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INTRODUCTION

Age-related cataracts are the leading cause of visual impairment worldwide.1 At present, surgery is the only therapy for cataracts, and because of its demo-graphics it has been estimated that delaying the

development of cataract by 10 years would decrease the need for cataract surgery by approximately 45%.2 Therefore, there is a need for protective agents that will delay cataract development and obviate sur-gery.

It is widely accepted that oxidation of lens proteins plays a key role in age-related cataract.3 Oxidative stress may induce post-translational modifications (PTMs) of lens crystallins, resulting in conformational changes and aggregation of these proteins and lead-ing to lens opacity and cataract formation.4,5 As one of

Current Eye Research, 35(7), 620–630, 2010Copyright © 2010 Informa Healthcare USA, Inc.ISSN: 0271-3683 print/ 1460-2202 onlineDOI: 10.3109/02713681003768211

ORIGINAL ARTICLE

α-Lipoic Acid Alters Post-Translational Modifications and Protects the Chaperone

Activity of Lens α-Crystallin in Naphthalene-Induced Cataract

Yan Chen1, Lu Yi1, GuoQuan Yan2, YanWen Fang1, YongXiang Jang1, XinHua Wu1, XinWen Zhou2, and LiMing Wei2

1Department of Ophthalmology, Eye and ENT Hospital of Fudan University, Shanghai, China2Department of Chemistry & Research Center of Proteome, Fudan University, Shanghai, China

ABSTRACT

Purpose: To evaluate whether α-lipoic acid (LA) inhibits lens opacity of naphthalene-induced cata-ract by altering post-translational modifications (PTMs) and protecting the chaperone activity of α-crystallins.Methods: Forty-five Sprague-Dawley rats were divided into three groups: control, naphthalene, and naphthalene plus LA. Cataracts were induced by oral administration of 1 g naphthalene/kg body weight/day. Rats in the naphthalene plus LA group were also fed 30 mg LA/day. The develop-ment of naphthalene-initiated cataract was monitored every week by slit lamp microscopy for nine weeks, then the lens proteins were separated by HPLC, and peaks corresponding to α-crystallins were resolved on 2-DE. The spots of 2-DE were subjected to mass spectrometry to identify PTMs. Chaperone activity of α-crystallins was measured by heat-induced aggregation of βL-crystallin.Results: The lenses of rats fed with naphthalene plus LA exhibited less light scattering than that fed with only naphthalene at three weeks after treatment (P < 0.01). C-terminal truncated αA crystallin was detected in naphthalene-induced cataract and was abrogated by LA treatment. Several other post-translational modifications were identified including methylation, phosphorylation, acetyla-tion, carbamylation, and oxidation.Conclusions: Our data are the first to show PTM changes induced by naphthalene in rat lenses. Our findings also indicate that LA can inhibit naphthalene-induced lens opacity by altering PTM and protecting the chaperone activity of α-crystallins.

KEYWORDS: α-Lipoic acid; Cataract; α-Crystallins; Molecular chaperone; Post-translational modifications

Received 05 November 2009; accepted 28 February 2010

Correspondence: Yi Lu, Department of Ophthalmology, Eye and ENT Hospital of Fudan University, 83 Fen Yang Road, Shanghai 200031, China. E-mail: luyi_eent@yahoo.com.cn

05 November 2009

28 February 2010

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the major structural protein components, α-crystallins represent 35% of all crystallins. α-Crystallins can act as a molecular chaperone to protect other crystallins against aggregation, and this activity is thought to play an important role in maintaining lens transparency.6,7 α-Crystallins consist of two subunits, αA and αB, usu-ally present in a molar ratio of αA to αB of three to one.8 Post-translational modifications of α-crystallins may decrease their chaperone activity by varying extents, resulting in increased aggregation of other crystallins and cataract formation8; however, the cor-relation between PTMs and the chaperone activity of α-crystallins has not been fully clarified. With the development of the powerful proteomic technique of mass spectroscopy, it has recently become possible to perform a comprehensive analysis of PTMs of these proteins.

As a novel biological antioxidant, α-lipoic acid (LA) has been reported to prevent cataract develop-ment in rats treated with l-buthionine sulfoximine and streptozotocin.9,10 LA acts not only directly by radical quenching and metal chelation, but also indirectly through the recycling of other antioxidants, such as ascorbic acid, vitamin E, and glutathione.11,12 Moreover, the long-term safety of LA has been verified.13,14

The aim of this study was to evaluate whether addition of LA would inhibit lens opacity by alter-ing PTMs of α-crystallins and protecting its chaper-one activity. We used naphthalene-induced cataract in rats as a model for investigating oxidative insults that are linked to human age-related cataract. The metabolism of naphthalene in laboratory animals has been extensively studied.15–17 After being rapidly absorbed from the gut, naphthalene is oxidized in the liver by cytochrome P-450 to an epoxide that can be conveyed into lens. In lens, naphthalene epoxide was converted enzymically into 1,2-dihydroxynaphthalene. 1,2-Dihydroxynaphthalene is readily autoxidizable in neutral solution to form 1,2-dihydronaphthoquinone, which can catalytically oxidize ascorbate acid, gluta-thione (GSH), and other antioxidative molecules and lead to impairment of antioxidative capacity of lens. Naphthalene cataractogenesis in rats has been used as a valuable animal model to study the etiology of senile cataractogenesis in humans because its toxic mechanism is oxidative stress, which is thought to be analogous to age-related cataract.18,19

MATERIALS AND METHODS

Animals and Induction of Cataracts

Forty-five 6-week-old female Sprague–Dawley rats (average weight 150 g) were purchased from the Chi-

nese Scientific Academy. Rats were maintained at the laboratory animal center of Shanghai Medical Univer-sity. All procedures were carried out in accordance with Chinese legislation on the use and care of labora-tory animals and the ARVO statement for the Use of Animals in Ophthalmic and Vision Research and were approved by the respective university committees for animal experiments. The rats were housed in hanging steel wire cages with free access to natural diet and tap water and kept on a fixed length light/dark cycle. The rats were weighed and randomly assigned to three groups with 15 animals in each group as follows: A, control group; B, naphthalene group; and C, naph-thalene plus LA group. For the induction of cataracts, naphthalene solution (10%) was prepared in warm par-affin oil by heating at 60°C for 30 min. This naphtha-lene solution was administered via gastric irrigation to rats in experimental groups B and C using an 18 gauge lavage needle (1 g/kg body weight/day). The dietary regimen of the three groups was as follows: Group A was kept on a natural diet and given the same volume of paraffin oil only; Group B was kept on a natural diet supplemented with 10% naphthalene; Group C was kept on a natural diet supplemented with 10% naphthalene, and LA (30 mg/rat/day) was mixed with powdered food and given as a daily dietary supple-ment beginning three days after the initial administra-tion of naphthalene.9,18 Treatment was continued for nine weeks.

Lens Gray Scale Value Analytical Procedures

Rat lenses were examined every week by slit lamp microscope after pupillary dilation with tropicamide-phenylephrine ophthalmic solution (Santen Pharma-ceutical Co. Ltd., Shanghai, China). To take a picture of the lens, the rat’s eyes were kept facing straight ahead against the object lens of the slit lamp in a dark room using 10× amplification. The slit light was 2 mm wide with a 45° slope and focused on the quarter boundary of the lens. The light power of the slit lamp was mea-sured using a luminometer to ensure that it was the same value every time. Pictures of lenses were taken and densities were analyzed using lens gray scale value analysis software: The nuclear area was selected with the same size and position as Figure 1A shows, and the gray scale value of the selected area was measured according to the instruction of the software. As for the perinuclear area, four different positions with the same size were selected, and the average gray scale value of the four positions was regarded as the lens perinuclear opacity (Figure 1B). To evaluate the gray scale value of lens, the aqueous humor gray scale value was set as zero reference point, and the lens gray scale value

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was compared with this reference point (YiDe Medical Instrument Co. Ltd., Shanghai, China) (Figure 1).20

Purification of α-Crystallins

After nine weeks of treatment, lenses from all the rats in each group were obtained by excision from a posterior approach immediately after death, and the lens weight was recorded. Each whole lens was homogenized at a ratio of 1 mg of lens wet weight/90 μl buffer I containing 0.05 M phosphate (pH 7.0), 0.15 M NaCl, 1 mM EDTA, and 0.02% NaN3 (The 4th Reagent Factory of Shanghai, Shanghai, China) at 4°C.21,22 The homogenate was centrifuged at 15,000 g for 15 min to remove the water-insoluble material, then 1.5 ml of the supernatant in one rat lens was applied to a Shodex PROTEIN KW-803 column (SHOWA DENKO K.K, Tokyo, Japan), and eluted with 0.9% NaCl buffer at a flow rate of 0.5 ml/min. Elution was monitored at 280 nm using high performance liquid chromatogra-phy (HPLC) (LC-2010A, Shodex, Kyoto, Japan), and the peak fractions were collected for further use. At the same time, 60 lenses from 15-day-old rats were also purified using this method to obtain βL-crystallin.22

Electrophoresis and In-Gel Digestion

α-Crystallins from three rat lenses in each group were used to perform 2-DE maps. One-dimensional SDS-PAGE of lens proteins was performed on 0.75 mm thick 12% gels with protein standards of known molecular weight (MW) to identify βL-crystallin subunits by their MW.22 For 2-DE preparation, two-dimensional electrophoresis using a non-equilibrium pH gradient in the first dimension and 12% SDS-PAGE in the second dimension was conducted as described previously. 23,24 Gels were stained with silver staining, scanned using an Image Scanner

(GE Healthcare, London, UK), and analyzed with Image Master 2D software (GE Healthcare). An inten-sity ratio larger than 2.0 (p ≤ 0.05) or smaller than 0.5 (p ≤ 0.05) was set as the threshold that indicated a significant change.24

After the 2-DE gel was washed twice with water, selected spots were destained and digested with trypsin overnight, and the peptide extracts were dried under N2.

23,24

Identification of Protein Spots Using MALDI-TOF-MS

The digested proteins were desalted using ZipTip pipette tips prior to MALDI-TOF-MS analysis as described previously.23 Before sample identification, the mass spectrometer (MS) instrument was calibrated in external calibration mode using tryptic peptides of myoglobin. All tryptic digests were analyzed on a 4700 proteomics analyzer (Applied Biosystems, Beijing, China). For the α-crystallin sample, an MS/MS method (3800 laser shots per analysis, laser power set to 30% above the threshold for ion formation) was used, and only peaks with S/N ratios greater than 20 were selected from the peptide mass fingerprint spectrum. The search engine GPS Explorer software from Applied Biosystems with Mascot and the SwissProt (Version 050303) database were used for protein identification. The peptide mass tolerance was set to 0.6 Da, and the tandem mass tolerance was set to 0.8 Da.23,24

Confirmation of PTMs by Nano-LC-ESI- MS/MS

Selected peptides from the above preparation were resuspended with 5% acetonitrile in 0.1% formic acid, separated by nano-LC, and analyzed by online elec-trospray tandem mass spectrometry. The experiments

A B

FIGURE 1 Measurement of the lens gray scale value. The nuclear (A) and a representative perin-clear area (B; white arrow) is selected with fixed size and position, and the gray scale value of the selected area is shown on the top of the picture (white arrow).

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were performed on a Nano Aquity UPLC system (Waters Corporation, Massachusetts, USA) connected to an LTQ Orbitrap mass spectrometer (Thermo Elec-tron Corp., Pittsburgh, Pennsylvania, USA) equipped with an online nanoelectrospray ion source (Michrom Bioresources, Inc., Auburn, California, USA). Separa-tion of the peptides was achieved in a 15-cm reverse phase column (100 μm i.d., Michrom Bioresources, Inc.). The peptide mixtures were injected onto the trap-column with a flow rate of 10 μl/min and subsequently eluted with a gradient of 5–45% solvent B (95% ACN in 0.1% formic acid) over 60 min. The column flow rate was maintained at 500 nL/min and column tempera-ture was maintained at 35°C. Eluted peptides were analyzed by MS and data-dependent MS/MS acqui-sition, selecting the eight most abundant precursor ions for MS/MS with a dynamic exclusion duration of 60 s. The mass spectra were searched against the Rat International Protein Index (IPI) database using Bioworks software (Version 3.3.1; Thermo Electron Corp.) based on the SEQUEST algorithm. To reduce false positive identification results, a decoy database containing the reverse sequences was added to the database. The parameters for the SEQUEST search were as follows: partial trypsin (KR) cleavage with two missed cleavages was considered; the variable modifications were carbamylation (K), oxidation (M), phosphorylation (S, T), acetylation (K), methylation (C, H); peptide tolerance, 50 ppm; MS/MS tolerance, 1.0 Da. Positive protein identification was filtered by PeptideProphet with a p-value > 0.9,25 and a Protein-Prophet probability of 0.95 was used for the protein identification results.26

Assay for Chaperone Activity

α-Crystallins from six lenses in each group was used to examine chaperone activity. Heat-induced aggregation of βL-crystallin (200 μg/ml) separated from 15-day-old rats at 64°C was measured by the increase in light scattering at 340 nm in a spectrophotometer (Beckman DU800, California, USA). To test chaperone effects, 50 μg/ml α-crystallins were added to the βL-crystallin prior (5 min at 37°C) to heat-induced aggregation as described previously.22

Statistical Methods

Statistical comparisons were conducted with one-way ANOVA multiple-comparisons followed by LSD method (SPSS, version 11.0). p < 0.05 was considered significant. p < 0.01 was considered highly significant. Results are expressed as the means ± SE.

RESULTS

Estimation of Cataract Degree by Lens Gray Scale Value

Rats in the naphthalene group showed initial signs of cataract by three weeks, and formed perinuclear cataracts by six weeks. Development and progression of the opacity was much slower in the naphthalene plus LA group. Rats in this group showed initial signs of cataract by six weeks, and the rate of the opacity increase was much more gradual in this group. For the control group, there was no obvious cataract for-mation by nine weeks. Nuclear opacity progressed slowly in the three groups, and the difference in nuclear opacity between the three groups was not significant (Figure 2A).

The value of perinuclear gray scale in the naph-thalene group (95.6 ± 3.85 at nine weeks) was signifi-cantly higher than that of the naphthalene plus LA group (50.5 ± 2.93 at nine weeks) as well as that of the control group (26.6 ± 3.79 at nine weeks) from three weeks after intervention (p < 0.01). Although the value of perinuclear gray scale in the naphthalene plus LA group was higher than that of the control group from three weeks after intervention (p < 0.01), the difference between these two groups was much smaller than that between the naphthalene plus LA and the naphthalene group (Figure 2B). The value of nucleus gray scale in the naphthalene group (47.48 ± 2.81 at nine weeks) was slightly higher than that in the naphthalene plus LA group (43.55 ± 3.17 at nine weeks) as well as that in the control group (30.4 ± 1.96 at nine weeks) from three weeks after intervention (p < 0.01), and the value of nucleus gray scale in the naphthalene plus LA group was slightly higher than that in the control group (p < 0.01) (Figure 2C). However, the differences among the value of nucleus gray scale in the three groups were minor.

Identification of Proteins Spots on 2-DE Maps by MALDI-TOF-MS

To further evaluate post-translation modifications (PTMs) of α-crystallins, protein spots on 2-DE maps of α-crystallins were first identified by MALDI-TOF-MS analysis. α-Crystallins identified in our study were com-posed of αA, αB, and αA insert, as previously report-ed.27 The major protein spots migrated at 19–23 kDa, with isoelectric points (PI) between 5 and 8. These pat-terns of migration were consistent with the theoretical values for migration of αA-crystallin (19,779.9 Da, PI 5.78), αA-crystallin insert (22,433.1 Da, PI 6.35), and αB-crystallin (20,076.3 Da, PI 6.76) (Figure 3A–3C).

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Multiple spots at different PIs for the same protein are known as “charge trains,” and result from PTMs that alter the intrinsic charge of the protein. When the intensity of the major spots was compared, the original spots αA1, αAi1, and αB1 were the most abundant, and their intensity differed little among the three groups (p > 0.05). The intensity of αA2 and αA3 increased markedly in the naphthalene group compared with the control group (p < 0.01), but the intensity of these two spots was similar in the naphthalene plus LA group and the control group (p > 0.05). Two truncated proteins, T1 and T2, were present in the naphthalene and naph-thalene plus LA groups, but were noticeably absent in the control group (p < 0.01), and the intensity of T1

and T2 was lower in the naphthalene plus LA group compared with the naphthalene group (p < 0.05). These results suggest that the proteins underwent different PTM between the naphthalene group and the control group, and that LA abrogated these differences.

A representative MALDI-TOF mass spectrum of peptides generated from protein spot αA2 on 2-DE gels was identified as intact αA-crystallin. Zoom scan of the mass from 1631–1647 included mass 1641 matched with the m/z values for residues 158–173, indicating that the C-terminal extension is intact (Figure 4A). Meanwhile a MALDI-TOF mass spectrum of peptides from a protein spot T1 was identified as C-terminal truncated αA-crystallin (Figure 4B). Zoom scan of the mass from 1631–1647 lacked mass 1641, indicating that the C-terminal extension of 158–173 is truncated with 16 amino acid loss. The other spots of α-crystallins on the 2D gel were identified in the same way (Figure 3D–3F), and fragmentation of one peptide from residues 158–173 of spot T2 was also found to be absent because of C-terminal truncation of 16 amino acid.

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FIGURE 3 Identification of spots on 2-DE maps by mass spectroscopy of control group (A), naphthalene group (B), and naphthalene plus LA group (C). (αA1–6:αA-crystallin; T1-2: Truncation of αA-crystallin; αB1–4:αB-crystallin; αAi1–3:αA insert-crystallin.)

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FIGURE 2 (A) Slit lamp images for each group. (B) Trends of the perinuclear opacity of the three groups over time. Although the value of perinuclear gray scale in NAP+LA group shows an increase from three weeks, the change is much smaller than that in the NAP group (p < 0.01). (C) Trends of the nucleus opacity. The value of nucleus gray scale in the NAP+LA group was slightly smaller than in the NAP group (p < 0.01). (CTL, control group; NAP, naph-thalene group; NAP+LA, naphthalene plus LA group; βL, βL-crystallin.)

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FIGURE 4 (A) Full scan of a MALDI-TOF MS peptide mass fingerprint from spot αA2. The spectrum shows 13 of 15 peaks matched to the theoretical m/z values for residues in the sequence from rat αA crystallin. The 15 matching peptides covered 55% of the sequence. The asterisks denote the matched amino acid sequence. Zoom scan of the mass from 1631–1647 including peak 1641 matching the m/z values for residues 158–173 is also presented. (B) Full scan of a MALDI-TOF MS peptide mass fingerprint from spot T2. The spectrum shows 10 of 11 peaks matched to the theoretical m/z values for residues in the sequence from rat αA crystallin. The 11 matching peptides covered 47% of the sequence. Zoom scan of the mass from 1631–1647 lacking peak 1641 is also shown.

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Confirmation of PTMs by Nano-LC-ESI- MS/MS

We performed nano-LC-ESI-MS/MS to further con-firm PTMs of α-crystallins on 2DE maps spots as figure 5 showed. Our work identified several new sites of modification, as well as confirmed modifications that have been reported previously in rat and other mammalian lenses. Fourteen PTM sites of αA crystal-lin were identified with nine previously reported and five newly detected. Thirteen PTM sites of αB crystal-lin were identified with seven previously reported and five newly detected. To the best of our knowledge, it was the first report of PTMs of αA insert-crystallin, and thirteen PTM sites were identified in our study. Except that modifications of phosphorylation were a bit less than αA crystallin and modifications of oxida-tion were a little more than αA crystallin, no obvious difference was found between the two proteins. As for the “insert” residues 64–86 presented in the αA insert, three methylation and three oxidation modifications were found in the control group. The level of methyla-tion modifications decreased in the “insert” residues in the naphthalene group (Figure 6).

Figure 5 shows a representative MS/MS spectrum of S-phosphorylation peptide [VQSPGLDAGHSE] from amino acids 146–156 in spot αA2. In our study, phosphorylation of αA-crystallins increased slightly

on spots αA2 and αA3 as well as T1 and T2 compared with spot αA1, which may indicate that modifica-tion of phosphorylation increased in the naphthalene group, and LA abrogated such changes. The level of methylation decreased markedly for αA-crystallin on all other spots except that on spots αA1, αAins1, and αB1. This may suggest that the methylation level decreased due to oxidative stress and LA inhibited these changes.

Other modifications, such as acetylation, oxidation, and carbamylation, showed little change in our study; among these modifications, oxidation could be gener-ated during sample preparation.

Comparison of the Chaperone Activity of α-Crystallins

βL-crystallin without α-crystallins aggregated most rapidly (p < 0.01), while βL-crystallin with α-crystallins in the control group aggregated much slower com-pared with other groups (p < 0.01), and βL-crystallin with α-crystallins in the naphthalene plus LA group aggregated slower than the naphthalene group (p < 0.01). The result indicated that the chaperone activity of α-crystallins was decreased by exposure to naphthalene, and LA could decrease such insult (Figure 7).

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FIGURE 5 A nano-LC-ESI-MS/MS-spectrum of the phosphorylated peptide NQSPGLDAGHSE from residues 146–156 in spot αA2. The amino acid sequence is displayed above the spectrum. The y- and b-type product ions found experimentally are writ-ten above and below the sequence, respectively, and the space between the arrows demonstrates the neutral loss ion, which is expected for a peptide containing a phosphorylated serine.

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DISCUSSION

Our results provide quantitative evidence that α-lipoic acid (LA) can protect the lens against naphthalene-induced opacity in a rat model cataract system. Both slit lamp microscope and light-scattering measure-ments indicate that dietary LA is effective in delaying naphthalene-induced cataract.

It has been reported that LA may exert its protec-tive effect by preventing protein glycation28 and reduc-ing oxidative stress.11 It may also serve as a source of reduced sulphydryl groups, thus maintaining lens ascorbate and glutathione levels.11 Our study provides the first evidence that LA might inhibit lens opacity of oxidative stress-induced cataract by altering PTMs of α-crystallins in the lens and preserving their molecular chaperone activity.

α-Crystallins identified in our study were composed of αA, αB, and αA insert as previously reported.27 From the distribution of the multiple protein spots of α-crystallins on 2-DE gels and subsequent analysis by mass spectrometry, we found that α-crystallins was highly modified by exposure to naphthalene and LA could inhibit such alteration.

Flexibility of the C-terminal tail of α-crystallins, a feature shared by mammalian sHsps, is essential for its chaperone and thermostability functions.7,29 In our study, we detected a C-terminal truncation of 16 amino acids in the naphthalene group, which was abrogated in the naphthalene plus LA group. Truncation of 16 C-terminal amino acids as well as 10,11 and other C-terminal amino acids can be digested by overacti-vation of calpains and lead to profound decreases in the chaperone function of α-crystallins.30,31 In rodents,

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110 120 130 140 150 160 170

110 120 130 140 150 160 170 180 190

110 120 130 140 150 160 170

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10 20 40 10 50 60 70 80 90 100

FIGURE 6 Modification sites identified in lens α-crystallins by nano-LC-ESI-MS/MS-spectrum. Residues marked with a dia-mond have not previously been reported as modified. White on black shading indicates that the residue was modified, black on light gray indicates that the residue was not modified, and white on dark gray indicates that the residue was not observed. (αA1–6:αA-crystallin; T1-2: Truncation of αA-crystallin; αB1–4:αB-crystallin; αAi1–3:αA insert-crystallin. • oxidation; acetyla-tion; & phosphorylation; $ carbamylation; # methylation.)

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calpains may be activated by increased calcium, which is induced by oxidative stress and other detriments.22 Thus, we propose that the major cause of decreased chaperone function of α-crystallins in our study may be truncation of the C-terminus induced by oxidative stress. As we know, calpains do not play a major role in truncation of α-crystallins in human lenses, partial degradation of α-crystallin in human lenses during aging, and cataract could involve a protease other than calpains or a mechanism other than proteolysis.22 Further investigations should be conducted to verify whether LA could have the same effect on human cataract.

Our study showed that phosphorylation slightly increased in the naphthalene group, and LA abrogated these increases. Phosphorylation can lead to migration of spots on 2-DE maps to lower PI position, which could be one of the reasons for generating multiple spots at different PIs for the same a. -crystallin protein on 2-DE maps. As the effects of phosphorylation have not been clearly elucidated, it is difficult to estimate the significance of this finding for chaperone activity of α-crystallins. Studies suggest that chaperone activ-ity is decreased32 or increased33 upon phosphorylation, whereas others show little or no change.6,34

Our findings also suggested that the level of methylation decreased markedly in the naphthalene group, which can be abrogated by LA. S-methylation modification of cysteine for γ-crystallin may prevent formation of intermolecular disulfide cross-links, and thus may serve as protection from cataractogenesis.35 We did not detect methylation modification of cysteine for α -crystallins, but we did observe methylation of

histidine for this protein. Although the reasons for this difference in methylation between crystallins is not clear, our findings indicate that methylation may be a far more prevalent and important post-translational modification than previously thought.

All the ᾶ-crystallins in our study were N-terminally acetylated. Despite its wide occurrence, the biological role of N-terminal acetylation remains obscure.36 It has been reported that N-acetylated terminal methionine of α-crystallin can be oxidized to methionine sulfoxide in vivo. Oxidation of the N-terminal methionine, which is exposed on the outside of polypeptide, can negatively affect the function of the protein.37 We were surprised to find that all the N-acetylated terminal methionines were oxidized in our study. As modifi-cation of oxidation can be generated during sample preparation, the results of this modification may not be credible. Moreover, since every type of modifications has its special characteristics, which need different mass spectrometry analysis techniques, further, more detailed, analysis should be carried out to study vari-ous PTMs of α-crystallins.

It has not been reported of PTMs of αA insert-crystallin as far as we know. αA-Crystallin and αA insert-crystallin are derived from the αA-crystallin gene via alternative splicing. They are identical except for the presence of a polypeptide, 23 amino acids long, residues 64–86 that are present in αA insert-crystallin, encoded by the “insert” exon. Evolutionary logic would suggest that the insertion of a 23 amino acid peptide in the middle of αA-crystallin, a protein evolv-ing more slowly than either histone H1, cytochrome c, or hemoglobin, would lead to appreciable structural and functional changes. Bhat suggested that the “insert peptide” in aA insert-crystallin led to the property of inhibiting growth of the bacterial host that were dif-ferent from αA-crystallin. But Anke reported that the mutant proteins were readily incorporated into the normal large water soluble αA-crystallin complexes, and the insert did not disturb the integrity of these complexes. He suggested that this viable αA-crystallin mutant thus mimicked the origins and effects of exon duplication, which was a common consequence of exon shuffling in mammalian genome evolution.38,39 By now, the real relation between αA-crystallin and αA insert-crystallin is still needed to be clarified. In our study, except that modifications of phosphorylation were a bit less than αA-crystallin and modifications of oxida-tion were a little more than αA crystallin, no obvious difference was found between the two proteins. For the insert residues, three methylation and three oxidation modifications were found in the control group. We do not know the exact reason for the changes of the modi-fications. Oxidation could be generated during sample preparation. As methylation may serve as a protection

400βLβL+NAPβL+NAP+LAβL+CTL

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FIGURE 7 Comparison of the chaperone activity of α-crythallin in the three groups. βL-crystallin with α-crystallins in naphthalene plus LA group aggregated slower than naphthalene group (p < 0.01).

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α-Lipoic Acid Alters Post-Translational Modifications and Protects the Chaperone Activity of Lens 629

© 2010 Informa Healthcare USA, Inc.

factor for cataractogenesis, probably the “insert” resi-due is important for maintaining the stability of the protein and protecting the lens from cataract.35 Further studies should be carried out to discover the relation of structure and function between αA-crystallin and αA insert-crystallin.

As a kind of molecular chaperon.e., α-crystallins has the activity to protect βL-crystallin from thermal-induced aggregation and renature chemically dena-turated proteins.6,7,40 By measuring thermal-induced aggregation of βL-crystallin, our findings showed that the chaperone activity of α-crystallins was inhibited by naphthalene, and LA could decrease such insult. Our findings showed that the chaperone activity of α-crystallins was inhibited by naphthalene, and LA could decrease such insult. As a novel biological antioxidant, LA may protect α-crystallins chaperone activity by reducing the oxidative stress that induces molecular modifications and results in decreased chaperone activity.

In conclusion, our study provides the first evidence that LA may inhibit lens opacity of naphthalene-in-duced cataract by altering PTMs of α-crystallins in the lens and preserving their molecular chaperone activity. Besides, we verified the C-terminal truncation of 16 amino acids that may be induced by calpains in oxida-tive stress, and we also identified some other novel modifications of α-crystallins in rats.

ACKNOWLEDGMENTS

The authors thank Dr. Laney Weber (Ph D) for provid-ing helpful writing revision. This work was supported by ShangHai Committee for Science and Technology (08411963300 to L.Y).

Declaration of interest: The authors report no conflict of interest. The authors alone are responsible for the content and writing of the paper.

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