Scholarly article on topic 'An Increase in Phosphorylation and Truncation of Crystallin With the Progression of Cataracts'

An Increase in Phosphorylation and Truncation of Crystallin With the Progression of Cataracts Academic research paper on "Clinical medicine"

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Current Therapeutic Research
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{cataract / crystallin / "mass spectrometry" / phosphorylation / truncation}

Abstract of research paper on Clinical medicine, author of scientific article — Hui-Ju Lin, Chien-Chen Lai, Shiuan-Yi Huang, Wei-Yi Hsu, Fuu-Jen Tsai

Abstract Background Cataracts are the leading cause of blindness worldwide; however, there is no evidence regarding the direct formation of cataracts. At present, there is no treatment method other than surgery to prevent the formation or progression of cataracts. Objective Understanding the protein changes during various stages of cataracts might help realize the mechanism of the formation and progression of cataracts. Methods Lens materials were collected from cataract surgery. Cataracts were classified according to lens opacity using the gradation of the Lens Opacities Classification System. Lens proteins were separated by 2-dimensional polyacrylamide gel electrophoresis. Protein spots were visualized by Coomassie blue staining, and expression patterns were analyzed. Protein spots of interest were excised from 2-dimensional polyacrylamide gel electrophoresis gels, digested in situ with trypsin, and analyzed by mass spectrometry and liquid chromatographic tandem mass spectrometry. Results Crystallin was the major protein in the cataract lens, and αA, βB1, αB, and βA4 were the dominant types. Crystallin αB and βA4 increased with the formation of lens opacity. Moreover, phosphorylation and truncation of these proteins increased with the progression of cataracts. Conclusion Crystallin αB and βA4 and phosphorylation and truncation of crystallin in the lens might contribute to the formation of cataracts. In contrast, acetylation was not dominant in the progression of cataracts and did not play major role in the formation of cataracts.

Academic research paper on topic "An Increase in Phosphorylation and Truncation of Crystallin With the Progression of Cataracts"

ELSEVIER

An Increase in Phosphorylation and Truncation of Crystallin With the Progression of Cataracts $

Hui-Ju Lin, MD,1,2'3,y Chien-Chen Lai, PhD,2At Shiuan-Yi Huang, MS,2 Wei-Yi Hsu, MS,2 Fuu-Jen Tsai, MD, PhD2,3*

1 Department of Ophthalmology, China Medical University Hospital, Taichung, Taiwan

2 Department of Medical Science, China Medical University Hospital, Taichung, Taiwan

3 Department of Chinese Medicine, China Medical University, Taichung, Taiwan

4 Institute of Molecular Biology, National Chung Hsing University, Taichung, Taiwan

ARTICLE INFO ABSTRACT

Background: Cataracts are the leading cause of blindness worldwide; however, there is no evidence regarding the direct formation of cataracts. At present, there is no treatment method other than surgery to prevent the formation or progression of cataracts.

Objective: Understanding the protein changes during various stages of cataracts might help realize the mechanism of the formation and progression of cataracts.

Methods: Lens materials were collected from cataract surgery. Cataracts were classified according to lens opacity using the gradation of the Lens Opacities Classification System. Lens proteins were separated by 2-dimensional polyacrylamide gel electrophoresis. Protein spots were visualized by Coomassie blue staining, and expression patterns were analyzed. Protein spots of interest were excised from 2-dimensional polyacrylamide gel electrophoresis gels, digested in situ with trypsin, and analyzed by mass spectrometry and liquid chromatographic tandem mass spectrometry. Results: Crystallin was the major protein in the cataract lens, and aA, pB1, aB, and pA4 were the dominant types. Crystallin aB and pA4 increased with the formation of lens opacity. Moreover, phosphorylation and truncation of these proteins increased with the progression of cataracts. Conclusion: Crystallin aB and pA4 and phosphorylation and truncation of crystallin in the lens might contribute to the formation of cataracts. In contrast, acetylation was not dominant in the progression of cataracts and did not play major role in the formation of cataracts.

© 2013. The Authors. Published by Elsevier Inc. All rights reserved.

Contents lists available at SciVerse ScienceDirect

Current Therapeutic Research

journal homepage: www.elsevier.com/locate/cuthre

CrossMark

Article history: Accepted 11 October 2012

Key words:

cataract

crystallin

mass spectrometry

phosphorylation

truncation

Introduction

The effect of cataracts on vision is often described as being similar to looking through a waterfall or a piece of waxed paper. Poor vision from cataracts affects 80% of people aged >75 years.1 This disease causes clouding of the eye lens, which reduces the amount of incoming light and deteriorates vision. Daily functions such as reading or driving a car may become difficult or impossible.2

Thus, patients may require frequent change in eyeglass prescrip-tions.3 It is estimated that 200 million people have cataracts

$This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

* Address correspondence to: Fuu-Jen Tsai, MD, PhD, Department of Medical Genetics and Pediatrics, China Medical University Hospital, No. 2 Yuh-Der Road, Taichung 404, Taiwan.

E-mail address: irisluu2396@gmail.com (F.-J. Tsai). yThese authors contributed equally to this work.

worldwide.4,5 Data from the National Institutes of Health indicate that >350,000 cataract surgeries are performed annually in the United States.

The lens is the clear part of the eye that helps to focus light and images on the retina.6 The retina is the light-sensitive tissue at the back of the eye. In a normal eye, light passes through the transparent lens to the retina. Once it reaches the retina, light is changed into nerve signals that are sent to the brain.7 The lens must be clear for the retina to receive a sharp image. If the lens is cloudy as a result of cataracts, the image will be blurred. The lens is mostly made of water and protein. The proteins are arranged to let light pass through and focus on the retina. Sometimes, some lens proteins clump together and begin to cloud a small area of the lens. Over time, the cells accumulate and cause the lens to cloud, thereby resulting in blurred or fuzzy images.4,8 Cataracts are the leading cause of visual loss among adults >55 years old. Cataract surgery costs Medicare more money than any other medical procedure, with 60% of those who initially qualify for Medicare already having cataracts.9 Most people are concerned regarding the time of onset of cataracts and not about its

0011-393X/$-see front matter © 2013. The Authors. Published by Elsevier Inc. All rights reserved. http ://dx.doi.org/10.1016/j.curtheres.2012.10.003

occurrence. Hence, preventative steps at an early stage in life may lead to good eye health and prevent cataracts.10 Many factors influence vision and cataract development, for example, age, nutrition, heredity, medications, toxins, health habits, sunlight exposure, and head trauma. Cataracts can also be caused by high blood pressure, kidney disease, diabetes, or direct trauma to the eye.11,12 Although cataract surgeries have advanced progressively, cataracts are still the leading cause of blindness and are a profound economic cost to society.13-15 In this study, we used 2-dimensional polyacrylamide gel electrophoresis (2D-PAGE) to identify the proteins that change during the formation of lens opacity and liquid chromatographic tandem mass spectrometry (LC/MS/MS) to evaluate post-translation modifications of the proteins. Investigating the protein changes during various stages of cataracts elucidated the mechanism of the formation of cataracts and might be helpful in designing new therapies.

Materials and Methods

Lens materials were obtained during cataract surgery. All patients in this study received serial ophthalmic examinations, including intraocular pressure (IOP), visual acuity, and retinal examination. Patients with ocular diseases other than cataracts were excluded. Patients (38 women and 42 men), aged 56 to 85 years (mean, 72 years), were followed up after 3 to 24 weeks (mean, 5 weeks). The study was performed according to the tenets of the Declaration of Helsinki for research involving human participants.

Patients with Stage 2 to 5 cataracts were enrolled in the study. The patients did not have any systemic diseases or eye diseases other than cataracts. They underwent phacoemulsification surgery using a phacoemulsification machine (Universal II; Alcon, Houston, Texas). Lens materials were collected into bags by the machine after quaking the lens into small particles by ultrasound. Lens opacity was classified according to the Lens Opacities Classification System (LOCS) before surgery.16,17 This classification involves comparison of the slit lamp view of the lens to a color plate of LOCS III standards. LOCS uses standard reference photographs taken during slit lamp examination. The extent of opacification of cortical (C) and posterior subcapsular (P) changes was defined, and color changes of the nucleus as well as the intensity of nuclear opalescence was noted using LOCS. Lens opacity was scored according to the description in LOCS, for example, N0 and NIV were denoted as 0 and 4, respectively. After adding the scores of the 4 parts (nuclear color, nuclear opacity, cortical cataracts, and posterior subcapsule), a total score (0-15) was obtained. These scores were used to categorize cataracts into 5 stages: score 0, Stage 1; scores 1 to 4, Stage 2; scores 5 to 8, Stage 3; scores 9 to 12, Stage 4; and scores 13 to 15, Stage 5.

Two-dimensional Gel Electrophoresis

Sample Preparation and Running of Gels

An aliquot containing 100 mg of protein sample was diluted with 350 mL of rehydration buffer containing 8-M urea, 4% [3-(3-cholamidopropyl)dimethylammonio]-1- propanesulfonate], 65-mM dithioerythritol (DTE), 0.5% ampholytes, and a trace of bromophenol blue. An immobilized pH gradient (17 cm; pH 3-10; ReadyStrip IPG strip; Bio-Rad, Tokyo, Japan) was hydrated overnight, and the samples were focused for a total of 60 kVh (PROTEAN IEF cell; Bio-Rad) at 20°C and then stored at - 80°C. Strips were equilibrated with 3 mL of an equilibrium solution containing 50-mM Tris-hydrocholride (pH 8.8), 6-M urea, 30%

glycerol, 2% sodium dodecyl sulfate (SDS), a trace of bromophenol blue, and DTE (1% w/v) for 20 minutes, followed by equilibration for 20 minutes in the same solution containing iodoacetamide (IAA; 2.5% w/v) instead of DTE. The strips were transferred to the tops of 12% PAGE and held in position with molten 0.5% agarose in running buffer containing 25-mM Tris, 0.192-M glycine, and 0.1% SDS. The gels were run at 16 mA for 30 minutes, followed by 50 mA for 4 to 5 hours.

Detection of Protein Spots and Data Analysis

The gels were routinely stained with Coomassie blue and then scanned using a GS-800 imaging densitometer with PDQuest software (version 7.1.1; Bio-Rad). To evaluate intra- and inter-sample variability, the gels were analyzed as follows: protein spots from each gel were detected and matched automatically to generate a master gel image from the matched gel sets. Finally, the intensity of the spots was compared between gels. Data were exported to Microsoft Excel (Microsoft Inc, Redmond, Washington) for creating correction and spot intensity graphs.

In-Gel Digestion

The procedure of Terry et al18 was slightly modified and used for in-gel digestion of proteins from the Coomassie blue-stained gels for nanoscale capillary LC/MS/MS. In brief, each spot of interest on the Coomassie blue-stained gel was sliced into 1-mm cubes. The proteins in these gels were reduced and methylated with 50-mM DTE and 100-mM IAA in 50-mM ammonium bicarbonate. The gel pieces were washed 2 times with 50% v/v acetonitrile (ACN) in 100-mM ammonium bicarbonate buffer (pH 8.0) for 10 minutes at room temperature. They were then soaked in 100% ACN for 5 minutes, dried in a lyophilizer for 20 to 30 minutes, and rehydrated in 50-mM ammonium bicarbonate buffer (pH 8.0) containing 10 mg/mL trypsin (Promega, Madison, Wisconsin) until fully immersed. After incubating for 16 to 20 hours at 30°C, the remaining trypsin solution was transferred into a new microtube. The gel pieces were resuspended with 50% ACN in 5.0% formic acid (FA) for 60 minutes, and then concentrated to dryness.

Nanoelectrospray Mass Spectrometry

Nanoscale capillary LC/MS/MS was used to analyze the proteins involved in the development of cataracts. The Ultimate Capillary LC System (LC Packings, Amsterdam, the Netherlands) coupled to a QSTARXL quadrupole-time of flight (Q-TOF) mass spectrometer (Applied Biosystem/MDS Sciex, Foster City, California) was used for analysis. Nanoscale capillary LC separation was performed on a reverse phase C18 column (15 cm x 75 mm inner diameter) with a flow rate of 200 nL/min and a 60-minute linear gradient of 5% to 50% buffer B. Buffer A contained 0.1% FA in 5% aqueous ACN, and buffer B contained 0.1% FA in 95% aqueous ACN. The nano-LC tip for online LC/MS was a PicoTip (FS360-20-10-D-20; New Objective, Cambridge, Massachusetts). Data acquisition was performed using automatic information dependent acquisition (IDA; Applied Biosystem/MDS Sciex). Automatic IDA finds the most intense ions in TOF MS spectra and then performs optimized MS/MS analysis on these ions. The product ion spectra generated by nano-LC/MS/MS were searched against National Center for Biotechnology Information (NCBI) databases for exact matches using the ProID program (Applied Biosystem/MDS Sciex) and the MASCOT search program (MASCOT search program; Matrix Science, Inc, Boston, Massachusetts). A mammalian taxonomy restriction was used, and the mass tolerance of both

3.2 4 5 5.5 6.0 7.0 8.0 9.0

Figure 1. Spots 1 to 44 existed in Stage 2 cataracts. MW, molecular weight.

precursor and fragment ions was set to + 0.3 Da. Carbamido-methyl cysteine was set as a fixed modification, whereas phosphorylation of serine, threonine, and tyrosine, and other modifications were set as variable modifications. All identified phosphopeptides were confirmed by manual interpretation of the spectra.

Results

Lens materials from various stages were prepared for 2D-PAGE. To identify protein expression, master gels were computed from scanned images of quartet silver-stained gels. The scanned gel images were processed using the Proteomics Software System developed by Xzillion (Frankfurt am Main, Germany). The master gel was computed by registering and jointly segmenting multiple registered replicates. Algorithmic details can be found in the SEQUEST algorithm (version C1; Thermo Fisher Scientific, Waltham, Massachusetts) incorporated

M.W (kDa)

Figure 3. Spots 92 to 129 existed in Stage 4 cataracts. MW, molecular weight.

into the ThermoFinnigan BIOWORKS software (version 3.0; Thermo Fisher Scientific). Spot volumes were determined by modeling optical density of individual spot segments using 2D Gaussian analysis. To correct for variability due to gel electro-phoresis, quartet gels were run for each cataract stage. In addition, spots expressed in < 50% of the gels were disregarded. Furthermore, upregulation of proteins was considered significant when the corresponding spot volumes were increased by more than twofold. Representative master gels showed proteins expressed in different stages of cataracts. The proteins varied between 10 and 120 kDa in size and had isoelectric point (pI) values ranging from 5 to 9 (Figures 1-4).

No patient with Stage 1 cataracts underwent surgery; therefore, data were collected from patients with Stage 2 to 5 cataracts. Spots 1 to 44 were expressed in Stage 2 (Figure 1 and Tables I and II). After analysis, the major proteins in Stage 2 were crystallin PB1, aB, a A, and PA4. Eleven spots were identified as crystallin PB1. Of these, 5 were phosphorylated, 1 was acetylated, and none was truncated. In addition, 11 protein spots were identified as

Table I

Proteins identified from mass spectrometry.

Table I (continued)

Spot Accession Protein Identification MW No. No. (kDa) /pI

1 P02489

2 P02489

3 P02489

4 P02489

5 P02489

6 P02489

7 P02489

8 P02489

9 P53673

10 P53673

11 P53673

12 P53673

13 P02489

14 P53673

15 P53674

16 P05813

17 P53673

18 P07320

19 P53674

20 P53674

21 P53674

22 P53674

23 P53674

24 P53674

25 P53674

26 P53674

27 P53674

28 P53674

29 P43320

30 P07315

31 P07320

32 P07315

33 P02511

34 P02511

35 P02511

36 P02511

37 P02511

38 P02511

39 P02511

40 P02489

41 Q01469

42 P02489

43 P02489

44 P02489

45 P02489

46 P02489

47 P02489

48 P02489

49 P22914

50 P53674

51 P02489

52 P02489

53 P05813

54 P05813

55 P53674

56 P05813

57 P53674

58 P05813

59 P05813

60 P02511

61 P43320

62 P53674

63 P53674

64 P53674

65 P53674

66 P43320

67 P05813

68 P53674

69 P53674

70 P02511

71 P02511

a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain b-Crystallin A4 b-Crystallin A4 b-Crystallin A4 b-Crystallin A4 a-Crystallin A chain b-Crystallin A4 b-Crystallin B1 b-Crystallin A3 b-Crystallin A4 g-Crystallin D b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B2 g-Crystallin C g-Crystallin D g-Crystallin C a-Crystallin B chain a-Crystallin B chain a-Crystallin B chain a-Crystallin B chain a-Crystallin B chain a-Crystallin B chain a-Crystallin B chain a-Crystallin A chain Fatty acid-binding protein

a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain a-Crystallin A chain b-Crystallin S b-Crystallin B1 a-Crystallin A chain a-Crystallin A chain b-Crystallin A3 b-Crystallin A3 b-Crystallin B1 b-Crystallin A3 b-Crystallin B1 b-Crystallin A3 b-Crystallin A3 a-Crystallin B chain b-Crystallin B2 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B1 b-Crystallin B2 b-Crystallin A3 b-Crystallin B1 b-Crystallin B1 a-Crystallin B chain a-Crystallin B chain

19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 19.9/5.77 22.2/5.82 27.9/8.59 25.1/5.81 22.2/5.82 20.6/7.15 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 23.2/6.54 20.7/7.04 20.6/7.15 20.7/7.04 20.1/6.76 20.1/6.76 20.1/6.76 20.1/6.76 20.1/6.76 20.1/6.76 20.1/6.76 19.9/5.77 15.0/6.84

19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 20.9/6.43 27.9/8.59 19.9/5.77 19.9/5.77 25.1/5.81 25.1/5.81 27.9/8.59 25.1/5.81 27.9/8.59 25.1/5.81 25.1/5.81 20.1/6.76 23.2/6.54 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 23.2/6.54 25.1/5.81 27.9/8.59 27.9/8.59 20.1/6.76 20.1/6.76

Spot Accessio

Score Post- translational Modification No. No.

72 P02511

448 Ac Pi 73 P02511

464 Ac Pi 74 P05813

511 Ac Pi 75 P07320

375 Pi 76 P02511

561 Ac Pi 77 P02489

642 Ac Pi 78 P53673

383 Pi 79 P02489

365 Ac 80 P53673

526 Pi 81 P53673

407 Pi 82 P53673

510 83 P53673

546 Pi 84 P53673

475 86 P02489

413 87 P02489

1068 Pi 88 P02489

665 Ac Pi 89 P02489

541 90 P02489

519 91 P02489

1077 Ac Pi 92 P02489

928 93 P53674

1177 Pi 94 P53674

1145 95 P53674

1073 96 P53674

1215 Pi 97 P53674

1135 Pi 98 P53674

1081 Pi 99 P53674

932 100 P05813

926 101 P05813

679 102 P53674

650 103 P53674

450 104 P05813

665 105 P53674

515 106 P05813

607 Pi 107 P53674

591 Pi 108 P53674

694 Pi 109 P53674

565 110 P53674

407 111 P53674

338 112 P53674

409 Ac 113 P05813

207 114 115 P05813 P07315

441 116 P02511

309 117 P02511

402 118 P02489

354 Ac Pi 119 P53673

481 Ac Pi 120 P53673

557 Ac Pi 121 P53673

661 Ac Pi 122 P02489

515 Ac Pi 123 P53673

586 124 P53673

597 Ac Pi 125 P53673

564 Ac Pi 126 P53673

569 Pi 127 P53673

708 Ac Pi 128 P53673

1105 Ac Pi 129 P02489

753 Pi 130 P53673

1051 Ac Pi 131 P02489

688 Pi 132 P02489

847 Pi 133 P02489

595 Pi 134 P02489

437 Ac Pi 135 P02489

1033 Pi 136 P53673

637 137 P53673

1022 Pi 138 P02489

1082 Pi 139 P53673

703 140 P02489

640 141 P02489

1058 Pi 142 P02489

1366 Pi 143 P02489

791 144 P02489

497 145 P02489

Protein Identification

(kDa) /pI

Score Post-

translational Modification

a-Crystal a-Crystal b-Crystal g-Crystal a-Crystal a-Crystal b-Crystal a-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal g-Crystal a-Crystal a-Crystal a-Crystal b-Crystal b-Crystal b-Crystal a-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal b-Crystal a-Crystal b-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal b-Crystal b-Crystal a-Crystal b-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal a-Crystal

lin B chain lin B chain lin A3 lin D

lin B chain lin A chain lin A4 lin A chain lin A4 lin A4 lin A4 lin A4 lin A4 lin A chain lin A chain lin A chain lin A chain lin A chain lin A chain lin A chain lin B1 lin B1 lin B1 lin B1 lin B1 lin B1 lin B1 lin A3 lin A3 lin B1 lin B1 lin A3 lin B1 lin A3 lin B1 lin B1 lin B1 lin B1 lin B1 lin B1 lin A3 lin A3 lin C

lin B chain lin B chain lin A chain lin A4 lin A4 lin A4 lin A chain lin A4 lin A4 lin A4 lin A4 lin A4 lin A4 lin A chain lin A4 lin A chain lin A chain lin A chain lin A chain lin A chain lin A4 lin A4 lin A chain lin A4 lin A chain lin A chain lin A chain lin A chain lin A chain lin A chain

20.1/6.76 20.1/6.76 25.1/5.81 20.6/7.15 20.1/6.76 19.9/5.77 22.2/5.82 19.9/5.77 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 25.1/5.81 25.1/5.81 27.9/8.59 27.9/8.59 25.1/5.81 27.9/8.59 25.1/5.81 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 27.9/8.59 25.1/5.81 25.1/5.81 20.7/7.04 20.1/6.76 20.1/6.76 19.9/5.77 22.2/5.82 22.2/5.82 22.2/5.82 19.9/5.77 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 22.2/5.82 19.9/5.77 22.2/5.82 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 22.2/5.82 22.2/5.82 19.9/5.77 22.2/5.82 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77 19.9/5.77

510 466 427 216 239 373

511 442 523 478 436 362 364 309 278 201 234 247 135 155 432 718 337 945 889 1009 691 551 1071 1029 608 1214 621 786 997 923 1161 927 876 551 475 465 170 239 206 192 447 539 296 424 475 481 444

512 396

485 402

513 494 641 707 506 517 570 579 553 460 409 329 553 755 641

Ac Pi Pi

Ac Pi Ac Pi Ac Pi Ac Pi Ac Pi

Ac Pi Pi

Ac Pi Ac Pi

Ac Pi Pi

Table I (continued)

Spot Accession Protein Identification MW Score Post-

No. No. (kDa) /pi translational Modification

146 P53673 P-Crystallin A4 22.2/5.82 372

147 P02489 a-Crystallin A chain 19.9/5.77 308

149 P53673 P-Crystallin A4 22.2/5.82 405

150 P02489 a-Crystallin A chain 19.9/5.77 439 Pi

151 P07320 g-Crystallin D 20.6/7.15 458

152 P02511 a-Crystallin B chain 20.1/6.76 775 PiAc

153 P07320 g-Crystallin D 20.6/7.15 445

154 P02511 a-Crystallin B chain 20.1/6.76 882 Pi

155 P02511 a-Crystallin B chain 20.1/6.76 948 PiAc

156 P43320 P-Crystallin B2 23.2/6.54 1292 Ac

157 P43320 P-Crystallin B2 23.2/6.54 1346 PiAc

158 P02511 a-Crystallin B chain 20.1/6.76 693

159 P02511 a-Crystallin B chain 20.1/6.76 489

160 P02511 a-Crystallin B chain 20.1/6.76 895 PiAc

161 P02511 a-Crystallin B chain 20.1/6.76 982 PiAc

162 P02511 a-Crystallin B chain 20.1/6.76 964 Pi

163 P02511 a-Crystallin B chain 20.1/6.76 268

164 P02511 a-Crystallin B chain 20.1/6.76 228 Pi

166 P02511 a-Crystallin B chain 20.1/6.76 758

167 P02511 a-Crystallin B chain 20.1/6.76 821 Pi

169 P02511 a-Crystallin B chain 20.1/6.76 282

170 P02511 a-Crystallin B chain 20.1/6.76 307

171 P02489 a-Crystallin A chain 19.9/5.77 314

172 P02489 a-Crystallin A chain 19.9/5.77 255 Ac

173 P02489 a-Crystallin A chain 19.9/5.77 284

175 P02489 a-Crystallin A chain 19.9/5.77 143

Ac, acetylation; MW, molecular weight; Pi, phosphorylation; PiAc, phosphorylation, acetylation; pi, isoelectric point.

crystallin aB in Stage 2. Of these, 5 were phosphorylated, 1 was both phosphorylated and acetylated, and none was truncated. Eleven spots were identified as crystallin aA. Six of these were phosphorylated, 6 were acetylated, 5 were both phosphorylated and acetylated, and all were truncated. Eight spots were identified as crystallin PA4. Of these, 3 were phosphorylated and none was acetylated. However, all crystallin PA4 proteins were truncated. Among the protein spots in Stage 2, 46.3% were phosphorylated (Tables I and II); the ratio of phosphorylated to nonphosphorylated proteins was 17:24 (41.46% phosphorylated proteins). Acetylated proteins were more abundant than nonacetylated ones; the ratio of acetylated to nonacetylated proteins was 8:33. Only 19.5% of crystallin proteins in Stage 2 were acetylated. The ratio of truncated to nontruncated proteins was 19:22 (46.3% truncated) (Tables I and II). The proportions of phosphorylated and truncated proteins were high in Stage 2, whereas that of acetylated proteins was low.

The major proteins associated with Stage 3 (Figure 2 and Tables

1 and II) were crystallin aA, PB1, PA3, and PA4. Thirteen proteins were identified as crystallin aA, 6 of which were phosphorylated, 7 were acetylated, and 5 were both phosphorylated and acetylated. All crystallin aA proteins in Stage 3 were truncated. Ten proteins were identified as crystallin PB1. Of these, 8 were phosphorylated,

2 were acetylated, and none was truncated. Six proteins were identified as crystallin PA3. Of these, 5 were phosphorylated, 1 was acetylated, and all 6 were truncated. Six proteins in Stage 3 were identified as crystallin PA4. None of these were phosphorylated or acetylated; however, they were all truncated. In Stage 3, the ratio of phosphorylated to nonphosphorylated proteins was 19:16 (54.2% phosphorylated). The ratio of acetylation was lower than that of phosphorylation, with 10:25 (28.6%) proteins carrying acetyl groups (Tables I and II). The ratio of truncated to nontrun-cated proteins was 25:10, indicating that more than half of the proteins in Stage 3 were truncated (71.4%; Tables I and II). The phosphorylation ratio in Stage 3 was higher than that in Stage 2

(Stage 3 to Stage 2, 54.3%:46.3%). Similarly, the acetylation ratio in Stage 3 was also higher than that in Stage 2 (Stage 3 to Stage 2, 28.6%:19.5%). In contrast, the truncation ratio was increased in Stage 3 (Stage 3 to Stage 2, 71.4%:46.3%) (Tables I and II).

The major lens proteins associated with Stage 4 were crystallin PB1 and PA4 (Figure 3 and Table II). Sixteen proteins were identified as crystallin PB1. Of these, 4 were phosphorylated, and none was acetylated or truncated. In addition, 9 proteins were identified as crystallin PA4, none of which was modified or truncated. The ratio of phosphorylated to nonphosphorylated proteins in Stage 4 was 4:21. The prevalence of modifications such as phosphorylation (16%), acetylation (0%), and truncation (0%) decreased in Stage 4 (Tables I and II).

The major protein components associated with Stage 5 cataract lenses (Figure 4 and Tables I and II) were crystallin aA, PA4, and aB. Of these, 19 proteins were identified as truncated crystallin aA, and 11 of them were phosphorylated. Fourteen proteins were identified as crystallin aB. Of these, 8 were phosphorylated, 4 were acetylated, and none was truncated. Six proteins were identified as crystallin PA4, 1 of which was phosphorylated, and none was acetylated or truncated. In Stage 5, the ratio of phosphorylated to nonphosphorylated proteins was 20:19, that of acetylated to nonacetylated proteins was 4:35, and that of truncated to nontruncated proteins was 19:20. The prevalence of phosphorylation (51.3%) and truncation (48.7%) increased again in Stage 5, whereas that of acetylated crystallin proteins (10.3%) remained low (Table II).

Discussion

Cataract and intraocular lens surgery is progressing at an astonishing speed.19,20 Nevertheless, cataracts are still the leading cause of blindness worldwide,4,5,13 especially in underdeveloped countries where cataract surgery is not widely available.15 Where it is available, cataract surgery continues to be expensive, representing a significant cost to health services in many nations.21 The etiology of cataracts involves induction of free radicals and superoxide-mediated damage to lens proteins by ultraviolet (UV) light.22,23 The mechanisms of this disease remain elusive, and preventative medicines have not yet been discovered.20 In this study, we used proteomic analyses to determine differential expression and post-translational modifications of lens proteins during the development of cataracts.

Crystallins were identified as the most differentially expressed proteins in cataract lenses. The pI of human lens proteins was distributed from 5 to 9, and the molecular weight was between 10 and 120 kDa. Phosphorylation and truncation were increased in the early stages of lens clouding, indicating that these modifications of crystallins might contribute to the formation of catar-acts.24-27 Acetylation of crystallins was not as marked as phosphorylation in the opaque lens, although acetylation increased with the progression of cataracts. In contrast, phos-phorylation, truncation, and acetylation decreased in Stage 4. This might indicate that the modification of crystallin proteins is not important in maintaining lens opacity in late-stage cataracts. In addition, microscopic structures of lenses begin to deteriorate in Stage 4; however, modification and truncation of crystallin proteins were not predominant in this stage. The major components of human lens crystallin were aA, PB1, aB, and PA4. The abundance of crystallin PA4 did not change with the progression of cataracts, and it remained the major component in every stage. Crystallin PB1 was also a dominant component in Stages 2 to

4 human cataract lenses; however, it disappeared in Stage

5 cataracts. These data suggested that crystallin PB1 is important in the progression of lens opacity in early and middle stages.

Table II

The proteins expressed in cataracts.

Total Protein Number Proteins (no.)

Phosphorylation (%) Acetylation (%) Truncation (%)

44 47 38 46

ßB1 11 aB 11

aA 13 ßB1 10

ßB1 16 ßA4 9

aA 19 ßA4 14

aA 11 PA4 8 Others 3 19(46.3)

PA3 6 PA4 6 Others 12 19(54.3)

Others 13 4 (16)

aB 6 Others 7 20 (51.3)

8 (19.5) 10 (28.6) 0

4 (10.3)

19 (46.3) 25 (71.4) 0

19 (48.7)

Nevertheless, in severely opaque lenses, such as those in Stage 5, the basic structure had deteriorated to that of severe cataracts, such as morgagnian cataracts, and crystallin PB1 was totally absent. Crystallin aA was predominantly expressed in Stages 2, 3, and 5; however, it was not present in Stage 4. Therefore, crystallin aA might be involved in very early stages of cataract formation and later stages of severely opaque lenses. Very similar aB expression was noted in Stages 2 and 5.28-32

N-acetylcarnosine or carcinine eye drops resistant to enzymatic hydrolysis could act as pharmacological chaperones and decrease oxidative stress and excessive glycation in stress-related eyes such as cataracts. Ischemic diabetic retinopathy might protect against nuclear sclerotic cataracts, and these findings were consistent with the hypothesis that increased exposure to oxygen is responsible for nuclear cataract forma-tion.33 None of the patients in this study used these drugs regularly, but the relations of these drugs' antioxidative function and the crystalline changes noted in this study are worthy of advanced studies.34 In contrast, 2 well-known drugs, corticoster-oids35 and the antipsychotic drug quetiapine,36 can induce cataracts; none of our patients received these drugs for > 1 month; therefore, they were not the issues of our study. Cataracts are also classified by their location, with the posterior type usually due to steroid and diabetes mellitus.35,37 To decrease the special cataract type-induced bias in the study, the score of LOCS classification focus in any part, and the difference of any 2 parts over 4 (denoted as 0 and 4 by the 2 parts), were excluded from this study. To study the crystalline expression of different cataracts is also an important issue and worthy of study in the future. Other special type cataracts, such as traumatic cataracts, congenital cataracts, and exfoliation syndrome, were excluded in this study to obtain the simple information of natural progressing cataracts. In conclusion, crystallin protein levels and post-translational modifications were changeable during the progression of cataracts.

Conclusions

Crystallin protein levels and post-translational modifications were changeable during the progression of cataracts. Understanding these protein dynamics during the formation of cataracts might help in designing distinct treatments for this disease.

Acknowledgments

This study was supported by the grants from China Medical Medical University (DMR-99-093, DMR-100-097 and DMR-101-074, Taichung, Taiwan). We gratefully acknowledge Emily Hsieh and Chia Ming Wu from Department of Medical Genetics, China Medical University Hospital, Taichung, Taiwan for their help with the experiment. Dr. Lin contributed to the data collection, literature search, and writing. Dr. Lai contributed to the study design and data interpretation. Drs. Huang and Hsu contributed to the data collection. Dr. Tsai contributed to the study design.

Conflicts of Interest

The authors have indicated that they have no conflicts of interest regarding the content of this article.

References

[1] Bernth-Peterson P. Visual functioning in cataract patients. Methods of measuring results. Acta Ophthalmol. 1981;59:198-205.

[2] Knudtson MD, Klein BE, Klein R. Age-related eye disease, visual impairment and survival: the Beaver Dam Eye study. Arch Ophthalmol. 2006;124:243-249.

[3] McGinty SJ, Truscott RJ. Presbyopia. The first stage of nuclear cataract? Ophthalmic Res. 2006;38:137-148.

[4] Sapkota YD, Pokharel GP, Nirmalan PK, et al. Prevalence of blindness and cataract surgery in Gandaki Zone, Nepal. Br J Ophthalmol. 2006;90:411-416.

[5] Dineen B, Foster A, Faal H. A proposed rapid methodology to assess the prevalence and causes of blindness and visual impairment. Ophthalmic Epidemiol. 2006;13:31-34.

[6] Atchison DA. Optical models for human myopic eyes. Vision Res. 2006;46:2236-2250.

[7] Artal P, Benito A, Tabernero J. The human eye is an example of robust optical design. J Vis. 2006;6:1-7.

[8] Chen KH, Cheng WT, Li MJ, et al. Calcification of senile cataractous lens determined by Fourier transform infrared (FTIR) and Raman microspectros-copies. J Microsc. 2005;219:36-41.

[9] Javitt JC, Steinberg EP, Sharkey P, et al. Cataract surgery in one eye or both: a billion dollar per year issue. Ophthalmology. 1995;102:1583-1593.

[10] Giligson A. Doubts about lutein. CMAJ. 2006;174:662.

[11] Lu M, Cho E, Taylor A, et al. Prospective study of dietary fat and risk of cataract extraction among US women. Am J Epidemiol. 2005;161:948-959.

[12] Bojarskiene F, Cerniauskiene LR, Paunksnis A, Luksiene DI. Association of metabolic syndrome components with cataract. Medicina (Kaunas). 2006;42:115-122.

[13] Vijaya L, George R, Arvind H, et al. Prevalence and causes of blindness in the rural population of the Chennai Glaucoma study. Br J Ophthalmol. 2006;90:407-410.

[14] Ceklic L, Latinovic S, Aleksic P. Cataract as a leading cause of visual disability and blindness in the region of Eastern Sarajevo and Eastern Herzegovina. Med Pregl. 2005;58:449-452.

[15] Adegbehingbe BO, Fajemilehin BR, Ojofeitimi EO, Bisiriyu LA. Blindness and visual impairment among the elderly in Ife-Ijesha zone of Osun State, Nigeria. Indian J Ophthalmol. 2006;54:59-62.

[16] Chylack LTJ, Wolfe JK, Singer DM, et al. The Lens Opacities Classification System III. The Longitudinal Study of Cataract Study Group. Arch Ophthalmol. 1993;111:831-836.

[17] Hall AB, Thompson JR, Deane JS, Rosenthal AR. LOCS III versus the Oxford clinical cataract classification and grading system for the assessment of nuclear, cortical and posterior subcapsular cataract. Ophthalmic Epidemiol. 1997;4:179-194.

[18] Terry DE, Umstot E, Desiderio DM. Optimized sample-processing time and peptide recovery for the mass spectrometric analysis of protein digests. J Am SocMass Spectrom. 2004;15:784-794.

[19] Dada T, Muralidhar R, Sethi HS. Insertion of a foldable hydrophobic IOL through the trabeculectomy fistula in cases with Microincision cataract surgery combined with trabeculectomy. BMC Ophthalmol. 2006;6:14.

[20] Gray CS, Karimova G, Hildreth AJ, et al. Recovery of visual and functional disability following cataract surgery in older people: Sunderland Cataract Study. J Cataract Refract Surg. 2006;32:60-66.

[21] Taylor HR, Pezzullo ML, Keeffe JE. The economic impact and cost of visual impairment in Australia. Br J Ophthalmol. 2006;90:272-275.

[22] Yuh H, Chien-Lin L, Fu-Yung H. Oxidation-induced structural alterations and its effect on chaperone function of rat lensa-crystallin. J Chi Chem Soc. 1998;45:425-431.

[23] Hegde KR, Varma SD. Combination of glycemic and oxidative stress in lens: implications in augmentation of cataract formation in diabetes. Free Radic Res. 2005;39:513-517.

[24] Horwitz J. Alpha-crystallin can function as a molecular chaperone. Proc Natl AcadSciUSA. 1992;89:10449-10453.

[25] Nicholl ID, Quinlan RA. Chaperone activity of alpha-crystallins modulates intermediate filament assembly. EMBO J. 1994;13:945-953.

[26] Perng MD, Muchowski PJ, van Den IP, et al. The cardiomyopathy and lens cataract mutation in alphaB-crystallin alters its protein structure, chaperone activity, and interaction with intermediate filaments in vitro. J Biol Chem. 1999;274:33235-33243.

[27] Chen Y, Yi L, Yan GQ, et al. Decreased chaperone activity of alpha-crystallins in naphthalene-induced cataract possibly results from C-terminal truncation. JInt Med Res (England). 2010;38:1016-1028.

[28] Moreau KL, KingJA. Cataract-causing defect of a mutant g-crystallin proceeds through an aggregation pathway which bypasses recognition by the a-crystallin chaperone. PLoS One. 2012;7:e37256.

[29] Ma Z, Piszczek G, Wingfield PT, et al. The G18V CRYGS mutation associated with human cataracts increases gammaS-crystallin sensitivity to thermal and chemical stress. Biochemistry. 2009;48:7334-7341.

[30] Kondo T, Ishiga-Hashimoto N, Nagai H, et al. An increase in apoptosis and reduction in aB-crystallin expression levels in the lens underlie the catar-actogenesis of Morioka cataract (MCT) mice. Med Mol Morphol. 2011;44:221-227.

[31] Zampighi GA, Zampighi L, Lanzavecchia S. The three-dimensional distribution of aA-crystalline in rat lenses and its possible relation to transparency. PLoS One. 2011;6:e23753.

[32] Wang L, Zhao WC, Yin XL, et al. Lens proteomics: analysis of rat crystallins when lenses are exposed to dexamethasone. Mol Biosyst. 2012;8:888-901.

[33] Holekamp NM, Bai F, Shui YB, et al. Ischemic diabetic retinopathy may protect against nuclear sclerotic cataract. Am J Ophthalmol (United States). 2010;150:p543-550:e1.

[34] Babizhayev MA. Structural and functional properties, chaperone activity and posttranslational modifications of alpha-crystallin and its related subunits in the crystalline lens: n-acetylcarnosine, carnosine and carcinine act as alpha-crystallin/small heat shock protein enhancers in prevention and dissolution of cataract in ocular drug delivery formulations of novel therapeutic agents. Recent Pat Drug Deliv Formul. 2012;6:107-148.

[35] Spencer R, Andelman S. "Steroid cataracts. Posterior subcapsular cataract formation in rheumatoid arthritis patients on long term steroid therapy.'' Arch Ophthalmol. 1965;74:38-41.

[36] Shahzad S, Suleman MI, Shahab H, et al. Cataract occurrence with antipsy-chotic drugs. Psychosomatics. 2002;43:354-359.

[37] Hashim Z, Zarina S. Osmotic stress induced oxidative damage: possible mechanism of cataract formation in diabetes. J Diabetes Complications. 2012;26:275-279.