Current Eye Research, 2014; 39(5): 431–438 ! Informa Healthcare USA, Inc. ISSN: 0271-3683 print / 1460-2202 online DOI: 10.3109/02713683.2013.851705
ORIGINAL ARTICLE
Zinc Finger Protein in Severe Dry Eye Syndrome Seung Hoon Lee1,2, Min-Yi Park1, Kyoung Woo Kim1, Sung Wook Wee1 and Jae Chan Kim1 Department of Ophthalmology, College of Medicine, Chung-Ang University Hospital, Heukseok-dong, Dongjak-Gu, Seoul, South Korea and 2Major in Biomedical science, Department of Medicine, Graduate School, Chung-Ang University, Seoul, Korea
ABSTRACT Purpose: Zinc finger protein known to induce squamous metaplasia and regulate vitamin A expression has been few investigated as tear protein. We investigated tear protein variations in patients with dry eye syndrome (DES). Materials and methods: Tears from healthy subjects as control and patients with DES were collected. Tear proteins were separated by one-dimensional electrophoresis. The protein bands were analyzed by nano liquid chromatography–mass spectrometry. Results: Compared to the controls, significant down-regulation of lactoferrin and lysozyme was detected, while significant up-regulation was observed for serum albumin in patients with DES. DES grade 4 patients showed different protein patterns. Zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B were detected in DES grade 4 patients. Conclusions: Tear protein changes are valuable to diagnosis DES. Zinc finger proteins may be associated with pathophysiology of severe DES. Further studies are needed to better understand the relationship of zinc finger proteins in tear of patients with DES. Keywords: Dry eye syndrome, electrophoresis, nano liquid chromatography–mass spectrometry, tear protein, zinc finger protein
INTRODUCTION
These methods have confirmed the variation of tear proteins related to ocular diseases like DES.9–11 Several investigations reported the alterations in epidermal growth factor, lipocalin-1 and lipophilin-A proteins in tears of patients with DES.12,13 The up-regulation of various inflammatory cytokines has been related to severity of DES classified by clinical parameters. Levels of interleukin (IL)-1b, IL-6 and IL-8 are elevated according to DES severity.14–16 Zinc finger proteins are members of a multigene family encoding zinc-mediated nucleic acid binding proteins characterized by the coordination of zinc ions to stabilize structure.17,18 Zinc finger proteins regulate gene expression by interacting with target DNA sequences.19 It is widely assumed that
Dry eye syndrome (DES) is caused by inflammatory cytokines in tears and ocular surface.1,2 DES is recognized as an immune disease, and inflammation of ocular surface has an important role in the pathogenesis of DES.3,4 Severe DES induces damage to the corneal surface, keratinization of the ocular surface and squamous metaplasia of the conjunctival surface.5,6 Although the prevalence of DES is high and several diagnostic criteria of DES have been reported, there are no uniform criteria for the diagnosis of DES due to the lack of correlation between diagnostic tools and clinical symptoms.7,8 Electrophoresis and mass spectrometry have been applied to investigate the changes in tear proteins.
20 14
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
1
Received 2 July 2013; revised 16 September 2013; accepted 1 October 2013; published online 11 November 2013 Correspondence: Jae Chan Kim, Department of Ophthalmology, College of Medicine, Chung-Ang University Hospital 224-1, Heukseok-dong, Dongjak-Gu, Seoul 156-755, Republic of Korea. Tel: +82-2-6299-1689. Fax: +82-2-825-1666. E-mail: [email protected]
431
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
432 S. H. Lee et al. zinc finger proteins induce squamous metaplasia. Basonuclin, a zinc finger protein of corneal stratified squamous epithelia, is a transcriptional factor for squamous epithelium.20,21 Zinc-finger E-box binding homeobox 1 is a transcriptional factor for small proline-rich protein 1B (SPRR1B) that induces squamous metaplasia in human corneal epithelium.22 DNA-binding proteins containing zinc fingers have roles in the expression of vitamin A related to DES.23 We hypothesized that tear proteins from patients with DES compared with healthy subjects are specially changed and zinc finger proteins may be related to DES. Therefore, we investigated the variation of tear proteins related to DES as a way of elucidating their role in the pathophysiology of DES.
MATERIALS AND METHODS Subjects All the subjects who volunteered for this study were from Chung-Ang University Hospital. The average age of the six normal control subjects was 34.1 13.9 years (range 25–62 years). The average age of the 24 DES subjects was 39.2 15 years (range 17–65 years). The normal control subjects did not have any ocular disease and eye-related symptoms and were not using eye medications. Diagnosis of DES was based on Delphi classification.24 The total 24 patients was diagnosed and further classified into 4 groups (6 subjects in each group) according to their severity based on Delphi classification. Classification was conducted by one ophthalmologist to exclude individual differences. The study protocol and informed consent were approved by the institutional review board of the Chung-Ang University Hospital, and the study conformed to the tenets of the Declaration of Helsinki.
room temperature. One-dimensional electrophoresis gels were stained with Coomassie Brilliant Blue R-250 for 4 h at room temperature. Densitometric analysis of each one-dimensional gel was carried out using ImageJ software (National Institutes of Health, Bethesda, MD) and evaluated as the relative percentage of each protein in the total gel band intensities. Protein levels are expressed as the mean standard error (SE).
Nano Liquid Chromatography-Tandem Mass Spectrometry Analysis Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using an Agilent 1100 Series nano-LC and LTQ-mass spectrometer (Thermo Electron, San Jose, CA). The capillary column used for LC-MS/MS analysis (150 mm 0.075 mm) was obtained from Proxeon (Odense, Denmark) and slurry packed in-house with ˚ pore size Magic C18 stationary phase 5 mm, 100 A (Michrom Bioresources, Auburn, CA). The mobile phase A for the LC separation was 0.1% formic acid in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was set up to give a linear increase from 5% B to 35% B in 50 min and from 40% B to 60% B in 20 min, and from 60% B to 80% B in 5 min. The flow rate was maintained at 300 nL/min after splitting. Mass spectra were acquired using data-dependent acquisition with full mass scan (400–1800 m/z) followed by MS/MS scans. Each MS/MS scan acquired was an average of one microscan on the LTQ. The temperature of the ion transfer tube was controlled at 200 C and the spray was at 1.5.0–2.0 kV. The normalized collision energy was set at 35% for MS/MS. Mass tolerances of 1.2 Da and 0.6 Da were used for precursor and fragment ions, respectively. Peptides were allowed to be variably oxidized at methionine residues and to be variably carboxyamidomethylated at cysteine residues.
Tear Collection and One-Dimensional Electrophoresis Statistical Analysis We used 75 ml hematocrit-capillary tubes for tear sampling and minimized ocular stimulation to avoid reflex tearing. Care was taken not to touch eye lashes. The tip of the microcapillary tube was located in the exterior of ocular surface. Approximately 10 ml of tears were collected from each subject. Tear samples were immediately centrifuged at 12,000 rpm for 10 min and stored at 72 C in the Eppendorf tube. Tear samples were mixed with 5 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer and boiled at 95 C for 5 min. Then, the tear proteins were loaded (30 mg/well) into each well of a gel and separated by 12% SDS-PAGE using an applied voltage of 100 V for 2.5 h at
Densitometric analysis of monodimensional electrophoresis gels was quantified by ImageJ software (Wayne Rasband, National Institutes of Health, USA) and evaluated as the relative percentage of each protein in the total gel band intensities. Protein levels were expressed as the mean standard error (SE). Statistical analysis was performed using oneway ANOVA followed by a post hoc pairwise comparison adjusted with a Bonferroni correction. Statistical analyses were performed using SPSS software version 19.0 (SPSS Inc., Chicago, IL). p Values less than 0.05 were considered to be statistically significant. Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome
433
TABLE 1 Demographics of the subjects.
Age (years) Age range TBUT (s)
Control (N = 6)
DES grade 1 (N = 6)
DES grade 2 (N = 6)
DES grade 3 (N = 6)
DES grade 4 (N = 6)
34.1 13.9 25–62 6.3 3.1
47.3 17.6 17–65 4.5 2.9
47.6 12.5 25–60 4.3 1.3
48.6 13.2 33–65 3.1 1.2
44.8 13.9 28–63 2.8 1.7
Age and TBUT are expressed as mean standard deviation. N = number of subjects and TBUT = tear break-up time.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
RESULTS Six normal control subjects and 24 patients with DES were enrolled in this investigation. Demographics of normal control group and each DES patient group are listed in Table 1. The mean age of normal control group was not significantly different from each DES patient group. One-dimensional electrophoresis was conducted to investigate the difference of protein between DES patients and normal controls. Marked differences were observed in DES grade 4 patients. Lane of normal controls and DES grade 1–3 patients showed similar electrophoretic pattern of tear proteins, which were in the 14–77 kDa range. In contrast, DES grade 4 samples routinely showed a different pattern of tear proteins (Figure 1). The densitometric analysis of onedimensional electrophoresis gel showed variations of protein band intensities between normal controls and patients with DES. Decrease in levels of lactoferrin (controls: 21.3 1.2; DES grade 1: 21.3 1.4; DES grade 2: 20.8 1.5; DES grade 3: 19.9 1.2; DES grade 4: 8.7 1.8; relative percentage, mean SE) and lysozyme (controls: 20.7 1.8; DES grade 1: 15.4 1.6; DES grade 2: 10.3 1.4; DES grade 3: 8.7 1.1; DES grade 4: 7.3 1.7) was observed in each grade of DES patients, compared to the controls. In addition, increase in levels of serum albumin (controls: 6.1 1.4; DES grade 1: 7.3 2.1; DES grade 2: 10.9 1.5; DES grade 3: 21.6 2.2; DES grade 4: 27.2 1.0) was detected also in each grade of DES group, compared to the controls. Protein levels of lysozyme were showed a significant difference between each of DES grade 1 to 4 group and healthy subjects. Protein levels of serum albumin were showed a significant difference between each of DES grade 2, 3 and 4 group and healthy subjects. Protein levels of lactoferrin revealed statistically significant difference only between DES grade 4 group and healthy subjects (Figure 3). The proteins from the bands only observed in DES grade 4 patient group lane were analyzed by nano LC-MS/MS analysis. The protein identities are summarized in Table 2, which presents data on protein score, protein mass, protein coverage and p value. Ten distinct proteins were identified in all tear samples of DES grade 4 patients including zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B. !
2014 Informa Healthcare USA, Inc.
FIGURE 1 One-dimensional electrophoresis gel of coomassie stained proteins. All lane contain 30 mg of total protein precipitated from tears collected by capillary. Protein band C was only observed in tears of DES grade 4 patients. (A) Lactoferrin; (B) lysozyme; (C) serum albumin, zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B.
LC-MS/MS spectra for zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B are presented in Figure 3. Figure 3(A) shows the MS/MS spectrum of peptide ion (KMVEDRQSGDLEK) found in gij50657091 (zincfinger motif-enhancer binding-protein-1 gamma). Figure 3(B) shows the MS/MS spectrum of peptide ion (DQDESDSDTEGEK) found in gij6683500 (bromodomain adjacent to zinc finger domain 2B. The proteins from the A and B bands were analyzed by nano LC-MS/MS analysis. The protein identities are summarized in Table 2, which presents data on protein score, protein mass, protein coverage and p-value. Twelve distinct proteins were identified in band A including lactoferrin and sixteen distinct proteins were identified in band B including lysozyme. In the LC-MS/MS results, protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Ions scores were 10 Log
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
434 S. H. Lee et al.
FIGURE 2 LC-MS/MS spectra of peptide fragment representing proteins observed in DES grade 4 patients. (A) Zinc-finger motifenhancer binding-protein-1 gamma peptide ion (KMVEDRQSGDLEK). (B) Bromodomain adjacent to zinc finger domain 2B peptide ion (DQDESDSDTEGEK). Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome
435
TABLE 2 Identification of the protein detected in SDS-PAGE using LC-MS/MS method. Protein score
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
Protein name Band A in gij187122 lactoferrin gij48425709 Chain A, Structure Of Human Diferric Lactoferrin At 2.5a Resolution Using Crystals Grown At Ph 6.5 gij17066105 Titin gij13654237 DNA-dependent protein kinase catalytic subunit isoform 1 gij34328016 KIAA0614 protein gij238236 transmembrane secretory component gij223069 protein Tro alpha1 H,myeloma gij223099 Ig Aalpha1 Bur gij70058 Ig alpha-2 chain C region gij229537 Ig A H gij2135473 Ig alpha-2 chain - human (fragment) gij10835403 Chain H, Crystal Structure Of An Unliganded (Native) Fv From A Human Igm Anti-Peptide Antibody
Protein mass (Da)
SDS-PAGE 3693 78,409 3186 76,194
Sequence coverage (%)
Calculated pI value
No. matched peptide
73% 66%
8.5 8.47
475 411
150 118
3,816,172 469,084
2% 3%
6.01 6.75
79 20
113 112 1001 934 888 870 660 529
328,672 83,313 51,072 50,613 36,573 51,108 36,557 13,203
4% 7% 29% 31% 38% 32% 38% 24%
5.96 5.58 8.77 9.34 7.64 9.27 5.85 8.61
16 4 203 224 147 186 174 20
71% 36% 68% 65%
5.39 8.26 9.28 9.14
279 25 11 10
60% 45% 5% 93% 90% 90%
9.28 9.38 5.63 9.28 9.28 9.28
10 13 12 334 335 335
90% 90% 90%
9.28 9.28 9.28
334 333 332
91%
9.28
334
90% 90%
9.28 9.14
332 332
60% 61% 58% 68% 61% 7% 2% 7% 4%
6.05 5.96 5.99 6.14 6.97 6.02 6.01 5.95 8.52
258 253 239 167 161 12 77 13 9
3%
5.27
25
Band B in SDS-PAGE gij4504963 lipocalin-1 precursor 1037 19,250 gij4505821 prolactin-inducible protein precursor 294 16,572 gij4930014 Chain A, T11a Mutant Human Lysozyme 255 14,671 gij11514208 Chain A, Crystal Structure Of Mutant Human 245 14,601 Lysozyme Substituted At Left-Handed Helical Positions gij4930021 Chain A, T43v Mutant Human Lysozyme 228 14,699 gij30714 lysozyme precursor (EC 3.2.1.17) 212 16,555 gij20521976 KIAA1731 protein 91 233,738 gij4930014 Chain A, T11a Mutant Human Lysozyme 858 14,671 gij17942568 Chain A, G127a Human Lysozyme 852 14,715 gij6729881 Chain A, Verification Of Spmp Using Mutant 851 14,761 Human Lysozymes gij17942566 Chain A, G129a Human Lysozyme 840 14,715 gij14278473 Chain A, Buried Polar Mutant Human Lysozyme 839 14,703 gij9955033 Chain A, Crystal Structure Of Mutant Human 837 14,733 Lysozyme Substituted At The Surface Positions gij11513937 Chain A, Crystal Structure Of Mutant Human 837 14,658 Lysozyme Substituted At Left-Handed Helical Positions gij17942570 Chain A, G72a Human Lysozyme 827 14,715 gij13399625 Chain A, Mutant Human Lysozyme (Q86d) 826 14,687 Band C in SDS-PAGE gij28592 serum albumin 2480 69,365 gij11493459 PRO2619 2335 56,782 gij178345 alloalbumin Venezia 2330 69,226 gij7770217 PRO2675 1412 32,574 gij6650826 PRO2044 1370 29,248 gij179665 complement component C3 266 187,162 gij17066105 Titin 128 3,816,172 gij6683500 bromodomain adjacent to zinc finger domain 2B 96 220,863 gij50657091 Zinc-finger motif-Enhancer binding-Protein-1 92 159,470 gamma gij24417711 nesprin-2 92 796,260
Zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B were detected in tears of DES grade 4 patients.
(P), where P was the probability that the observed match is a random event. Individual ions score higher than 44 indicated identity or extensive homology (p50.05).
DISCUSSION Perturbation of tear proteins may induce the development of ocular disease, which makes tear proteins !
2014 Informa Healthcare USA, Inc.
an interesting target for ocular disease diagnosis. Our study revealed increased serum albumin and decreased lactoferrin and lysozyme in the tears of patients with DES compared to healthy individuals. These results are similar to previous research.13,25,26 Lactoferrin and lysozyme are components of the innate immune system and have antimicrobial, antibacterial and antiviral activity.27–32 Serum albumin is considered to be derived from the plasma
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
436 S. H. Lee et al.
FIGURE 3 Relative density of protein bands in one-dimensional electrophoresis gel. Relative density was calculated the relative percentage of each protein of total density in each lane. Values are presented as mean standard error (%). p50.05 is considered significant. A: Lactoferrin; B: Lysozyme; C: Serum albumin.
and to leak into the tear fluid from conjunctival blood vessels,33 and is thus a potentially significant inflammatory marker indicating vascular leakage. Up-regulated permeability spurs development of inflammation.34 Therefore, inflammation may be induced at the ocular surface in patients with DES. The common diagnostic tests for dry eye are the Schirmer test and measurement of tear break-up time (TBUT). However, the Schirmer test suffers from error and has low reproducibility35 and TBUT can destabilize the tear film because of instillation of fluorescein.36 On the other hand, diagnostic testing using tear proteins can be effective. Tear proteins analyzed by electrophoretic separation and MS reveals distinct patterns associated with specific disease.13,37 In addition, electrophoresis is a good diagnostic tool for those at high risk of dry eye38 and electrophoretic separation and MS have been used to investigate for tear protein of patients with DES.37,39 Although SDS-PAGE and LC-MS/MS is a high cost measurement, precise diagnosis of DES through SDSPAGE and LC-MS/MS can provide a prompt
treatment to DES patients. Presently, one-dimensional electrophoresis was conducted to investigate difference between tear proteins of patients with DES and normal controls. A specific protein pattern was evident in DES grade 4 patients. Zinc finger proteins stabilized by zinc ion binding cause squamous metaplasia and regulate vitamin A expression. As squamous metaplasia of the ocular surface is an indication of DES and vitamin A deficiency is an essential cause of xerophthalmia40–43, zinc finger proteins may be related to DES. Association of zinc finger proteins and DES has not been investigated previously. In this study, zinc finger proteins detected in tears of patients with dry eye severity grade 4 were zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B. RREB1 encoding zinc-finger motif-enhancer bindingprotein-1 gamma is a gene that has been associated with inflammatory and apoptosis signaling pathways.43 Human bromodomain containing proteins have been demonstrated in pathological conditions including inflammation.45 Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome In conclusion, measurement of tear proteins may be an important diagnostic tool in DES. In addition, zinc finger protein may be related to the pathogenesis of DES and may be used to indicate severe DES. Further investigations will be necessary to conclusively investigate the relationship of zinc finger proteins in tears of patients with DES.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
ACKNOWLEDGEMENTS We wish to acknowledge technical support from Yonsei Proteome Research Center (www.proteomix. org).
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This research was supported by the Biomedical science Scholarship Grants, Department of Medicine, Chung-Ang University in 2013.
REFERENCES 1. Bhavsar AS, Bhavsar SG, Jain SM. A review on recent advances in dry eye: pathogenesis and management. Oman J Ophthalmol 2011;4:50–56. 2. Rolando M, Barabino S, Mingari C, Moretti S, Giuffrida S, Calabria G. Distribution of conjunctival HLA-DR expression and the pathogenesis of damage in early dry eye. Cornea 2005;24:951–954. 3. Stern ME, Gao J, Siemasko KF, Beuerman RW, Pflugfelder SC. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004;78:409–416. 4. Dana MR, Hamrah P. Role of immunity and inflammation in corneal and ocular surface disease associated with dry eye. Adv Exp Med Biol 2002;506:729–738. 5. Lemp MA. Advances in understanding and managing dry eye disease. Am J Ophthalmol. 2008;146:350–356. 6. De Paiva CS, Villarreal AL, Corrales RM, Rahman HT, Chang VY, Farley WJ, et al. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma. Invest Ophthalmol Vis Sci 2007;48: 2553–2560. 7. Brewitt H, Sistani F. Dry eye disease: the scale of the problem. Surv Ophthalmol 2001;45:S199–S202. 8. Schein OD, Mun˜oz B, Tielsch JM, Bandeen-Roche K, West S. Prevalence of dry eye among the elderly. Am J Ophthalmol 1997;124:723–728. 9. Lemp MA. Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21: 221–232. 10. Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I. Diagnostic potential of tear proteomic patterns in Sjogren’s syndrome. J Proteome Res 2005;4:820–825. 11. Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, et al. SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 2005;46:863–876. !
2014 Informa Healthcare USA, Inc.
437
12. Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, et al. Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 2003;136: 291–299. 13. Versura P, Nanni P, Bavelloni A, Blalock WL, Piazzi M, Roda A, et al. Tear proteomics in evaporative dry eye disease. Eye (Lond) 2010;24:1396–1402. 14. Huang JF, Zhang Y, Rittenhouse KD, Pickering EH, McDowell MT. Evaluations of tear protein markers in dry eye disease: repeatability of measurement and correlation with disease. Invest Ophthalmol Vis Sci 2012;53: 4556–4564. 15. Na KS, Mok JW, Kim JY, Rho CR, Joo CK. Correlations between tear cytokines, chemokines, and soluble receptors and clinical severity of dry eye disease. Invest Ophthalmol Vis Sci 2012;53:5443–5450. 16. Enrı´quez-de-Salamanca A, Castellanos E, Stern ME, Ferna´ndez I, Carren˜o E, Garcı´a-Va´zquez C, et al. Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis 2010;16: 862–873. 17. Chowdhury K, Rohdewohld H, Gruss P. Specific and ubiquitous expression of different Zn finger protein genes in the mouse. Nucleic Acids Res 1988;16:9995–10011. 18. Krishna SS, Majumdar I, Grishin NV. Structural classification of zinc fingers: survey and summary. Nucleic Acids Res 2003;31:532–550. 19. Laity JH, Lee BM, Wright PE. Zinc finger proteins: new insights into structural and functional diversity. Curr Opin Struct Biol 2001;11:39–46. 20. Tseng H. Basonuclin, a zinc finger protein associated with epithelial expansion and proliferation. Front Biosci 1998;3: D985–D988. 21. Tseng H, Green H. Basonuclin: a keratinocyte protein with multiple paired zinc fingers. Proc Natl Acad Sci U S A. 1992;89:10311–10315. 22. Li S, Gallup M, Chen YT, McNamara NA. Molecular mechanism of proinflammatory cytokine-mediated squamous metaplasia in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2010;51:2466–2475. 23. Brody T. Nutritional biochemistry. San Diego: Academic Press; 1999. 24. Kaercher T, Bron AJ. Classification and diagnosis of dry eye. Dev Ophthalmol 2008;41:36–53. 25. Zhou L, Beuerman RW, Chan CM, Zhao SZ, Li XR, Yang H, et al. Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics. J Proteome Res 2009;8:4889–4905. 26. Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, et al. Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 2003;136:291–299. 27. Yang PZ, Zhang XF, Zhang JS, Wu YD, Shi RH. The longitudinal observation of the effect of antibiotics on human tear lysozyme activity. Yan Ke Xue Bao 1989;5: 60–64. 28. Kirkpatrick CH, Green I, Rich RR, Schade AL. Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: relation to host-defense mechanisms in chronic mucocutaneous candidiasis. J Infect Dis 1971;124:539–544. 29. Valenti P, Antonini G. Lactoferrin: an important host defence against microbial and viral attack. Cell Mol Life Sci 2005;62:2576–2587. 30. Ward PP, Paz E, Conneely OM. Multifunctional roles of lactoferrin: a critical overview. Cell Mol Life Sci 2005;62: 2540–2548. 31. Legrand D, Elass E, Carpentier M, Mazurier J. Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 2005;62:2549–2559.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
438 S. H. Lee et al. 32. Gustot A, Raussens V, Dehousse M, Dumoulin M, Bryant CE, Ruysschaert JM, et al. Activation of innate immunity by lysozyme fibrils is critically dependent on cross-beta sheet structure. Cell Mol Life Sci 2013;70:2999–3012. 33. Fukuda M, Fullard RJ, Willcox MD, Baleriola-Lucas C, Bestawros F, Sweeney D, et al. Fibronectin in the tear film. Invest Ophthalmol Vis Sci 1996;37:459–467. 34. Sack RA, Tan KO, Tan A. Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 1992;33:626–640. 35. Lucca JA, Nunez JN, Farris RL. A comparison of diagnostic tests for keratoconjunctivitis sicca: lactoplate, Schirmer, and tear osmolarity. CLAO J 1990;16:109–112. 36. Mengher LS, Bron AJ, Tonge SR, Gilbert DJ. A non-invasive instrument for clinical assessment of the pre-corneal tear film stability. Curr Eye Res 1985;4:1–7. 37. Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, et al. SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 2005;46:863–876. 38. Chiva A. Electrophoresis of tear proteins as a new diagnostic tool for two high risk groups for dry eye:
39.
40. 41. 42. 43.
44.
45.
computer users and contact lens wearers. J Med Life 2011; 4:228–233. Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I. Diagnostic potential of tear proteomic patterns in Sjogren’s syndrome. J Proteome Res 2005;4:820–825. McLaren DS. Vitamin A deficiency disorders. J Indian Med Assoc 1999;97:320–323. Sommer A. Vitamin a deficiency and clinical disease: an historical overview. J Nutr 2008;138:1835–1839. Tseng SC. Topical tretinoin treatment for dry-eye disorders. Int Ophthalmol Clin 1987;27:47–53. Li S, Nikulina K, De Voss J, Wu AJ, Strauss EC, Anderson MS, et al. Small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia in dry eye disease. Invest Ophthalmol Vis Sci 2008;49:34–41. Kurji KH, Cui JZ, Lin T, Harriman D, Prasad SS, Kojic L, et al. Microarray analysis identifies changes in inflammatory gene expression in response to amyloid-beta stimulation of cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2010;51:1151–1163. Sanchez R, Zhou MM. The role of human bromodomains in chromatin biology and gene transcription. Curr Opin Drug Discov Devel 2009;12:659–665.
Notices of Correction: A transposition of Figure legends 2 and 3, and an inaccuracy in the declaration of interest, were corrected following ahead-of-print online publication.
Current Eye Research
ORIGINAL ARTICLE
Zinc Finger Protein in Severe Dry Eye Syndrome Seung Hoon Lee1,2, Min-Yi Park1, Kyoung Woo Kim1, Sung Wook Wee1 and Jae Chan Kim1 Department of Ophthalmology, College of Medicine, Chung-Ang University Hospital, Heukseok-dong, Dongjak-Gu, Seoul, South Korea and 2Major in Biomedical science, Department of Medicine, Graduate School, Chung-Ang University, Seoul, Korea
ABSTRACT Purpose: Zinc finger protein known to induce squamous metaplasia and regulate vitamin A expression has been few investigated as tear protein. We investigated tear protein variations in patients with dry eye syndrome (DES). Materials and methods: Tears from healthy subjects as control and patients with DES were collected. Tear proteins were separated by one-dimensional electrophoresis. The protein bands were analyzed by nano liquid chromatography–mass spectrometry. Results: Compared to the controls, significant down-regulation of lactoferrin and lysozyme was detected, while significant up-regulation was observed for serum albumin in patients with DES. DES grade 4 patients showed different protein patterns. Zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B were detected in DES grade 4 patients. Conclusions: Tear protein changes are valuable to diagnosis DES. Zinc finger proteins may be associated with pathophysiology of severe DES. Further studies are needed to better understand the relationship of zinc finger proteins in tear of patients with DES. Keywords: Dry eye syndrome, electrophoresis, nano liquid chromatography–mass spectrometry, tear protein, zinc finger protein
INTRODUCTION
These methods have confirmed the variation of tear proteins related to ocular diseases like DES.9–11 Several investigations reported the alterations in epidermal growth factor, lipocalin-1 and lipophilin-A proteins in tears of patients with DES.12,13 The up-regulation of various inflammatory cytokines has been related to severity of DES classified by clinical parameters. Levels of interleukin (IL)-1b, IL-6 and IL-8 are elevated according to DES severity.14–16 Zinc finger proteins are members of a multigene family encoding zinc-mediated nucleic acid binding proteins characterized by the coordination of zinc ions to stabilize structure.17,18 Zinc finger proteins regulate gene expression by interacting with target DNA sequences.19 It is widely assumed that
Dry eye syndrome (DES) is caused by inflammatory cytokines in tears and ocular surface.1,2 DES is recognized as an immune disease, and inflammation of ocular surface has an important role in the pathogenesis of DES.3,4 Severe DES induces damage to the corneal surface, keratinization of the ocular surface and squamous metaplasia of the conjunctival surface.5,6 Although the prevalence of DES is high and several diagnostic criteria of DES have been reported, there are no uniform criteria for the diagnosis of DES due to the lack of correlation between diagnostic tools and clinical symptoms.7,8 Electrophoresis and mass spectrometry have been applied to investigate the changes in tear proteins.
20 14
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
1
Received 2 July 2013; revised 16 September 2013; accepted 1 October 2013; published online 11 November 2013 Correspondence: Jae Chan Kim, Department of Ophthalmology, College of Medicine, Chung-Ang University Hospital 224-1, Heukseok-dong, Dongjak-Gu, Seoul 156-755, Republic of Korea. Tel: +82-2-6299-1689. Fax: +82-2-825-1666. E-mail: [email protected]
431
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
432 S. H. Lee et al. zinc finger proteins induce squamous metaplasia. Basonuclin, a zinc finger protein of corneal stratified squamous epithelia, is a transcriptional factor for squamous epithelium.20,21 Zinc-finger E-box binding homeobox 1 is a transcriptional factor for small proline-rich protein 1B (SPRR1B) that induces squamous metaplasia in human corneal epithelium.22 DNA-binding proteins containing zinc fingers have roles in the expression of vitamin A related to DES.23 We hypothesized that tear proteins from patients with DES compared with healthy subjects are specially changed and zinc finger proteins may be related to DES. Therefore, we investigated the variation of tear proteins related to DES as a way of elucidating their role in the pathophysiology of DES.
MATERIALS AND METHODS Subjects All the subjects who volunteered for this study were from Chung-Ang University Hospital. The average age of the six normal control subjects was 34.1 13.9 years (range 25–62 years). The average age of the 24 DES subjects was 39.2 15 years (range 17–65 years). The normal control subjects did not have any ocular disease and eye-related symptoms and were not using eye medications. Diagnosis of DES was based on Delphi classification.24 The total 24 patients was diagnosed and further classified into 4 groups (6 subjects in each group) according to their severity based on Delphi classification. Classification was conducted by one ophthalmologist to exclude individual differences. The study protocol and informed consent were approved by the institutional review board of the Chung-Ang University Hospital, and the study conformed to the tenets of the Declaration of Helsinki.
room temperature. One-dimensional electrophoresis gels were stained with Coomassie Brilliant Blue R-250 for 4 h at room temperature. Densitometric analysis of each one-dimensional gel was carried out using ImageJ software (National Institutes of Health, Bethesda, MD) and evaluated as the relative percentage of each protein in the total gel band intensities. Protein levels are expressed as the mean standard error (SE).
Nano Liquid Chromatography-Tandem Mass Spectrometry Analysis Nano liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis was performed using an Agilent 1100 Series nano-LC and LTQ-mass spectrometer (Thermo Electron, San Jose, CA). The capillary column used for LC-MS/MS analysis (150 mm 0.075 mm) was obtained from Proxeon (Odense, Denmark) and slurry packed in-house with ˚ pore size Magic C18 stationary phase 5 mm, 100 A (Michrom Bioresources, Auburn, CA). The mobile phase A for the LC separation was 0.1% formic acid in deionized water and the mobile phase B was 0.1% formic acid in acetonitrile. The chromatography gradient was set up to give a linear increase from 5% B to 35% B in 50 min and from 40% B to 60% B in 20 min, and from 60% B to 80% B in 5 min. The flow rate was maintained at 300 nL/min after splitting. Mass spectra were acquired using data-dependent acquisition with full mass scan (400–1800 m/z) followed by MS/MS scans. Each MS/MS scan acquired was an average of one microscan on the LTQ. The temperature of the ion transfer tube was controlled at 200 C and the spray was at 1.5.0–2.0 kV. The normalized collision energy was set at 35% for MS/MS. Mass tolerances of 1.2 Da and 0.6 Da were used for precursor and fragment ions, respectively. Peptides were allowed to be variably oxidized at methionine residues and to be variably carboxyamidomethylated at cysteine residues.
Tear Collection and One-Dimensional Electrophoresis Statistical Analysis We used 75 ml hematocrit-capillary tubes for tear sampling and minimized ocular stimulation to avoid reflex tearing. Care was taken not to touch eye lashes. The tip of the microcapillary tube was located in the exterior of ocular surface. Approximately 10 ml of tears were collected from each subject. Tear samples were immediately centrifuged at 12,000 rpm for 10 min and stored at 72 C in the Eppendorf tube. Tear samples were mixed with 5 sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDSPAGE) sample buffer and boiled at 95 C for 5 min. Then, the tear proteins were loaded (30 mg/well) into each well of a gel and separated by 12% SDS-PAGE using an applied voltage of 100 V for 2.5 h at
Densitometric analysis of monodimensional electrophoresis gels was quantified by ImageJ software (Wayne Rasband, National Institutes of Health, USA) and evaluated as the relative percentage of each protein in the total gel band intensities. Protein levels were expressed as the mean standard error (SE). Statistical analysis was performed using oneway ANOVA followed by a post hoc pairwise comparison adjusted with a Bonferroni correction. Statistical analyses were performed using SPSS software version 19.0 (SPSS Inc., Chicago, IL). p Values less than 0.05 were considered to be statistically significant. Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome
433
TABLE 1 Demographics of the subjects.
Age (years) Age range TBUT (s)
Control (N = 6)
DES grade 1 (N = 6)
DES grade 2 (N = 6)
DES grade 3 (N = 6)
DES grade 4 (N = 6)
34.1 13.9 25–62 6.3 3.1
47.3 17.6 17–65 4.5 2.9
47.6 12.5 25–60 4.3 1.3
48.6 13.2 33–65 3.1 1.2
44.8 13.9 28–63 2.8 1.7
Age and TBUT are expressed as mean standard deviation. N = number of subjects and TBUT = tear break-up time.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
RESULTS Six normal control subjects and 24 patients with DES were enrolled in this investigation. Demographics of normal control group and each DES patient group are listed in Table 1. The mean age of normal control group was not significantly different from each DES patient group. One-dimensional electrophoresis was conducted to investigate the difference of protein between DES patients and normal controls. Marked differences were observed in DES grade 4 patients. Lane of normal controls and DES grade 1–3 patients showed similar electrophoretic pattern of tear proteins, which were in the 14–77 kDa range. In contrast, DES grade 4 samples routinely showed a different pattern of tear proteins (Figure 1). The densitometric analysis of onedimensional electrophoresis gel showed variations of protein band intensities between normal controls and patients with DES. Decrease in levels of lactoferrin (controls: 21.3 1.2; DES grade 1: 21.3 1.4; DES grade 2: 20.8 1.5; DES grade 3: 19.9 1.2; DES grade 4: 8.7 1.8; relative percentage, mean SE) and lysozyme (controls: 20.7 1.8; DES grade 1: 15.4 1.6; DES grade 2: 10.3 1.4; DES grade 3: 8.7 1.1; DES grade 4: 7.3 1.7) was observed in each grade of DES patients, compared to the controls. In addition, increase in levels of serum albumin (controls: 6.1 1.4; DES grade 1: 7.3 2.1; DES grade 2: 10.9 1.5; DES grade 3: 21.6 2.2; DES grade 4: 27.2 1.0) was detected also in each grade of DES group, compared to the controls. Protein levels of lysozyme were showed a significant difference between each of DES grade 1 to 4 group and healthy subjects. Protein levels of serum albumin were showed a significant difference between each of DES grade 2, 3 and 4 group and healthy subjects. Protein levels of lactoferrin revealed statistically significant difference only between DES grade 4 group and healthy subjects (Figure 3). The proteins from the bands only observed in DES grade 4 patient group lane were analyzed by nano LC-MS/MS analysis. The protein identities are summarized in Table 2, which presents data on protein score, protein mass, protein coverage and p value. Ten distinct proteins were identified in all tear samples of DES grade 4 patients including zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B. !
2014 Informa Healthcare USA, Inc.
FIGURE 1 One-dimensional electrophoresis gel of coomassie stained proteins. All lane contain 30 mg of total protein precipitated from tears collected by capillary. Protein band C was only observed in tears of DES grade 4 patients. (A) Lactoferrin; (B) lysozyme; (C) serum albumin, zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B.
LC-MS/MS spectra for zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B are presented in Figure 3. Figure 3(A) shows the MS/MS spectrum of peptide ion (KMVEDRQSGDLEK) found in gij50657091 (zincfinger motif-enhancer binding-protein-1 gamma). Figure 3(B) shows the MS/MS spectrum of peptide ion (DQDESDSDTEGEK) found in gij6683500 (bromodomain adjacent to zinc finger domain 2B. The proteins from the A and B bands were analyzed by nano LC-MS/MS analysis. The protein identities are summarized in Table 2, which presents data on protein score, protein mass, protein coverage and p-value. Twelve distinct proteins were identified in band A including lactoferrin and sixteen distinct proteins were identified in band B including lysozyme. In the LC-MS/MS results, protein scores were derived from ions scores as a non-probabilistic basis for ranking protein hits. Ions scores were 10 Log
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
434 S. H. Lee et al.
FIGURE 2 LC-MS/MS spectra of peptide fragment representing proteins observed in DES grade 4 patients. (A) Zinc-finger motifenhancer binding-protein-1 gamma peptide ion (KMVEDRQSGDLEK). (B) Bromodomain adjacent to zinc finger domain 2B peptide ion (DQDESDSDTEGEK). Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome
435
TABLE 2 Identification of the protein detected in SDS-PAGE using LC-MS/MS method. Protein score
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
Protein name Band A in gij187122 lactoferrin gij48425709 Chain A, Structure Of Human Diferric Lactoferrin At 2.5a Resolution Using Crystals Grown At Ph 6.5 gij17066105 Titin gij13654237 DNA-dependent protein kinase catalytic subunit isoform 1 gij34328016 KIAA0614 protein gij238236 transmembrane secretory component gij223069 protein Tro alpha1 H,myeloma gij223099 Ig Aalpha1 Bur gij70058 Ig alpha-2 chain C region gij229537 Ig A H gij2135473 Ig alpha-2 chain - human (fragment) gij10835403 Chain H, Crystal Structure Of An Unliganded (Native) Fv From A Human Igm Anti-Peptide Antibody
Protein mass (Da)
SDS-PAGE 3693 78,409 3186 76,194
Sequence coverage (%)
Calculated pI value
No. matched peptide
73% 66%
8.5 8.47
475 411
150 118
3,816,172 469,084
2% 3%
6.01 6.75
79 20
113 112 1001 934 888 870 660 529
328,672 83,313 51,072 50,613 36,573 51,108 36,557 13,203
4% 7% 29% 31% 38% 32% 38% 24%
5.96 5.58 8.77 9.34 7.64 9.27 5.85 8.61
16 4 203 224 147 186 174 20
71% 36% 68% 65%
5.39 8.26 9.28 9.14
279 25 11 10
60% 45% 5% 93% 90% 90%
9.28 9.38 5.63 9.28 9.28 9.28
10 13 12 334 335 335
90% 90% 90%
9.28 9.28 9.28
334 333 332
91%
9.28
334
90% 90%
9.28 9.14
332 332
60% 61% 58% 68% 61% 7% 2% 7% 4%
6.05 5.96 5.99 6.14 6.97 6.02 6.01 5.95 8.52
258 253 239 167 161 12 77 13 9
3%
5.27
25
Band B in SDS-PAGE gij4504963 lipocalin-1 precursor 1037 19,250 gij4505821 prolactin-inducible protein precursor 294 16,572 gij4930014 Chain A, T11a Mutant Human Lysozyme 255 14,671 gij11514208 Chain A, Crystal Structure Of Mutant Human 245 14,601 Lysozyme Substituted At Left-Handed Helical Positions gij4930021 Chain A, T43v Mutant Human Lysozyme 228 14,699 gij30714 lysozyme precursor (EC 3.2.1.17) 212 16,555 gij20521976 KIAA1731 protein 91 233,738 gij4930014 Chain A, T11a Mutant Human Lysozyme 858 14,671 gij17942568 Chain A, G127a Human Lysozyme 852 14,715 gij6729881 Chain A, Verification Of Spmp Using Mutant 851 14,761 Human Lysozymes gij17942566 Chain A, G129a Human Lysozyme 840 14,715 gij14278473 Chain A, Buried Polar Mutant Human Lysozyme 839 14,703 gij9955033 Chain A, Crystal Structure Of Mutant Human 837 14,733 Lysozyme Substituted At The Surface Positions gij11513937 Chain A, Crystal Structure Of Mutant Human 837 14,658 Lysozyme Substituted At Left-Handed Helical Positions gij17942570 Chain A, G72a Human Lysozyme 827 14,715 gij13399625 Chain A, Mutant Human Lysozyme (Q86d) 826 14,687 Band C in SDS-PAGE gij28592 serum albumin 2480 69,365 gij11493459 PRO2619 2335 56,782 gij178345 alloalbumin Venezia 2330 69,226 gij7770217 PRO2675 1412 32,574 gij6650826 PRO2044 1370 29,248 gij179665 complement component C3 266 187,162 gij17066105 Titin 128 3,816,172 gij6683500 bromodomain adjacent to zinc finger domain 2B 96 220,863 gij50657091 Zinc-finger motif-Enhancer binding-Protein-1 92 159,470 gamma gij24417711 nesprin-2 92 796,260
Zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B were detected in tears of DES grade 4 patients.
(P), where P was the probability that the observed match is a random event. Individual ions score higher than 44 indicated identity or extensive homology (p50.05).
DISCUSSION Perturbation of tear proteins may induce the development of ocular disease, which makes tear proteins !
2014 Informa Healthcare USA, Inc.
an interesting target for ocular disease diagnosis. Our study revealed increased serum albumin and decreased lactoferrin and lysozyme in the tears of patients with DES compared to healthy individuals. These results are similar to previous research.13,25,26 Lactoferrin and lysozyme are components of the innate immune system and have antimicrobial, antibacterial and antiviral activity.27–32 Serum albumin is considered to be derived from the plasma
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
436 S. H. Lee et al.
FIGURE 3 Relative density of protein bands in one-dimensional electrophoresis gel. Relative density was calculated the relative percentage of each protein of total density in each lane. Values are presented as mean standard error (%). p50.05 is considered significant. A: Lactoferrin; B: Lysozyme; C: Serum albumin.
and to leak into the tear fluid from conjunctival blood vessels,33 and is thus a potentially significant inflammatory marker indicating vascular leakage. Up-regulated permeability spurs development of inflammation.34 Therefore, inflammation may be induced at the ocular surface in patients with DES. The common diagnostic tests for dry eye are the Schirmer test and measurement of tear break-up time (TBUT). However, the Schirmer test suffers from error and has low reproducibility35 and TBUT can destabilize the tear film because of instillation of fluorescein.36 On the other hand, diagnostic testing using tear proteins can be effective. Tear proteins analyzed by electrophoretic separation and MS reveals distinct patterns associated with specific disease.13,37 In addition, electrophoresis is a good diagnostic tool for those at high risk of dry eye38 and electrophoretic separation and MS have been used to investigate for tear protein of patients with DES.37,39 Although SDS-PAGE and LC-MS/MS is a high cost measurement, precise diagnosis of DES through SDSPAGE and LC-MS/MS can provide a prompt
treatment to DES patients. Presently, one-dimensional electrophoresis was conducted to investigate difference between tear proteins of patients with DES and normal controls. A specific protein pattern was evident in DES grade 4 patients. Zinc finger proteins stabilized by zinc ion binding cause squamous metaplasia and regulate vitamin A expression. As squamous metaplasia of the ocular surface is an indication of DES and vitamin A deficiency is an essential cause of xerophthalmia40–43, zinc finger proteins may be related to DES. Association of zinc finger proteins and DES has not been investigated previously. In this study, zinc finger proteins detected in tears of patients with dry eye severity grade 4 were zinc-finger motif-enhancer binding-protein-1 gamma and bromodomain adjacent to zinc finger domain 2B. RREB1 encoding zinc-finger motif-enhancer bindingprotein-1 gamma is a gene that has been associated with inflammatory and apoptosis signaling pathways.43 Human bromodomain containing proteins have been demonstrated in pathological conditions including inflammation.45 Current Eye Research
Zinc Finger Protein in Severe Dry Eye Syndrome In conclusion, measurement of tear proteins may be an important diagnostic tool in DES. In addition, zinc finger protein may be related to the pathogenesis of DES and may be used to indicate severe DES. Further investigations will be necessary to conclusively investigate the relationship of zinc finger proteins in tears of patients with DES.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
ACKNOWLEDGEMENTS We wish to acknowledge technical support from Yonsei Proteome Research Center (www.proteomix. org).
DECLARATION OF INTEREST The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This research was supported by the Biomedical science Scholarship Grants, Department of Medicine, Chung-Ang University in 2013.
REFERENCES 1. Bhavsar AS, Bhavsar SG, Jain SM. A review on recent advances in dry eye: pathogenesis and management. Oman J Ophthalmol 2011;4:50–56. 2. Rolando M, Barabino S, Mingari C, Moretti S, Giuffrida S, Calabria G. Distribution of conjunctival HLA-DR expression and the pathogenesis of damage in early dry eye. Cornea 2005;24:951–954. 3. Stern ME, Gao J, Siemasko KF, Beuerman RW, Pflugfelder SC. The role of the lacrimal functional unit in the pathophysiology of dry eye. Exp Eye Res 2004;78:409–416. 4. Dana MR, Hamrah P. Role of immunity and inflammation in corneal and ocular surface disease associated with dry eye. Adv Exp Med Biol 2002;506:729–738. 5. Lemp MA. Advances in understanding and managing dry eye disease. Am J Ophthalmol. 2008;146:350–356. 6. De Paiva CS, Villarreal AL, Corrales RM, Rahman HT, Chang VY, Farley WJ, et al. Dry eye-induced conjunctival epithelial squamous metaplasia is modulated by interferon-gamma. Invest Ophthalmol Vis Sci 2007;48: 2553–2560. 7. Brewitt H, Sistani F. Dry eye disease: the scale of the problem. Surv Ophthalmol 2001;45:S199–S202. 8. Schein OD, Mun˜oz B, Tielsch JM, Bandeen-Roche K, West S. Prevalence of dry eye among the elderly. Am J Ophthalmol 1997;124:723–728. 9. Lemp MA. Report of the National Eye Institute/Industry Workshop on Clinical Trials in Dry Eyes. CLAO J 1995;21: 221–232. 10. Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I. Diagnostic potential of tear proteomic patterns in Sjogren’s syndrome. J Proteome Res 2005;4:820–825. 11. Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, et al. SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 2005;46:863–876. !
2014 Informa Healthcare USA, Inc.
437
12. Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, et al. Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 2003;136: 291–299. 13. Versura P, Nanni P, Bavelloni A, Blalock WL, Piazzi M, Roda A, et al. Tear proteomics in evaporative dry eye disease. Eye (Lond) 2010;24:1396–1402. 14. Huang JF, Zhang Y, Rittenhouse KD, Pickering EH, McDowell MT. Evaluations of tear protein markers in dry eye disease: repeatability of measurement and correlation with disease. Invest Ophthalmol Vis Sci 2012;53: 4556–4564. 15. Na KS, Mok JW, Kim JY, Rho CR, Joo CK. Correlations between tear cytokines, chemokines, and soluble receptors and clinical severity of dry eye disease. Invest Ophthalmol Vis Sci 2012;53:5443–5450. 16. Enrı´quez-de-Salamanca A, Castellanos E, Stern ME, Ferna´ndez I, Carren˜o E, Garcı´a-Va´zquez C, et al. Tear cytokine and chemokine analysis and clinical correlations in evaporative-type dry eye disease. Mol Vis 2010;16: 862–873. 17. Chowdhury K, Rohdewohld H, Gruss P. Specific and ubiquitous expression of different Zn finger protein genes in the mouse. Nucleic Acids Res 1988;16:9995–10011. 18. Krishna SS, Majumdar I, Grishin NV. Structural classification of zinc fingers: survey and summary. Nucleic Acids Res 2003;31:532–550. 19. Laity JH, Lee BM, Wright PE. Zinc finger proteins: new insights into structural and functional diversity. Curr Opin Struct Biol 2001;11:39–46. 20. Tseng H. Basonuclin, a zinc finger protein associated with epithelial expansion and proliferation. Front Biosci 1998;3: D985–D988. 21. Tseng H, Green H. Basonuclin: a keratinocyte protein with multiple paired zinc fingers. Proc Natl Acad Sci U S A. 1992;89:10311–10315. 22. Li S, Gallup M, Chen YT, McNamara NA. Molecular mechanism of proinflammatory cytokine-mediated squamous metaplasia in human corneal epithelial cells. Invest Ophthalmol Vis Sci 2010;51:2466–2475. 23. Brody T. Nutritional biochemistry. San Diego: Academic Press; 1999. 24. Kaercher T, Bron AJ. Classification and diagnosis of dry eye. Dev Ophthalmol 2008;41:36–53. 25. Zhou L, Beuerman RW, Chan CM, Zhao SZ, Li XR, Yang H, et al. Identification of tear fluid biomarkers in dry eye syndrome using iTRAQ quantitative proteomics. J Proteome Res 2009;8:4889–4905. 26. Ohashi Y, Ishida R, Kojima T, Goto E, Matsumoto Y, Watanabe K, et al. Abnormal protein profiles in tears with dry eye syndrome. Am J Ophthalmol 2003;136:291–299. 27. Yang PZ, Zhang XF, Zhang JS, Wu YD, Shi RH. The longitudinal observation of the effect of antibiotics on human tear lysozyme activity. Yan Ke Xue Bao 1989;5: 60–64. 28. Kirkpatrick CH, Green I, Rich RR, Schade AL. Inhibition of growth of Candida albicans by iron-unsaturated lactoferrin: relation to host-defense mechanisms in chronic mucocutaneous candidiasis. J Infect Dis 1971;124:539–544. 29. Valenti P, Antonini G. Lactoferrin: an important host defence against microbial and viral attack. Cell Mol Life Sci 2005;62:2576–2587. 30. Ward PP, Paz E, Conneely OM. Multifunctional roles of lactoferrin: a critical overview. Cell Mol Life Sci 2005;62: 2540–2548. 31. Legrand D, Elass E, Carpentier M, Mazurier J. Lactoferrin: a modulator of immune and inflammatory responses. Cell Mol Life Sci 2005;62:2549–2559.
Curr Eye Res Downloaded from informahealthcare.com by University of Newcastle Upon Tyne on 12/20/14 For personal use only.
438 S. H. Lee et al. 32. Gustot A, Raussens V, Dehousse M, Dumoulin M, Bryant CE, Ruysschaert JM, et al. Activation of innate immunity by lysozyme fibrils is critically dependent on cross-beta sheet structure. Cell Mol Life Sci 2013;70:2999–3012. 33. Fukuda M, Fullard RJ, Willcox MD, Baleriola-Lucas C, Bestawros F, Sweeney D, et al. Fibronectin in the tear film. Invest Ophthalmol Vis Sci 1996;37:459–467. 34. Sack RA, Tan KO, Tan A. Diurnal tear cycle: evidence for a nocturnal inflammatory constitutive tear fluid. Invest Ophthalmol Vis Sci 1992;33:626–640. 35. Lucca JA, Nunez JN, Farris RL. A comparison of diagnostic tests for keratoconjunctivitis sicca: lactoplate, Schirmer, and tear osmolarity. CLAO J 1990;16:109–112. 36. Mengher LS, Bron AJ, Tonge SR, Gilbert DJ. A non-invasive instrument for clinical assessment of the pre-corneal tear film stability. Curr Eye Res 1985;4:1–7. 37. Grus FH, Podust VN, Bruns K, Lackner K, Fu S, Dalmasso EA, et al. SELDI-TOF-MS ProteinChip array profiling of tears from patients with dry eye. Invest Ophthalmol Vis Sci 2005;46:863–876. 38. Chiva A. Electrophoresis of tear proteins as a new diagnostic tool for two high risk groups for dry eye:
39.
40. 41. 42. 43.
44.
45.
computer users and contact lens wearers. J Med Life 2011; 4:228–233. Tomosugi N, Kitagawa K, Takahashi N, Sugai S, Ishikawa I. Diagnostic potential of tear proteomic patterns in Sjogren’s syndrome. J Proteome Res 2005;4:820–825. McLaren DS. Vitamin A deficiency disorders. J Indian Med Assoc 1999;97:320–323. Sommer A. Vitamin a deficiency and clinical disease: an historical overview. J Nutr 2008;138:1835–1839. Tseng SC. Topical tretinoin treatment for dry-eye disorders. Int Ophthalmol Clin 1987;27:47–53. Li S, Nikulina K, De Voss J, Wu AJ, Strauss EC, Anderson MS, et al. Small proline-rich protein 1B (SPRR1B) is a biomarker for squamous metaplasia in dry eye disease. Invest Ophthalmol Vis Sci 2008;49:34–41. Kurji KH, Cui JZ, Lin T, Harriman D, Prasad SS, Kojic L, et al. Microarray analysis identifies changes in inflammatory gene expression in response to amyloid-beta stimulation of cultured human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci 2010;51:1151–1163. Sanchez R, Zhou MM. The role of human bromodomains in chromatin biology and gene transcription. Curr Opin Drug Discov Devel 2009;12:659–665.
Notices of Correction: A transposition of Figure legends 2 and 3, and an inaccuracy in the declaration of interest, were corrected following ahead-of-print online publication.
Current Eye Research
