while food and protein restriction appear to lower the susceptibility of β- and γ-crystallins towards aggregation, vitamin restriction tends to increase the aggregation. Chaperone activity of α-crystallin is affected (improved) by only vitamin restriction.
Cataract, the opacification of the eye lens, is the leading cause of blindness worldwide . Age-related or senile cataract is the most common type of cataract and contributes to the largest number of blind population [1,2]. The age-adjusted prevalence of cataract in India is three times that of the United States . Though the etiology of cataract is not fully understood, the major risk factors include nutritional deficiencies/inadequacies, diabetes, exposure to sun ultraviolet (UV) light, environmental toxicants, heavy smoking, drugs, and possibly severe diarrhea [2,3]. There is no single universally accepted pharmacological agent that has emerged to either inhibit or reverse the progression of cataract. However, a delay of 10 years in the onset of cataract by any means would be expected to halve the number of cataract extractions .
It has been well demonstrated that overall nutrition can affect the outcome of various disease states, including cataract [4-8]. Long-term restriction of calorie intake extends the mean and maximum life span of a variety of species [7,8] and is associated with retardation of many age-related debilities [4-8]. Both oxidative lesions in DNA and oxidatively damaged proteins have been shown to accumulate during aging [9,10]. Beneficial effects of dietary restriction have been hypothesized to be through prolonged maintenance of cellular homeostasis by limiting oxidative damage and preserving antioxidant defense mechanisms during aging [11,12].
About 35% of the wet weight (95% of dry weight) of the lens is made up of proteins, of which 90% is in soluble form and constituted by the organ specific crystallins, α-, β-, and γ-crystallins [2,13]. The short ordered arrangement of crystallins provides the required refractive index for lens transparency . These proteins remain in situ for the lifetime of the organism and are subject to extensive damage such as tryptophan oxidation, increased protein-bound browning compounds, fluorophores and protein cross-links . A fraction of these cross-links represent oxidatively produced disulfide bonds but the majority are non-disulfide, covalent cross-links [15,16]. Although there is a battery of other posttranslational modifications, accumulation of large amounts of insoluble protein derived from the otherwise soluble protein due to aggregation is the major biochemical mechanism in cataractogenesis [2,13]. In recent years, one of the major crystallins, α-crystallin, has been shown to function like a molecular chaperone by preventing the aggregation of other proteins, including β- and γ-crystallins, under various stress conditions [17-21]. However, a decreased chaperone-like activity of α-crystallin during aging has been demonstrated [22,23]. This molecular chaperone function of α-crystallin may be instrumental in the maintenance of lens transparency as shown by α-crystallin knock out mouse studies [24,25].
Earlier studies have clearly demonstrated the beneficial effects of dietary calorie restriction in slowing cataract formation in rodents [6,7,26]. It is believed that postponement of cataractogenesis by dietary restriction is in part because of improved antioxidant status and/or enhanced protein editing. Lens epithelial cells derived from calorie restricted mice were more resistant to H2O2-induced cellular damage than those of adlibitum fed mice . Loss of γ-crystallin, supposedly a marker for cataractogenesis, was shown to be ameliorated by calorie restriction in the mouse lens . Furthermore, altered eye lens crystallin distribution, particularly the high molecular weight fraction, was observed in undernourished cataract subjects compared to well nourished cataract subjects, indicating an earlier and/ or faster insolubilization of lens proteins in undernourished cataract subjects [29,30]. Most of the studies focussed on the ultimate change in the lens, such as protein insolubilization/precipitation in cataract and its modulation by various factors, including diet restriction.
In this study, we have investigated how long-term diet (food, protein and vitamin) restriction influences the aggregation pattern of crystallins vis a vis α-crystallin chaperone activity in the clear normal rat lens in relation to cataractogenesis. While the susceptibility of lens crystallins to in vitro induced aggregation was less in food and protein restriction groups, it was more in the vitamin restricted group compared to controls. Chaperone activity of α-crystallin was not affected substantially by food and protein restriction but was improved by vitamin restriction.
Animals and diet
Thirty day old male WNIN rats having an average body weight of 60-80 g (obtained from the National Center for Laboratory Animal Sciences, National Institute of Nutrition) were randomized into four groups each with eight animals. The Control (C) group had free access to water and normal stock diet based on AIN-93 formula . The Food Restriction (FR) group was fed with the above diet but with 50% restricted intake to that of C group. The Protein Restriction (PR) group was fed with the above diet but with 75% restriction of protein (casein is limited to 7% in the diet instead of 28% and the rest of the calories are adjusted with starch). The Vitamin Restriction (VR) group was fed with the above diet but with 50% restriction of vitamins, in which all the vitamins were restricted (0.5% vitamin mixture was added to the diet instead of 1%). Diet composition of each group is given in Table 1. Animal care and protocols were in accordance with and approved by the Institutional Animal Ethics Committee. Animals were housed in individual cages in a temperature and humidity controlled room having a 12 h light/dark cycle. Animals were fed for 20 weeks. Food intake (daily), body weights (weekly), hemoglobin and serum proteins (monthly) were monitored. Eyes were examined once per month with slit lamp microscope (Kowa Portable, Japan) and were found to be clear and free from any ocular abnormalities.
Collection of lenses
Animals were killed at the end of 20 weeks after overnight fasting and eyes were removed. Lenses were dissected from the eyes by the posterior approach.
Isolation and purification of lens crystallins
Lenses from 2-3 animals were pooled and 10% homogenates were prepared in homogenization buffer (25 mM Tris-Cl, pH 8.0 containing 0.5 mM EDTA and 100 mM NaCl). The homogenate was centrifuged at 10,000 x g for 30 min at 4 °C. The supernatant, referred to as the soluble fraction, was applied onto a Sephacryl S-300 HR (Pharmacia Biotech, Uppasala, Sweden) column equilibrated with the homogenization buffer. Peaks corresponding to αH-, αL-, βH-, βL-, and γ-crystallins (Figure 1) were pooled and dialysed extensively against the buffer. To avoid possible contamination of individual crystallins with other crystallins, 2-3 fractions between the peaks were discarded. Proteins were concentrated by ultrafiltration using 10 K filters (Millipore Pvt., Ltd., Bangalore, India).
Oxidation-induced aggregation of crystallins
Oxidation of crystallins was performed at 37 °C with an ascorbate-FeCl3-EDTA-H2O2 system according to previously described methods . Briefly, 0.5 mg/ml protein samples in 0.2 M phosphate buffer, pH 7.2 containing 1 mM ascorbate, 0.2 mM FeCl3, 0.6 mM EDTA and 1 mM H2O2 in a final volume of 1 ml were incubated for 24 h. Light scattering due to aggregation was measured at 400 nm in Cary 100 Spectrophotometer.
UV-induced aggregation of crystallins
The crystallin protein samples (0.5 mg/ml α-, 1 mg/ml β-, 0.3 mg/ml γ-crystallin) in 50 mM phosphate buffer, pH 7.2 were irradiated at 300 nm in a quartz cuvette in a final volume of 2 ml. The protein samples were subjected to constant and very gentle stirring during irradiation. The light source and irradiating conditions were described previously . Light scattering due to aggregation was monitored as described above.
Heat-induced aggregation of crystallins
Purified crystallins (0.3 mg/ml) or total soluble fractions in 50 mM phosphate buffer, pH 7.2 were heated at 65 °C using a circulating water bath . Light scattering due to aggregation was monitored as described above.
Chaperone activity of α-crystallin
The chaperone activity of α-crystallin was probed by measuring its ability to prevent the aggregation of substrate proteins denatured by reduction of disulfide bonds (insulin) or heat (βL-crystallin) [17,21]. Briefly, 0.4 mg/ml insulin in 50 mM phosphate buffer, pH 7.4 in the absence or presence of 0.5 mg/ml of α-crystallin was reduced with 20 mM DTT. Similarly, 0.3 mg/ml of βL-crystallin in 50 mM phosphate buffer in the absence or presence of 0.3 mg/ml α-crystallin was heated at 65 °C for 50 min. The aggregation of substrate proteins upon denaturation was monitored as described above.
Protein was estimated by the method of Lowry, using BSA as standard.
Statistical significance between control and each test group was assessed by the Student t test and p<0.05 was considered as significant.
While the body weights of FR and PR animals showed a significant decrease at all time points, VR group did not show any change in the body weight compared to the C group (Figure 2).
Lens and protein content
Lenses of all the animals were clear and transparent during the course of the experiment and at the end of the study as monitored by slit-lamp biomicroscopy. Further, there was no difference in lens weight and protein (total, soluble and insoluble) content between the groups as estimated by the Lowry method (Table 2). This was further confirmed by the bicinoconic acid method (BCA). The soluble proteins were separated and analyzed further for aggregation and chaperone properties.
Lens soluble fraction was resolved into αH-, αL-, βH-, βL-, and γ-crystallin peaks clearly on Sephacryl S-300 gel filtration column (Figure 1). The distribution of crystallins was further analysed by HPLC using TSK G-4000 SW and there was no difference in protein profile and percentage distribution of different crystallins between the groups (not shown).
Aggregation of crystallins
The implicit assumption is that the turbidity of proteins which develop in the test tube due to a variety of treatments such as oxidation, UV irradiation, elevated temperatures and glycation in a few minutes to few hours, represents an accelerated version of the situation which might arise in the lenses over greater periods of time due to the same insults. Therefore, susceptibility of different crystallins to aggregation induced by in vitro oxidation, UV irradiation and heat was studied by light scattering methods. The extent of aggregation between the groups was compared by using same amount of protein from all the groups for a given protein under the chosen aggregation treatment.
Oxidation of crystallins
As shown in Figure 3, the aggregation of βL-crystallin upon incubation with oxidation reaction mixture for 24 h is significantly less in FR and PR groups (68% and 58% respectively) compared to the C group. Aggregation of βL-crystallin isolated from the VR group, however, is markedly higher (132%) than the C group. The extent of aggregation of βH- and γ-crystallins due to oxidation was slightly lower than βL-crystallin and the aggregation trend was similar to that of βL-crystallin (data not shown). However, no measurable aggregation was found with αH- and αL-crystallins by oxidation (not shown), which is in agreement with earlier studies .
UVB irradiation of crystallin
Different crystallins were irradiated at 300 nm for 5 h and turbidity due to aggregation was measured at 400 nm. Figure 4 shows the aggregation pattern of γ-crystallin upon irradiation. UVB-induced aggregation of γ-crystallin isolated from the FR and PR groups was marginally less than that of the C group, while γ-crystallin aggregation was slightly higher in the VR group compared to the C group. Nevertheless, βH- and βL-crystallins showed similar aggregation patterns (less in FR and PR groups and high in VR group compared to C group) upon UVB irradiation, except that the duration of irradiation time required for observable aggregation was higher (8-10 h, data not shown). No light scattering due to irradiation was noticed with αH- and αL-crystallin .
Heat-induced aggregation of crystallin
βL-Crystallin isolated from the FR and PR groups showed significantly less aggregation at 65 °C compared to the C group, whereas it was substantially more in the VR group (Figure 5A). Very similar observations were found with βH-crystallin (not shown). In the case of γ-crystallin, compared to the C group, aggregation was less in all groups (Figure 5B). Nonetheless, aggregation of γ-crystallin was higher in the VR over the FR and PR groups.
Chaperone activity of α-crystallin
In view of the suggested physiological role of α-crystallin in maintaining lens transparency, it is pertinent to determine the effect of dietary restriction on α-crystallin chaperone activity in relation to the aggregation of other proteins. The chaperone activity of α-crystallin was studied by assessing its ability to prevent the aggregation of substrate proteins denatured by either reducing agent or heat.
DTT-induced aggregation of insulin
Reduction of disulfide bonds connecting insulin A and B chains leads to the unfolding and aggregation of the B chain. This aggregation can be suppressed by α-crystallin, which binds to a non-native conformer of the B-chain . Figure 6A shows the relative chaperone activity of α-crystallin isolated from the C, FR, PR, and VR groups. Food and protein restriction for 20 weeks did not affect the chaperone activity under native conditions with insulin as substrate. Interestingly, vitamin restriction appears to have improved the α-crystallin chaperone activity (Figure 6A). An unrelated protein, lysozyme, did not slow down the aggregation of the insulin B-chain indicating the specificity of α-crystallin in suppressing insulin aggregation (Figure 6A).
Heat-induced aggregation of βL- and γ-crystallin
We have assessed the chaperone activity of α-crystallin against heat-induced aggregation of βL- and γ-crystallins (isolated from normal stock rats) as substrates. The chaperone activity of α-crystallin isolated from the FR and PR groups showed a marginally decreased chaperone activity compared to the C group against heat-induced aggregation of βL-crystallin (Figure 6B). However, the improved chaperone function of α-crystallin due to vitamin restriction is further confirmed in the βL-crystallin heat-induced aggregation assay (Figure 6B). Similar results were observed with heat-induced aggregation of γ-crystallin (not shown). Lysozyme was used as a positive control and it did not minimize heat-induced aggregation of βL-crystallin (Figure 6B).
Previous work has shown that dietary restriction (21-40% restriction of calories) delays cataract progression in both mid-age and old-age Emory mice [6,7,26]. It has been suggested that cataracts are due in part to the combined effects of prolonged oxidative stress, age-related decrements in antioxidant capabilities and decrements in proteolytic functions [26,35] and calorie restriction is believed to limit the oxidative stress and/or improve the proteolytic scavenging of damaged proteins [6,7,26,35]. However, the status of the crystallins prone to aggregation due to dietary restriction vis a vis chaperone function is not known. Secondly, calorie restriction studies in general have been conducted by feeding restricted amounts of the same diet given to ad libitum controls. Nevertheless, restricted animals receive proportionately less protein as well as all other dietary constituents. Although beneficial effects of vitamin restriction in general have not been reported, protein restriction has been shown to produce beneficial effects with regard to retardation of age-related debilities [6,9]. Therefore, we have investigated the effect of not only calorie (50%) restriction but also protein (75%) and vitamin (50%) restriction on aggregation and chaperone activity of crystallins.
As a general tendency the aggregation of β- and γ-crystallins due to oxidation, UVB irradiation and heat was less in food and protein restricted groups. This may not be surprising, as dietary restriction has been shown to limit the oxidative damage and also aid to repair the oxidatively damaged macromolecules, including proteins [4,9,11,12]. Although we have not determined the effect of diet restriction on lens protein modifications due to tissue limitation, it has been demonstrated earlier in other sources that protein or calorie restriction reduced accumulation of oxidatively damaged proteins (protein carbonyls) during oxidative stress of chronic irradiation . On the other hand, vitamin restriction has resulted in increased susceptibility of β- and γ-crystallins in all the in vitro induced aggregation assays (with exception of γ-crystallin in the heat-induced assay). It has not been demonstrated earlier as to how vitamin restriction affects lens protein aggregation patterns. Most of the water-soluble vitamins are involved in antioxidant defense system either as cofactors of the enzymes or directly as free radical scavengers. In addition, age-related decrements in the antioxidant system along with restricted supply of micronutrients can lead to increased oxidative damage to lens proteins, thereby rendering them more susceptible to aggregation. Moreover, previously we found a protective effect for antioxidant vitamins against UV-induced damage to lens macromolecules in vitro, including protein aggregation . Therefore, results of the present study suggest that unlike food and protein restriction, vitamin restriction may not be beneficial (rather deleterious) as far as lens protein aggregation is considered. Although we have not determined the extent of individual vitamin deficiency (inadequacy) created by 50% vitamin restriction, a definite inadequacy is very much possible, which is a likely situation in underdeveloped and as well as developing countries due to malnutrition. Our previous studies found increased protein modifications possibly due to increased oxidative stress and inadequate antioxidant micronutrients during lens browning and cataractogenesis [37-39].
It has been proposed that α-crystallin, one of the three major crystallins, acts as a chaperone in vivo to maintain lens clarity and that during aging α-crystallin loses this ability [17,19,22-24]. Cataract is associated with conformational changes and unfolding of proteins in the lens, which can arise directly as a result of posttranslational modifications [16,40]. Therefore, it is of considerable importance to investigate the effect of dietary restriction on α-crystallin chaperone activity.
Food and protein restriction for 20 weeks seems to have no effect on α-crystallin chaperone activity as assessed by its ability to suppress DTT-induced aggregation of insulin B-chain (Figure 6A). However, α-crystallin isolated from vitamin restricted animals has improved chaperone ability against insulin aggregation. The chaperone function of α-crystallin in preventing the aggregation of β- and γ-crystallins, which are present in abundant amounts in the lens, is obviously relevant in terms of lens transparency. Thus, we have also assessed the chaperone activity of α-crystallin against heat-induced aggregation of β- and γ-crystallin. While the α-crystallin of FR and PR groups has marginally reduced chaperone activity, α-crystallin from the VR group has increased chaperone activity compared to the C group against heat-induced aggregation of βL-crystallin (Figure 6B). Similar results were found in the heat-induced aggregation assay of γ-crystallin (not shown). However, the mechanism for this improved chaperone activity of α-crystallin by vitamin restriction is not known at present and requires further investigation. However, preliminary data indicate that increased chaperone activity due to vitamin restriction is not related to altered protein oxidation status (as a measure of protein carbonly and sulfydryl groups, unpublished data).
A number of studies have demonstrated the anti-aggregative role of α-crystallin using isolated proteins in solutions, making it unclear whether the same process occurs in the intact lens. To understand this question in the present study, we have investigated the stabilizing effect of α-crystallin in lens soluble homogenates, which is a complement of all the three crystallins. Lens total soluble protein fraction was heated at 65 °C for different lengths of time and light scattering was recorded. As expected, regardless of components of restriction, total soluble proteins showed a minimal light scattering (<0.35 OD at 400 nm) after heating for 60 min at 65 °C (Figure 7), compared to the heat-induced aggregation of either β-crystallin or γ-crystallin alone (Figure 5A,B). Compared to the C group, aggregation of total crystallins was lower in FR, PR and more in VR groups. The observation of lowered aggregation of total soluble proteins in FR and PR could be due two reasons; (i) food and protein restriction has not affected α-crystallin considerably (Figure 6) and, (ii) as such the aggregation of β/γ-crystallins was less in the FR and PR groups (Figure 3, Figure 4, and Figure 5). In isolation, the aggregation of β/γ-crystallins was more in the VR group, though markedly higher was the chaperone activity of α-crystallin in this group. Therefore, the enhanced stabilizing effect of α-crystallin (to compensate for the increased aggregation of β/γ-crystallins) could be the possible reason for the decreased aggregation of total soluble proteins in the VR group (Figure 7). However, the possible interference of small molecules, present in the soluble homogenate, on the aggregation of soluble homogenate cannot be completely ruled out.
Although, diet restriction has been shown to delay the onset or progression of cataracts [7,8,26], it did not affect either the biochemical parameters manifested in the protein profile or protein solubility measurements in clear lenses . In addition, animals fed different diets but with cataracts of comparable grades have very similar protein profile . Therefore, we deliberately did not prolong the study for a longer duration to observe cataract (beyond 12-15 months), but for a sufficient time (20 weeks) to observe the manifestation of respective diets in clear lenses. Epidemiological and experimental studies suggest that micronutrients including antioxidants play an important role in delaying and preventing cataract progression [38,39,41-43]. Recently we have shown the protective effect of dietary antioxidants against UVB-induced damage to the lens in vitro [44,45]. In this context, the subtle but very definite alterations in different crystallin fractions due to diet (food, protein and vitamin) restriction with respect to their aggregation and chaperone activity, despite no gross changes in protein profiles acquire significance.
We thank Dr. K.M. Nair and Mr. B. Sreedhar for the useful discussion and critical comments. MSK acknowledges Council of Scientific and Industrial Research for providing him research fellowship.
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