Proteomics analysis of water insoluble-urea soluble crystallins from normal and dexamethasone exposed lens

Reagents

Dex was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (200 mM). All reagents were from Sigma, St. Louis, MO.

Preparation of rat lens and drug treatments

Sprague-Dawley male rats (21-day-old) were provided by Animal Laboratories (Harbin Medical University, Harbin, China). Animals were handled in strict accordance with the Association for Research in Vision Ophthalmology Statement on the use of Animals in Ophthalmic and Vision Research. Rats were killed, and the lenses were dissected and flash frozen until use. All experiments using animals conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Rat lenses were carefully dissected from the surrounding ocular tissue and were immediately transferred into the culture in medium 199 (pH 7.2; Sigma), containing BSA (BSA; GibcoBRL, Grand Island, NY) and an antibiotic solution (GibcoBRL; 100 U/ml penicillin, 100 µg/ml streptomycin, and 0.25 µg/ml amphotericin B). Dex was added to final concentrations of 0 µM, 0.1 µM, 1 µM, 10 µM, and100 µM then added to the medium. Each Dex concentration group contained 6 lenses. Lenses were cultured for 48 h at 37 °C under 5% CO₂. The treatments were the same for all assays. The lenses were cultured and observed daily for 2 days and photographed under a stereomicroscope to record the development of opacity.

Isolation of WI-US lens crystallins

Each frozen lens was ground in liquid nitrogen and then dissolved in 1 ml of lysis buffer (20 mM sodium phosphate, pH 7.0, 1 mM EGTA, 100 mM NaCl, 1 mM PMSF, 1 μg/ml aprotinin, and 1 μg/ml leupeptin). Lens homogenates were centrifuged at 20,000× g for 45 min at 4 °C and supernatants were removed and collected. The supernatants were designated as water-soluble (WS) protein fractions, and the pellet was designated as the water-insoluble (WI) protein fraction. The WI protein fraction was suspended in a buffer (50 mM Tris-HCl, pH 7.9, containing 6 M urea and 5 mM dithiothreitol) and homogenized. The supernatants were designated as the WI-US protein fraction. The pellet was designated as the water-insoluble-urea-insoluble (WI-UI) protein fraction. The process was repeated twice to recover the WI-US and WI-UI protein fractions, and then combined with the above similar fractions. The WI-US protein concentration was determined according to the Bradford method.

2DE and protein identification by MALDI-TOF mass spectrometry

WI-US protein samples were processed by 2D clean-up kit then re-dissolved in a rehydration solution with immobilized pH gradient (IPG) buffer (8 M Urea, 2% CHAPS, 0.5% IPG buffer, 0.002% Bromophenol blue). Isoelectric focusing was performed using IPG gel strips (13 cm, pH 3–10), which were loaded with 300 ug of protein. Spot-picking and digestion were performed using preparative gels, and the gels were stained with Coomassie brilliant blue. After the preparative gels were scanned, protein spots of interest were excised from the gels and digested with trypsin. The tryptic peptides were extracted and dried completely by centrifugal lyophilization. The spots representing differential proteins were first identified by image analysis, and then further analyzed by matrix-assisted laser desorption/ionization-time of flight-tandem mass spectrometry (MALDI-TOF-MS/MS).

MALDI-TOF mass spectrometry and tadem TOF/TOF mass spectrometry were performed using a 4800 MALDI-TOF-TOF instrument (Applied Biosystems, Foster City, CA). Combined mass and mass/mass spectra were used to interrogate rat sequences in the International Protein Index database using the Mascot database search algorithms. Confident protein identification had a statistically significant protein score and the best ion score.

The Coomassie Blue–stained gel images (containing 3D views) were captured and image analysis was performed on a computer (ImageMaster 2D Platinum 6.0 software; GE Healthcare, Piscataway, NJ) to determine the percent at each spot that contributed to the total protein on the gel.

Western-blot

WI-US protein samples were collected from lenses after treatment with 4 concentrations of dexamethasone then treated with 5× SDS page buffer. Protein concentrations were determined using a bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Samples were electrophoresed on 12% Tris-glycinepolyacrylamide gels using a commercial apparatus (BIO-RAD Mini-PROTEAN Tetra cell; Bio-Rad Corporation, Hercules, CA) and then blotted on nitrocellulose membrane (Pall-Gelman, Ann Arbor, MI) using a transblot cell (BIO-RAD). Membranes were blocked in 3% BSA and probed with primary antibodies: a monoclonal αA -crystallin antibody (B-2, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA), a polyclonal αB -crystallin antibody (FL-175, 1:1,000; Santa Cruz Biotechnology). After washing, the membranes were then incubated with horseradish peroxidase-conjugated secondary antibody (1:5,000; Zsbio, Beijing, China) by standard protocols and visualized by diaminobenzidene (DAB; Tiangen Biotech Co. LTD, Beijing, China) stain using a gel documentation apparatus (FUJIFILM LAS-3000; Fujifilm Corporation, Tokyo, Japan). As an internal control, the membranes were re-probed with a 1:1,000 dilution of mouse anti-rat actine mAb (Beyotime Institute of Biotechnology, Jiangsu, China) using the same conditions described above. Images were captured as described above, and densitometric analyses were performed using an NIH Image (ver.163) with values normalized to the actine signal for each sample and expressed as multiples of increases (or decreases) relative to control samples (developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).

Analysis of immuno-histochemistry

At the end of the culture period, the nuclear regions of the lenses were placed in phosphate-buffered saline (PBS) and fixed overnight in 4% paraformaldehyde. The lenses were dehydrated, embedded in paraffin, and cut into 3-µm sections. For histology, de-waxed paraffin sections were washed with Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl [pH 7.4]) and incubated for 60 min in TBS containing 0.05% Triton X-100, 2% BSA; the sections were then incubated overnight with primary antibodies: a polyclonal αB-crystallin antibody (FL-175, 1:50; Santa Cruz Biotechnology). Afterwards, samples were washed with Tris-buffered saline then incubated for 1 h with horseradish peroxidase -conjugated secondary antibody (1:100; Zsbio, Bejing, China). Histologic images were photographed with a microscope (Eclipse TS100-F; Nikon, Tokyo, Japan).

Analysis of immuno-Fluorescence

At the end of the culture period, the nuclear portions of the lenses were frozen and cut into 5-µm sections. After being fixed with acetone, lens sections were washed with Tris-buffered saline (TBS; 10 mM Tris and 150 mM NaCl [pH 7.4]) and incubated for 60 min in TBS containing 0.05% Triton X-100, 2% BSA. The sections were then incubated overnight with primary antibodies: a monoclonal αA-crystallin antibody (B-2, 1:50; Santa Cruz Biotechnology), a polyclonal αB-crystallin antibody (FL-175, 1:50; Santa Cruz Biotechnology). Afterwards, samples were washed with Tris-buffered saline then incubated for 1 h with Cy3-conjugated or FITC-conjugated secondary antibody (1:100; Beyotime Institute of Biotechnology). Histologic images were photographed with confocal-fluorescence microscopy (CFM, Leica TCS SP5; Leica Microsystems Ltd, Wetzlar, Germany) or with a fluorescence microscope (Nikon, Eclipse TS100-F; Nikon Corporation, Tokyo, Japan).

Morphologic changes of apoptotic cells detected by electronic microscopy

For electron microscopy (EM) studies, the nuclear portions of the lenses were fixed in 4% glutaraldehyde (0.1 M sodium phosphate buffer, pH 7.4) and postfixed in 1% osmium tetroxide. The tissues were washed and dehydrated through a graded series of ethanol, cleared in propylene oxide, embedded in Epon mixture, and sectioned at 1 µm and 90 nm with an ultramicrotome. Semi-thin sections (1 µm) were stained with toluidine blue for light microscopy. Thin sections (90 nm) were stained with uranyl acetate and lead citrate, and the super thinly sliced structures of lenses were clearly observed by transmission electron microscopy (H-7650 JEM-1200EX; Japan Electron Optics Laboratory, Tokyo, Japan), then photographed and recorded.

Native-PAGE of WI-US crystallins

After isolating the WI-US protein from every lens group, we used Native-PAGE to detect expressional changes and percentage of α-, β-, and γ-crystallins in each group. WI-US protein samples were collected from normal lenses and from post-treatment lenses, with 4 concentrations of dexamethasone then treated with 2× native page buffer. Protein concentrations were determined using the bicinchoninic acid (BCA) assay (Pierce, Rockford, IL). Samples were electrophoresed on 6% native Tris-glycine polyacrylamide gels using a commercial apparatus (BIO-RAD Mini-PROTEAN Tetra cell) and the gels were stained with Coomassie brilliant blue.

Statistics

Data are shown as mean±SEM from triplicate samples. Each experiment was performed at least 3 times to confirm reproducibility. Statistical analysis was performed using ANOVA with Dunnett’s as post-hoc. A p-value of less than 0.05 was considered statistically significant. Statistical software, SPSS 17.0 (SPSS Inc. Chicago, IL), was used.

Induction of PSO by dexamethasone

To examine the effects of glucocorticoid on the lens, we developed an ex vivo rat lens model. We isolated rat lenses and treated them in organ culture to cause steroid-induced cataracts directly. The lenses were incubated with 0, 0.1, 1, and 10 µM Dex, and morphologic changes in the whole lenses were recorded photographically with a stereomicroscope. Lenses with posterior opacity were selected. The opacity first appeared at 48 h in the posterior region of lenses incubated with every concentrations of dexamethasone. The opacity (Figure 1B-E) in those lenses incubated with 10 or 100 µM Dex for 48 h were most obvious, whereas they were not detected in the anterior or equatorial regions. Untreated control lenses remained transparent for 48 h treatment of dexamethasone (Figure 1A).

Differential analysis of protein levels and spot identification

To investigate the crystallin changes in dexamethasone exposed lenses, 2DE mapping was performed (Figure 2). A greater difference in the amount of protein and a t-test score of less than 0.05 was considered to be significant. The peptides representing these spots were analyzed by MALDI-TOF mass spectrometry, and 25 spots were successfully identified (Table 1). A total of 7 protein spots were significantly different between normal and dexamethasone exposed lenses (Figure 3), and each was more or less abundant in the dexamethasone exposed lenses compared with the normal ones. The amino sequences of phosphorylated-αA-crystallin were not altered compared with amino sequences of proteins published in PubMed, and we present the MS spectra (PMF spectra) of it (Figure 4). Based on these analyses, seven unique proteins had different levels between normal and dexamethasone exposed lenses: αA-crystallin (spot 9), αB-crystallin (spot 16), βA3-crystallin (spot14), βB2-crystallin (spot 4), βA1-crystallin (spot 3), βA4-crystallin (spot 2) and βB1-crystallin (spot 1). For each unique protein, the seven spots were examined using 3D (Figure 3). We detected the differences of the intensities of the protein spots. As shown in Figure 3, which suggested that three unique proteins (βA1-crystallin, βA4 –crystallin and βB1-crystallin) were less abundant in the dexamethasone exposed lenses than in the controls (p<0.05). The intensity of proteins αA-crystallin, αB-crystallin tended to increase when lenses were exposed to 1 µM and 10 µM Dex (p<0.05); βB2-crystallin tended to increase when lenses were exposed to 1µM Dex (p<0.05); βA3-crystallin tended to increase when lenses were exposed to 100 µM Dex (p<0.05). The intensity histograms of modified protein spots were related to their overall intensity.

Western blot analysis

To further verify the 2DE and modified spots results, two important crystallins: αA-crystallin and αB-crystallin, were analyzed by western blot. The three crystallins were selected based on their important roles in maintaining transparency of lenses. By western blot, as shown in Figure 5, αA-crystallin and αB-crystallin were significantly (p<0.05) increased when the lens was exposed to 1 µM Dex and 10 µM Dex. αA-crystallin significantly (p<0.05) decreased when the lens was exposed to 100 µM Dex.

Inmmuno histology analysis on location of αA-crystallin and αB-crystallin

To characterize the effects of dexamethasone on cataract formation and location of αA-crystallin and αB-crystallin, organ-cultured rat lenses were examined histologically. The control lenses had normal cellular architecture and the lens fiber cells were regularly packed (Figure 6) 48 h after the culture with dexamethasone. After an incubation primer and second antibodies of αA-crystallin and αB-crystallin, figures of inmmuno histochemistry and inmmuno fluorescence showed that the quantities of αA-crystallin increased when the lens was exposed to 1 µM Dex, αB-crystallin increased when the lens was exposed to 10 µM Dex.

The morphologic changes of lens fiber cells detected by electronic microscopy

As shown in Figure 7, we used EM to observe in more detail the morphologic changes at the cellular level. After the lens was exposed to 1–100 µM Dex, the fiber cells in the posterior lens displayed a disordered arrangement, and the tight contact between fiber cell membranes separated. Circular lesions were located between the lens fiber cells, or the fiber cells, but not intracellularly. We also found that in lenses exposed to 1–100 µM Dex, the degree of crack between lens fiber cells was consistent with the degree of lens cataract.

Expressional changes of native crystallins

We used native-page to analyze expressional changes of α-crystallin. Figure 8 showed the bands of native α-crystallin isolated from lenses located on the top of the page and indicated that HMW were larger than 700 kDa. Three β-crystallin fractions (1, 2, 3) were isolated from lenses located in the middle of the page and the molecular weights ranged from 140 kDa to 232 kDa.

αA-crystallin

In the present study, the levels of αA-crystallin increased when lenses were exposed to 1 or 10 µM Dex compared with normal controls, and the western-blot figures showed decreased levels of αA-crystallin, which became more obvious as the lens was exposed to the highest concentration of Dex, perhaps because αA-crystallin gradually became water-insoluble-urea-insoluble or because the structure of it was destroyed and the antibody of αA-crystallin could not be combined with αA-crystallin effectively. A previous paper [17] reported that α-crystallin aggregates were approximately 600 kDa, and the γ-crystallin monomers of approximately 20 kDa molecular weight. Because we did not find obvious γ-crystallin (20 kDa), we could not be sure of native α-crystallin isolated from lenses exposed to 0–100 µM Dex located on the top of the page, and whether HMW of α-crystallin were larger than 700 kDa and native β-crystallin located on the middle of the page.

Although αA-crystallin increased under 1 or 10 µM Dex, figures of native-page showed that the percentage of α-crystallin gradually decreased when accompanied with increased concentrations of Dex; α-crystallin is an abundant protein in the eye lens of almost all vertebrates, reaching levels of up to 50% of water-soluble lens protein [18,19]. In our study, we also found that the percentage of α-crystallin was 53% in whole WI-US crystallins of normal lenses, but that it gradually decreased to 3% in whole WI-US crystallins after the lens was exposed to the highest concentration of Dex.

α-Crystallin belongs to the family of small heat-shock proteins (sHSP), and the α-crystallin domain is common to all the members of the sHSP superfamily [18,19]. Like other sHSP, α-crystallin functions like a molecular chaperone by suppressing the aggregation of other cellular proteins under various stress conditions [18,19].

In vitro, α-crystallin can protect β- and γ-crystallins and other proteins against denaturation and subsequent aggregation [20,21]. The mechanism of this protection involves binding of the partially denatured protein to a central region of an α-crystallin complex [22]. The α-crystallin may perform a similar function in vivo, binding to and protecting other crystallines against further denaturation [23]. In our study, we also found the expression of βA1-, βA4-, βB1-crystallin decreased under concentrations of Dex. After being attacked by Dex, the expressional levels decreased more than in normal controls, α-crystallin likely loses its chaperoning abilities, the remaining α-crystallin will probably be overwhelmed prematurely, thereby making damaged β-crystallin more prone to aggregated to WI-UI crystallins. The decreased expression of crystallins mentioned above are mainly because of the decreased expression of α-crystallin, which cannot protect the denaturation of them. The molecular chaperone properties of α-crystallin are believed to play a role in maintaining the transparency of the lens [23]. As expression levels of α-crystallin decreases, it may lose its chaperone abilities, thereby leading to a decrease in the ability to protect the denaturation of other proteins or crystallins when rat lenses are exposed to concentrations of Dex. Further, the electronic microscope showed that the intercellular space between lens fiber cells became larger, the degree of crack accompanied with increased concentration of Dex and mitochondrial were attacked to some extent when a lens was exposed to Dex.

We also found αA-crystallin became progressively and obviously acidified, phosphorylated in the lens WI-US fraction, with the phosphorylated form comprising one-third of the total αA when lenses were exposed to higher concentrations (1, 10, and 100µM) Dex. Phosphorylation of α -crystallins has already been well documented in rat lenses [24]. Further studies are required to determine which mechanism of α-crystallin phosphorylation predominates in rats. These studies are important, because phosphorylation of α-crystallin may cause dissociation of α-oligomers and reduction of chaperone-like activity [25]. This modification could cause the β- and γ-crystallin to partially unfold and further aggregate.

The expression of αA-crystallin further detected by western blot, immune fluorescence, and the results are consistent with the expressional levels detected by the 2-DE map.

αB-crystallin

Based on previous reports, which suggested that αB-crystallin was important for a chaperone function, it also appears to exhibit anti-apoptotic functions. We studied the expressional levels of αB-crystallin after lenses were exposed to concentrations of Dex. Our data suggests the levels of αB-crystallin increased when lenses were exposed to higher concentrations of Dex compared with normal controls, perhaps this was a reaction to stress for lenses placed under Dex. Because the expressional levels of α-crystallin detected by native-page showed it gradually decreased, accompanied by the increasing concentrations of Dex, the lens could not escape the fate of other crystallins being attacked and denatured, and the overall ability to protect other proteins decreased.

The expression of αB-crystallin was further detected by western blot, immuno histochemistry, and immune fluorescence, and the results are consistent with the expressional levels detected by 2DE mapping.

β-crystallines

The present literature suggests that β-crystallin oligomers exist and play a critical role in maintenance of lens transparency [26].

Excessive accumulation of modified β-crystalline in vivo may disrupt normal protein–protein interactions, diminishing their stability and thus contributing to the accumulation of insoluble β-crystalline during cataract development [27]. In our experiment, we also found that new protein spots appeared, compared with a normal lens, with increased concentrations of Dex: ßA3-, ßB1- (alkalization form), ßA2-, ßA1-, ßA4-, and ßB1-crystallins also experienced a decrease with increasing concentrations of Dex. This significant loss of ßA1-, ßA4-, and ßB1- crystallins may be more related to its greater tendency to undergo WI-UI during lens opacity. The figure of the native-page showed the percentages of β-crystallin gradually increased with increased concentrations of Dex, maybe because of newly appeared β-crystallin protein spots.

To detect the changes of lens fiber cells and the junctional condition of them, we further used EM to observe in more detail the morphologic changes at the cellular level. After a lens was exposed to 1–100 µM Dex, the fiber cells in the posterior lens displayed a disordered arrangement, and the tight contact between fiber cell membranes separated. Circular lesions were located between the lens fiber cells, or the fiber cell and capsules, but not intracellularly. We also found that when a lens was exposed to 1–100 µM Dex, the degree of crack between lens fiber cells was more serious depending on the degree of lens cataract. We speculated that this phenomenon may be caused by decreased normal β-crystallins and the appearance of new β-crystallin protein spots, which disorder the normal arrangement of lens fiber cells so that inter-cellular space became enlarged and the crack became more serious with increasing concentrations of Dex.

γ-crystallins

Newly appeared protein spots: γS-crystallin appeared after the lens was exposed to 10 µM and 100 µM Dex. γS-crystallin, the concentration of which rapidly increases with age of rat [28]. In our study, we also found newly γS-crystallin protein spots appeared when the lens was exposed to a high concentration of Dex. Because γ-crystallin could not be focused and divided well in a WI-US fraction, we could not compare the expression levels of γA- and γE/F-, respectively.

Some previous reports [29,30] have indicated that the incidence of cataract formation is higher in the lenses of patients given prednisolone in eye drops than in those who receive it systemically, suggests that glucocorticoid directly affects the pathogenesis of a lens. The changes in the relative abundance of crystallins would be expected to significantly alter the properties of lens fibers of the dexamethasone exposed lens. The gradually diminishing native α-crystallin, decreased expression of β-, βA1-, βA4-, and βB1- may favor dehydration and further insolubilization with a higher index of refraction. In the future, we should study the structural changes of crystallins when lenses are exposed to varying concentrations of Dex. During glucocorticoid-induced cataract development, our results showing multiple crystallins may be more vulnerable to glucocorticoid stress because of diminished important roles. The possible mechanisms are needed to further investigate.