Post-Translational Modification of Crystallins in Vitreous Body from

Sep 6, 2007 - ... αB-, βA4-, and βB2-crystallins could be correlated with uveitis progression via ... are intercorrelated with uveitis progression ...
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Post-Translational Modification of Crystallins in Vitreous Body from Experimental Autoimmune Uveitis of Rats Song-Chul Bahk,†,‡ Jung-Un Jang,†,§ Chang-Uk Choi,§ Sook-Hee Lee,†,§ Zee-Yong Park,| Ji-Yeon Yang,1† Jae-Duck Kim,§ Yun-Sik Yang,*,†,§ and Hun-Taeg Chung†,‡ Genome Research Center for Immune Disorders, School of Medicine, Wonkwang University, Iksan, Republic of Korea, Department of Microbiology and Immunology, School of Medicine, Wonkwang University, Iksan, Republic of Korea, Department of Ophthalmology, School of Medicine, Wonkwang University, Iksan, Republic of Korea, and Department of Life Science, Gwangju Institute of Science and Technology, Gwangju, Republic of Korea Received March 10, 2007

Experimental autoimmune uveitis (EAU) is a well-known animal model of posterior uveitis that is one of the major causes of blindness. EAU could be induced in susceptible animals (i.e., Lewis rat) by immune reactions using evolutionarily conserved retinal proteins, such as interphoto-receptor retinoid binding protein (IRBP), or epitaphs of the protein. First, we prepared the following four test groups that subsequently increased or decreased inflammation. (1) Normal control group, (2) IRBP-induced uveitis group, (3) Hemin-treated uveitis group, and (4) Sn(IV) protoporphyrin IX dichloride (SnPP)treated uveitis group. Second, in the vitreous bodies of Lewis rats, the infiltrated proteins were analyzed using two-dimensional electrophoresis (2-DE), MALDI-TOF/MS, and Micro LC/LC-MS/MS analysis. Finally, Western blotting was applied to confirm the relative amount of crystallins and phosphorylation sites of RB-crystallin. Thirty spots were identified in vitreous bodies, and 27 of these spots were members of the crystallin family. Unlike βA4- and B2-crystallins (that were significantly increased without truncation), RA- and B-crystallins were only truncated in EAU vitreous body. Taken as a whole, in the rat EAU model, we suggest that post-translational truncations of RA- and RB-crystallins, phosphorylation of RB-crystallin, and new production of βA4- and βB2-crystallins are intercorrelated with uveitis progression and inflammatory responses. Keywords: crystallin • experimental autoimmune uveitis (EAU) • vitreous body • truncation • phosphorylation

Introduction Experimental autoimmune uveitis (EAU) is a prototypic T-cell mediated autoimmune disease in which the target tissue is the neural retina. EAU has been produced by retinal proteins such as interphotoreceptor retinoid-binding protein (IRBP) and retinal soluble antigen (S-Ag, arrestin), rhodopsin, recoverin, phosducin, or their peptides.1-4 It is also induced by adoptive transfer of CD4+ MHC class II-restricted T-cells that is specific to those antigens.5 EAU is characterized by granuloma formation in the neural retina and by the destruction of photoreceptor cells that result in blindness.6 The pathogenesis of EAU serves as a model for the following sight-threatening diseases, such as Vogt-Koyanagi-Harada’s, Behcet’s disease, birdshot retinochoroidopathy, sympathetic ophthalmia, and ocular sarcoidosis.2 Posterior uveitis is an intractable disease in the field * To whom correspondence should be addressed: Prof. Yun-Sik Yang, Department of Ophthalmology, School of Medicine, Wonkwang University, Iksan, Chonbuk 570-749, Republic of Korea; E-mail, [email protected]; Tel, +82-63-850-1302; Fax, +82-63-855-1801. † Genome Research Center for Immune Disorders. ‡ Department of Microbiology and Immunology. § Department of Ophthalmology. | Gwangju Institute of Science and Technology. 10.1021/pr070133k CCC: $37.00

 2007 American Chemical Society

of ophthalmology. In spite of their many side effects, antiinflammatory agents (steroids or nonsteroidal anti-inflammatory drug, NSAID) and immunosuppressant agents have been used to ameliorate the inflammation of the uveitis. Unknown mechanism(s) of uveitis must be elucidated prior to a development of new therapeutic modalities. This study using the animal model is the other alternative way that substitutes human uveitis. Autoimmune reactions are directed toward retina-expressed protein after uveitic attack.7,8 The vitreous body is known to be associated with a secondary immune response in experimental rabbits’ uveitis.9 Furthermore, pars plana vitrectomy has beneficial effects to the inflammation and minimizes further uveitic attack.10 On the basis of this rationale, we assumed that the vitreous body might be a reservoir of inflammatory agents. Crystallins are well-known to be constitutive proteins of mammalian lenses. Recent studies, however, elucidated interesting new insights of crystallins. The proteins known as aldehyde dehydrogenase class 3 and transketolase were revealed as crystallins having moonlighting activities.11 In evolutionary track, ancestral enzymes may have been recruited as crystallins probably due to a regulatory change. Gene duplication is evidenced by the presence of two genes encoding RBJournal of Proteome Research 2007, 6, 3891-3898

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research articles crystallin/heat-shock protein 27 (hsp27), which are situated on separate chromosomes in mice and humans. The other crystallins such as -crystallin/lactate dehydrogenase, δ-crystallin/ R-enolase, τ-crystallin/R-enolase, and δ-crystallin/avian argininosuccinate lyase are encoded by single-copy genes.12 The η-crystallin is a retinal dehydrogenase that has acquired a role as a structural protein in the eye lens of elephant shrew.13 Interestingly, aldehyde dehydrogenase 3A1 (ALDH3A1) is a corneal crystallin having the diverse biological functions as the lens crystallin.14 On the other hand, overexpressed RB-crystallin blocks the activation of RAS. Eventually, the inactivated ERK1/2 attenuates calcimycin-induced apoptosis.15 The modifications of mouse RA-crystallin by truncation and phosphorylation were reported.16 Recently, quantitative Nterminal acetylation of RA-crystallin and variable C-terminal truncation were observed.17 The function of R-crystallin may be activated by the phosphorylation on serine residues by a cAMP-dependent or -independent manner.18 The RB-crystallin is phosphorylated in vivo at Ser 45,19 Ser 19, and Ser 59.20,21 The phosphorylations on two different sites of R-crystallins may be catalyzed by two different kinases.21 On the other hand, heme oxygenase-1 (HO-1) has been identified as a cytoprotective enzyme against oxidative injuries caused by various stimuli, including oxidative stress, ischemia reperfusion, and endotoxin.22 In an endogenous cellular protective system, HO-1 functions against ocular inflammation that is induced by LPS in vivo.23 The other study suggested that hyperglycemia in streptozotocin-induced diabetes leads to persistent inflammation and tissue damage following uveitis. That may be due to reduced levels of HO-1 in the ciliary body.24 Inflammation was reduced in the hemin-treated rats but deteriorated in the SnPP-treated ones. Previously, we proposed that crystallin family proteins might play a regulatory role in endotoxin-induced uveitis (EIU) progression.25 This study was designed to further refine dubious characters of vitreous crystallins that could regulate the posterior uveitis. Additionally, we analyzed the truncation and phosphorylation of the crystallins from EAU and uveitis treated with hemin or with SnPP through 2-DE, MALDI-TOF/MS, and Western blotting.

Materials and Methods Animals. Forty male Lewis rats (6-8 weeks old, 150-200 g) were procured from a local breeder (SLC, Japan). They were accommodated under specific pathogen-free conditions. All trials were performed in reference to the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were allocated into four groups; each groups contained 10 rats. The four groups were as follows: (1) normal control group, (2) EAU induced by peptides of IRBP, (3) condition of (2) with Hemin (HO-1 inducer) treatment, and (4) condition of (2) with SnPP (Sn(IV) protoporphyrin IX dichloride, HO-1 inhibitor) treatment. Induction of EAU. The rats were immunized by a subcutaneous injection into the hind footpad with 100 µg of IRBP 1169-1191 (PTARSVGAADGSSWEGVGVVPDV) peptide (Peptron, Korea) in the emulsion with Complete Freunds adjuvant (CFA, 1:1, v/v). Rats were given 10 µM/kg hemin (Sigma) or 10 µM/kg SnPP (Frontier Scientific Inc.) by intraperitoneal injection once daily from 5 to 20 days after immunization. Normal control rats received injections with the same volume of saline on the same schedule. 3892

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Sample Preparation. The rats were sacrificed by overdose of Ketamine and Xylazine mixture. The vitreous body close to the posterior lens surface was disunited. The vitreous bodies were obtained from the underlying retina. The vitreous bodies were washed twice with Tris-sorbitol buffer (pH 7.0) and solubilized with 1 mL of lysis buffer containing 8 M urea (Sigma), 2% (w/v) CHAPS (Sigma), 50 mM DTT (Sigma), 40 mM Tris, 2% (v/v) pH 3-10 carrier ampholytes (Bio-Rad), and a trace of bromophenol blue. For solubilization of proteins, tipprobe sonication was employed for 4 × 10 s with 3 min on ice between each cycle of sonication. Two-Dimensional Electrophoresis. 2-DE was performed with Amersham Biosciences system as described by Jungblut and Thiede.26 One milligram of whole vitreous lysate was added to each immobilized pH gradient (IPG) strip (Bio-Rad, 3-10 NL strip), which had been rehydrated in 9 M urea, 2% CHAPS, 50 mM DTT, 0.2% Bio-Lyte 3-10 ampholyte, and 0.001% bromophenol blue (Bio-Rad, ReadyPrep 2-D start kit rehydration/ sample buffer). The preisoelectric focusing and isoelectric focusing (IEF) were performed using premade 18 cm length IPG strips on the Multiphor II (Amersham Biosciences). The pre-IEF was performed linearly up to 500 V for 1 h and subsequently held at 500 V for 2 h. IEF was then performed with a linear increase up to 3500 V over 3 h and then held at 3500 V for 80 000 Vh. For the second dimension, the IPG strips were equilibrated in a buffer containing 375 mM Tris-Cl, pH 8.8, 20% glycerol, 2% SDS, and 6 M urea with 2% dithiothreitol (Bio-Rad, ReadyPrep 2-D starter kit Equilibration buffer I) for 30 min and then moved to Equilibration buffer II (Bio-Rad, 375 mM Tris-Cl (pH 8.8), 20% glycerol, 2% SDS, and 6 M urea with 2.5% iodoacetamide) for 1 h on a rocker. The strips were subjected to an electrophoresis on 15% SDS-PAGE using a protean Plus Dodeca Cell (Bio-Rad). The gel was stained with Coomassie Blue G250 (Bio-Rad). In-Gel Digestion and Mass Spectrometric Analysis. Excised spots were cut into smaller pieces and digested using trypsin (Promega) as previously described.27-29 For MALDI-TOF/MS analysis, the trypsin peptides were concentrated by poros R2 and oligo R3 column (Applied Biosystems) and eluted in R-cyano-4-hydroxycinnamic acid. Spectra were obtained using a Voyager DE PRO MALDI-TOF spectrophotometer (Applied Biosystems). Protein database searching was performed with Matrix Science (http://www.matrixscience.com) using monoisotopic peaks. Protein function was cited either from the ExPASy database (http://kr.expasy.org/), the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov), KEGG (http://www.genome.ad.jp/kegg/), or publications cited below. Digestion of Samples for Micro LC/LC-MS/MS Analysis. The samples were diluted with 50 mM ammomium bicarbonate (pH 8.0) and 8 M urea and then reduced by adding tris (2carboxyethyl) phosphine hydrochloride (TCEP) at room temperature for 30 min and carboxyamidomethylated in 10 mM iodoacetamide at room temperature for 30 min in the dark. Endoproteinase Lys-C (Promega) was added to a final substrate to enzyme ratio of 100:1 and incubated at 37 °C for 12 h. The Lys-C digested solution was diluted 4-fold with 50 mM ammomium bicarbonate (pH 8.0) followed by the addition of CaCl2 to 2 mM. Modified trypsin (Promega) was added to a final substrate to enzyme ratio of 100:1. The trypsin digestion was performed by incubating at 37 °C for 14 h. Microcapillary columns were constructed with 100 µm i.d. fused silica capillary tubing pulled to a 5 µm i.d. tip by using a CO2 laser puller (Sutter Instruments, P-2000). The capillary

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Figure 1. 2-DE maps of proteins from the vitreous body of normal control Lewis rat. A total of 30 spots were identifiable; 27 of these spots were confirmed as members of the crystallin family. The 2-DE was performed on 15% SDS-PAGE gel with 1 mg of protein. The isoelectrofocus range is pH 3-10. Gels were stained with Coomassie blue.

column was packed sequentially with 7.5 cm of 5 µm i.d. Polaris C18-A (Metachem) and 4.5 cm of 5 µm i.d. Partisphere strong cation exchange (Whatman), followed by another 3.5 cm of Polaris C18-A using a homemade high-pressure column loader. The columns were equilibrated with 5% acetonitrile/0.1% formic acid solution, and about 10-25 µg of protein digests were directly loaded onto the capillary column. The buffer solutions used to separate protein digests were 5% acetonitrile/ 0.1% formic acid (buffer A), 80% acetonitrile/0.1% formic acid (buffer B), and 500 mM ammonium acetate/5% acetonitrile/ 0.1% formic acid (buffer C). Six steps of SCX/RPLC peptide separation were performed. The first step of 120 min consisted of a 95 min gradient from 0 to 100% buffer B and 25 min hold at 100% buffer B. The next 5 steps of 120 min gradient were each with 55% buffer B from 15 to 120 min. The buffer C percentages in step 2 were 5% from 10 to 15 min and in steps 3-6 were as follows: 10, 25, 50, and 100% from 5 to 9 min, respectively. Peptides eluted from the capillary column were electrosprayed into a LCQ Deca XP Plus ion trap mass spectrometer (ThermoFinnigan) with the application of a distal 2.4 kV spray voltage at a flow rate of 250 µL/min. A cycle of one full scan (400-1400 m/z) followed by three data-dependent MS/MS scans at a 35% normalized collision energy was repeated throughout the LC separation. MS/MS spectra were searched against IPI’s combined protein database (downloaded on Nov. 2004) of mouse and rat using Bioworks (v.3.1, Turbo SEQUEST). The following options were selected for the entire searches: no enzyme, (1.5 Da for parent ions, carbamidomethylation of cysteines as a fixed modification, and oxidation of methionine as differential modification. DTASelect and Contrast were used to filter the search results, and the following Xcorr values and delta Cn values were applied to different charge states of peptides (1.8 for singly charged peptides, 2.2

for doubly charged peptides, 3.2 for triply charged peptides, and delta Cn of 0.08) with a minimum number of tryptic digest termini(NTT) requirement of one. All identified proteins are listed in the supplementary table (see Supporting Information). Western Blot. The samples were performed by SDS-PAGE on a 15% polyacrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad). The membranes were blocked in TBS-T (Tris buffered saline with 0.05% Tween 20, Sigma) buffer containing 5% skim milk (BD Biosciences). They were then incubated with anti RA-crystallin polyclonal antibody (Stressgen), anti RB-crystallin polyclonal antibody (Stressgen), anti β-crystallin monoclonal antibody (Stressgen), anti-phospho RBcrystallin (ser19) polyclonal antibody (Stressgen), anti-phospho RB-crystallin (ser45) polyclonal antibody (Stressgen), and antiphospho RB-crystallin (ser59) polyclonal antibody (Stressgen) as a dilution of 1:2000 in TBS-T with 3% skim milk for 2 h at room temperature. After washing three times in TBS-T, the membrane was incubated with anti rabbit IgG secondary antibody or anti mouse IgG secondary antibody at a dilution of 1:2000 in TBS-T with 1% skim milk for 1 h at room temperature. The final products were made visible with a BCL kit (Amersham Biosciences).

Results Identification of Proteins used MALDI-TOF/MS Analysis. Vitreous bodies were subjected to 2-DE, and protein spots were analyzed with the ImageMaster 2D Platinum Software (version 5.0, Amersham Bioscience). A total of 359 spots were detected on the 2-DE gel of normal vitreous body from Lewis rats. Among the 68 spots analyzed with a MALDI-TOF mass spectrometer (MS), 30 spots were identified as protein (Figure 1, Table 1), 27 of them belonging to the crystallin family (Table Journal of Proteome Research • Vol. 6, No. 10, 2007 3893

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Table 1. Protein Identification by Peptide Mapping Using MALDI-TOF/MS in Vitreous Body of Lewis Rat spot no.

Mr (kDa)

pI

sequence coverage (%)

searched/ matched

Mascot score

expecta

protein

SwissProt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

42.08 22.49 23.34 22.49 22.43 25.71 25.71 24.35 23.34 57.14 22.46 22.49 23.34 23.34 23.34 22.46 20.07 22.49 22.49 22.49 22.46 23.34 22.49 22.49 22.49 23.34 39.65 22.49 22.49 20.07

5.30 6.35 6.54 6.35 6.35 6.17 6.17 7.28 6.54 8.84 5.90 6.35 6.54 6.54 6.54 5.90 6.76 6.35 6.35 6.35 5.90 6.54 6.35 6.35 6.35 6.54 8.39 6.35 6.35 6.76

34 20 48 28 26 34 38 70 37 25 58 34 37 42 41 46 53 49 69 54 50 23 41 30 53 41 34 42 33 42

20/ 9 21/ 5 28/13 23/ 5 16/ 4 55/11 58/10 35/17 17/ 7 41/ 8 39/11 33/ 7 21/ 6 42/10 42/11 47/ 8 49/ 7 46/11 45/15 45/12 34/10 14/ 4 40/10 39/ 7 49/10 53/10 46/ 8 46/11 26/ 5 33/ 7

106 57 137 56 53 73 66 229 83 54 138 72 75 99 114 82 71 122 175 136 128 52 113 66 103 102 66 119 53 64

7.3e-06 0.014 1.3e-10 0.014 0.033 0.00031 0.0015 8e-20 3.1e-05 0.022 1e-10 0.00036 0.0002 8.1e-07 2.5e-08 4.5e-05 0.00049 4e-09 2e-14 1.6e-10 1e-09 0.045 3.2e-08 0.0016 3.2e-07 4e-07 0.0015 8e-09 0.032 0.0023

β-actin RA-crystallin βB2-crystallin RA-crystallin RA-crystallin βA3-crystallin βA3-crystallin βB3-crystallin βB2-crystallin Cytochrome P450 2G1 βA4-crystallin RA-crystallin βB2-crystallin βB2-crystallin βB2-crystallin βA4-crystallin RB-crystallin RA-crystallin RA-crystallin RA-crystallin βA4-crystallin βB2-crystallin RA-crystallin RA-crystallin RA-crystallin βB2-crystallin Aldolase A RA-crystallin RA-crystallin RB-crystallin

P53485 P24623 P62697 CRYAA_RAT CRYAA_RAT P14881 P14881 P02524 P62697 P10610 P56374 P24623 P62697 P62697 P62697 P56374 P23928 P24623 P24623 P24623 P56374 P62697 P24623 P24623 P24623 P62697 P05065 P24623 P24623 P23928

a

P < 0.05

1). As shown in 2-DE gel images (Figure 2), the expressed patterns of crystallins from the differently treated four groups were coherent; therefore, we further analyzed the matching individual spots of truncated crystallins (Figure 3). Truncation of RA- and RB-Crystallins in EAU Condition. Though 12 spots were identified as RA-crystallins (Table 1), 3 of them were analyzed in the 2-DE gel because the spots had different levels among the four groups. Truncated RA-crystallin (spot 28) was dominant in the SnPP-treated uveitis samples, but the untruncated full length of RA-crystallin (spot 23) was abundant in the other three groups (Figure 3A, C). Two spots were recognized as RB-crystallin (spot 17 and 30 of Table 1). Untruncated spot No. 17 of RB-crystallin had high density in the normal control group but truncated form (spot 30) was dominant in the IRBP-induced uveitis group. SnPPtreated uveitis samples were more truncated; on the other hand, the Hemin-treated uveitis sample appeared close to the normal control (Figure 3 B, D). Truncation of βA4- and βB2-Crystallins in Normal Condition. The three spots, 11, 16, and 21, were identified as βA4crystallin (Table 1). The truncated form of the crystallin was major in the normal control group. However, the full length of the βA4-crystallin (22.49 kDa) was dominant (spot 11) in the IRBP-induced uveitis group and the SnPP-treated uveitis group (Figure 4 A, C). The truncated form βA4-crystallin of the Hemin treated uveitis group was increased about four times than that of the two other groups, IRBP-induced uveitis group and SnPP treated uveitis group (Figure 4 C). Spot 11 revealed full length of βA4-crystallins; however, spots 16 and 21 were a lost sequence piece of the C-terminal part that was EWGSHAHTFQVQSVR, which had an average m/z of 1768.9 (data not shown). 3894

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A total of eight spots were recognized as βB2-crystallins (spot 3, 8, 9, 13, 14, 15, 22, and 26 of Table 1), but three of them were analyzed in this study as the RA-crystallin. The truncated form (spot 15) of βB2-crystallin was major in the normal control group. On the other hand, the intact form of βB2-crystallin (spot 9) was expressed in the IRBP-induced uveitis group, and they were more exaggerated in the SnPP-treated uveitis groups, but they went back to the normal control in the Hemin-treated uveitis group (Figure 4 B, D). Protein Identification with LC-MS/MS. We performed protein identification using micro LC/LC-MS/MS analysis for four groups of crude vitreous body proteins. There were 215 proteins in the normal control, 201 proteins of IRBP-induced uveitis, 185 proteins of SnPP treated uveitis-induced groups, and 155 proteins of IRBP with Hemin-treated uveitis groups identified (supplementary table). Various crystallin family proteins were detected in rat vitreous body, as RA-, RB-, βA1-, βA2-, βΑ3-, βΑ4-, βΒ1-, βΒ2-, βΒ3-, γA-, γB-, γC-, γD-, γE-, γF-, and γN-crystallin. R-Enolase 1, moonlight protein of τ-crystallin, was detected in all groups. Lactate dehydrogenase/ -crystallin was detected in normal vitreous. Transketolase as a moonlight protein of RB-crystallin was detected in IRBP and IRBP with SnPP-treated groups. Many heat shock proteins were presented in EAU-induced vitreous body as hsp 27, hsp 70, hsp 71, and hsp 89-R-δ-N. Hsp 27 and hsp 89-R-δ-N were commonly detected. Hemin-treated uveitis-induced groups were revealed as hsp 27, hsp 71, and hsp 86. Inflammationrelated proteins were detected as IL-4 induced protein 1 precursor, TGF β-inducible nuclear protein 1 (HUSSY-29), and NF-κB inhibitor in IRBP-induced uveitis vitreous body. Vimentin was a commonly detected. T-cell receptor R-chain was only detected in Hemin treated uveitis group. Interestingly, Glu-

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Figure 2. 2-DE gel images of the vitreous body. (A) Normal control, (B) IRBP-induced uveitis vitreous body, (C) Hemin-treated uveitisinduced vitreous body, and (D) SnPP-treated uveitis-induced vitreous body. Protein samples (1 mg) were loaded on 15% SDS-PAGE gel. The isoelectrofocus ranges are pH 3-10. Gels were stained with Coomassie blue.

tathione S-transferase was detected in SnPP-treated uveitis vitreous body. Glucose phosphate isomerase 1 (GPI) was presented in IRBP-induced uveitis group and SnPP-treated uveitis group. Western Blotting for Phosphorylation. We performed Western blotting of anti RA-, RB-, or β-crystallins for verification of crystallin expression in each group. RA-crystallin was expressed similarly in the three groups except in SnPP-treated uveitis group (Figure 5A). It is suitable to the RA-crystallin spots of the 2-DE (Figure 3A). RB-crystallin was detected in almost the same patterns for all four groups (Figure 5B); however, phosphorylated RB-crystallins were variably expressed. Serine-19phosphorylation of RB-crystallin was more expressed in the IRBP-induced uveitis group and Hemin-treated uveitis group than the normal control, but it was less expressed in the SnPPtreated uveitis group (Figure 6A). Twenty kiloDaltons of serine45-phosphorylated RB-crystallin was decreased in the SnPPtreated uveitis group, but the 17 kDa band was increased (Figure 6B). Serine 59-phosphorylated crystallin was increased in the IRBP-induced uveitis group; however, it disappeared in the SnPP-treated uveitis group (Figure 6C). Interestingly, β-crystallins were increased in the IRBP-induced uveitis group compared to the normal control, and they were more increased in the SnPP-treated uveitis group, but they were similar to normal control in the Hemin-treated uveitis group (Figure 5C).

Discussion The crystallins (R, β, and γ) have been known to be structural proteins of mammalian lens. However, the significance of crystallins is recently recognized as functional proteins that increase intracellular stability by reason of their chaperone activity and possibly due to the interaction with cell signaling pathways.15,30,31 In crystallins, the post-translational modifications, such as truncation and phosphorylation and their related functions, are not well-defined yet. Furthermore, the heat shock protein RB-crystalline is ubiquitously expressed in several human diseases32-34 and known to mediate a pro- or antiinflammatory reaction.35 On the basis of the known functions of HO-1,36,97 we induced EAU in Lewis rats with peptides of IRBP then treated them with HO-1 inducer or HO-1 inhibitor to define a regulatory mechanism of crystallins. Through the analysis of using 2-DE, MALDI-TOF/MS, Western blotting, and micro LC/LC-MS/MS (supplementary table), four groups of crude vitreous proteins were identified. In IRBP-induced EAU, we found that RA-, RB-, βA4-, and βB2-crystallins function through structural modifications by truncation or by phosphorylation. In healthy normal vitreous body, the truncated βA4- and βB2-crystallins were abundantly detected. In inflammatory condition of EAU, however, the full lengths of βA4- and βB2crystallins were the abundant ones, and this result was Journal of Proteome Research • Vol. 6, No. 10, 2007 3895

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Figure 3. (A) RA-crystallins spot images. (B) RB-crystallin spot images. (C) Bar graph of RA-crystallin spot density. (D) Bar graph of RB-crystallin spot density. Normal, normal control vitreous body; IRBP, IRBP-induced uveitis vitreous body; IRBP+SnPP, SnPP treated uveitis-induced vitreous body; IRBP+Hemin, Hemin-treated uveitis-induced vitreous body.

Figure 4. (A) βA4-crystallins spot images. (B) βB2-crystallin spot images. (C) Bar graph of βA4-crystallin spot density. (D) Bar graph of βB2-crystallin spot density. Normal, normal control vitreous body; IRBP, IRBP-induced uveitis vitreous body; IRBP+SnPP, SnPP treated uveitis-induced vitreous body; IRBP+Hemin, Hemin treated uveitis-induced vitreous body.

consistent with our previous study of endotoxin-induced uveitis.25 The data suggest that overexpression of βA4- and βB2crystallin and the truncation of the RA- and RB-crystallin are correlated with the IRBP-induced inflammation. These findings were supported by the results of Western blotting with polyclonal antibodies (against RA- and RB-crystallin) and monoclonal antibody against β-crystallin (Figure 5). Unlike the uveitic vitreous body that was treated with SnPP, the induction of HO-1 resulted in a similar pattern with the expressions of normal control. But it is unclear whether the reduced inflammatory response of Hemin-treated groups is due to tissue3896

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specific chaperone activity or to a signal transduction that is possibly mediated by HO-1. It is also plausible that the peptides, degraded βA4- and βB2-crystallin in EAU condition, could provide signals that activate the immune system or they may down-regulate inflammatory responses in retinal tissue or uveitic vitreous body. Phosphorylation is known to activate the target proteins functionally.18 Unexpectedly, more phosphorylations of RBcrystalline were observed in the Hemin-treated uveitic vitreous body than in the IRBP-induced and SnPP-treated uveitic vitreous bodies. The phosphorylations of RB-crystallin (in the

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Crystallins in Vitreous of EAU

Figure 5. Western blotting of vitreous bodies for (A) anti-RA-crystallin polyclonal antibody, (B) anti-RB-crystallin polyclonal antibody, and (C) anti-β-crystallin monoclonal antibody. Lane 1, normal control vitreous body; lane 2, IRBP-induced uveitis vitreous body; lane 3, SnPP treated uveitis-induced vitreous body; lane 4, Hemin treated uveitis-induced vitreous body.

Figure 6. Western blotting of rat vitreous body for (A) antiphospho (ser19) RB-crystallin polyclonal antibody, (B) antiphospho (ser45) RB-crystallin polyclonal antibody, and (C) antiphospho (ser59) RB-crystallin polyclonal antibody. Lane 1,normal control vitreous body, lane 2; IRBP-induced uveitis vitreous body; lane 3,SnPP treated uveitis-induced vitreous body, lane 4; Hemin treated uveitis-induced vitreous body.

phosphorylation was also enhanced by HO-1. New production of βA4- and βB2-crystallins was increased whereas the truncated ones were degraded (Figure 7). In the IRBP-induced EAU model system, we conclude that RA-, RB-, βA4-, and βB2crystallins might function in uveitic vitreous body through structural modifications of truncation and phosphorylation. The property of crystallins that increase intracellular stability would be a therapeutic target for the treatment of chronic eye inflammatory diseases. Abbreviations: EAU, experimental autoimmune uveitis; HO1, heme oxygenase-1; HSP, heat-shock protein; IRBP, interphotoreceptor retinoid-binding protein.

Acknowledgment. This study was supported by the grants from Korea Health 21 R&D Project (Ministry of Health and Welfare, Republic of Korea, A010251 & A030003) and partially by Wonkwang University. Supporting Information Available: Protein identification using micro LC/LC-MS/MS analysis for four groups of crude vitreous proteins showed 215 proteins in the normal control, 201 proteins of IRBP-induced uveitis, 185 proteins of SnPP treated uveitis-induced groups, and 155 proteins of IRBP with Hemin treated uveitis-induced groups. This material is available free of charge via the Internet at http://pubs.acs.org. References

Figure 7. Schematic diagram of crystallin changes of vitreous body from normal to EAU. RA- and RB-crystallins are truncated, and phosphorylated RB-crystalline is increased in the EAU. The phosphrylation is also enhanced by HO-1. New production of βA4- and βB2-crystallins is increased, whereas the truncated ones are degraded.

ser19, ser45, and ser59) did not exactly correspond to the degree of inflammation (in normal control, IRBP-induced, SnPPtreated, and Hemin-treated uveitis vitreous bodies). The discrepancy between phosphorylations of RB-crystallin and the status of inflammation indicates that HO-1 causes induction of RB-crystallin phosphorylation. This observation suggests that the induction of HO-1 could be cytoprotective through antiinflammatory effects; however, additional works are needed to refine immunological regulatory functions of crystallin. In summary, RA- and RB-crystallins were truncated, and the RB-crystallin was phosphorylated in the EAU conditions. The

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