Proteomic analysis of the tear film in patients with keratoconus

Isabel Lema, David Brea, Raquel Rodríguez-González, Elío Díez-Feijoo, Tomás Sobrino, Isabel Lema, David Brea, Raquel Rodríguez-González, Elío Díez-Feijoo, Tomás Sobrino

Abstract

Purpose: To identify proteins differentially expressed between the tear film of keratoconus (KC) patients and control subjects using two dimensional electrophoresis (2-DE) and mass spectrometry-based techniques.

Methods: Twenty two patients (44 eyes) diagnosed with bilateral KC and 22 control subjects (44 eyes) were studied in a prospective case-control study. Keratoconus screening programs and Orbscan II topographies were performed on all participants. Tear samples were collected by the Schirmer I method using filter paper. Proteins were extracted from the Schirmer strips and separated by 2-DE. Comparison of protein patterns was performed using PDQuest Software and protein differences were identified by mass spectrometry. Finally, results were validated by western-blot.

Results: Four spots were identified to be differentially expressed between KC patients and control subjects. Three of them were more expressed in healthy subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG. Differences in ZAG seem controversial in two different spots because different posttranslational modifications, however, analysis of both spots revealed that globally, ZAG is overexpressed in healthy subjects. Founded differences in ZAG, lactoferrin, and IGKC expression were subsequently validated by western blot.

Conclusions: IGKC protein, ZAG, and lactoferrin are under-expressed in the tears of patients diagnosed with bilateral KC compared with healthy subjects. These differences could contribute to the knowledge of the pathophysiology of this disease.

Figures

Figure 1
Figure 1
Master gel created by combining spots from all gels of keratoconus patients and control subjects.
Figure 2
Figure 2
Two-dimensional gel fragments showing differential expressed proteins between keratoconus patients and control subjects. Three of them were more expressed in control subjects and they were identified as zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain). The other spot was more expressed in KC patients and it was identified as ZAG.
Figure 3
Figure 3
A typical profile of molecular mass of tryptic fragments of zinc- α2-glycoprotein (ZAG) during analysis by MALDI-TOF mass spectrometry.
Figure 4
Figure 4
Western-blot analysis of the identified proteins showing a significant decrease in protein expression of zinc-α2-glycoprotein (ZAG), lactoferrin, and IGKC (immunoglobulin kappa chain) in KC patients compared to control subjects. O.D.: optical density; A.U.: arbitrary units; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; *p

References

    1. Krachmer JH, Feder RS, Belin MW. Keratoconus and related noninflammatory corneal thinning disorders. Surv Ophthalmol. 1984;28:293–322.
    1. Rabinowitz YS. Keratoconus. Surv Ophthalmol. 1998;42:297–319.
    1. Dobbins KR, Price FW, Jr, Whitson WE. Trends in the indications for penetrating keratoplasty in the midwestern United States. Cornea. 2000;19:813–6.
    1. Tuft SJ, Moodaley LC, Gregory WM, Davison CR, Buckley RJ. Prognostic factors for the progression of keratoconus. Ophthalmology. 1994;101:439–47.
    1. Kaldawy RM, Wagner J, Ching S, Seigel GM. Evidence of apoptotic cell death in keratoconus. Cornea. 2002;21:206–9.
    1. Erie JC, Patel SV, McLaren JW, Nau CB, Hodge DO, Bourne WM. Keratocyte density in keratoconus. A confocal microscopy study(a). Am J Ophthalmol. 2002;134:689–95.
    1. Wilson SE, He YG, Weng J, Li Q, McDowall AW, Vital M, Chwang EL. Epithelial injury induces keratocyte apoptosis: hypothesized role for the interleukin-1 system in the modulation of corneal tissue organization and wound healing. Exp Eye Res. 1996;62:325–7.
    1. Rahi A, Davies P, Ruben M, Lobascher D, Menon J. Keratoconus and coexisting atopic disease. Br J Ophthalmol. 1977;61:761–4.
    1. Chandler JW. Immunology of the ocular surface. Int Ophthalmol Clin. 1985;25:13–23.
    1. Bonini S, Lambiase A, Sgrulletta R, Bonini S. Allergic chronic inflammation of the ocular surface in vernal keratoconjunctivitis. Curr Opin Allergy Clin Immunol. 2003;3:381–7.
    1. Zhou L, Sawaguchi S, Twining SS, Sugar J, Feder RS, Yue BY. Expression of degradative enzymes and protease inhibitors in corneas with keratoconus. Invest Ophthalmol Vis Sci. 1998;39:1117–24.
    1. Collier SA, Madigan MC, Penfold PL. Expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) and MMP-2 in normal and keratoconus corneas. Curr Eye Res. 2000;21:662–8.
    1. Lema I, Duran JA. Inflammatory molecules in the tears of patients with keratoconus. Ophthalmology. 2005;112:654–9.
    1. de Freitas Campos C, Cole N, Van Dyk D, Walsh BJ, Diakos P, Almeida D, Torrecilhas A, Laus JL, Willcox MD. Proteomic analysis of dog tears for potential cancer markers. Res Vet Sci. 2008;85:349–52.
    1. Ananthi S, Chitra T, Bini R, Prajna NV, Lalitha P, Dharmalingam K. Comparative analysis of the tear protein profile in mycotic keratitis patients. Mol Vis. 2008;14:500–7.
    1. de Souza GA, Godoy LM, Mann M. Identification of 491 proteins in the tear fluid proteome reveals a large number of proteases and protease inhibitors. Genome Biol. 2006;7:R72.
    1. Zadnik K, Barr JT, Gordon MO, Edrington TB. Biomicroscopic signs and disease severity in keratoconus. Collaborative Longitudinal Evaluation of Keratoconus (CLEK) Study Group. Cornea. 1996;15:139–46.
    1. Shevchenko A, Wilm M, Vorm O, Mann M. Mass spectrometric sequencing of proteins from silver-stained polyacrylamide gels. Anal Chem. 1996;68:850–8.
    1. Green-Church KB, Nichols KK, Kleinholz NM, Zhang L, Nichols JJ. Investigation of the human tear film proteome using multiple proteomic approaches. Mol Vis. 2008;14:456–70.
    1. Flanagan JL, Willcox MD. Role of lactoferrin in the tear film. Biochimie. 2009;91:35–43.
    1. Puddu P, Valenti P, Gessani S. Immunomodulatory effects of lactoferrin on antigen presenting cells. Biochimie. 2009;91:11–8.
    1. Choe YH, Lee SW. Effect of lactoferrin on the production of tumor necrosis factor-alpha and nitric oxide. J Cell Biochem. 1999;76:30–6.
    1. Mattsby-Baltzer I, Roseanu A, Motas C, Elverfors J, Engberg I, Hanson LA. Lactoferrin or a fragment thereof inhibits the endotoxin-induced interleukin-6 response in human monocytic cells. Pediatr Res. 1996;40:257–62.
    1. Håversen L, Ohlsson BG, Hahn-Zoric M, Hanson LA, Mattsby-Baltzer I. Lactoferrin down-regulates the LPS-induced cytokine production in monocytic cells via NF-kappa B. Cell Immunol. 2002;220:83–95.
    1. Shoham A, Hadziahmetovic M, Dunaief JL, Mydlarski MB, Schipper HM. Oxidative stress in diseases of the human cornea. Free Radic Biol Med. 2008;45:1047–55.
    1. Russell ST, Zimmerman TP, Domin BA, Tisdale MJ. Induction of lipolysis in vitro and loss of body fat in vivo by zinc-alpha-(2)-glycoprotein. Biochim Biophys Acta. 2004;1636:59–68.
    1. Hassan MI, Waheed A, Yadav S, Singh TP, Ahmad F. Zinc alpha 2-glycoprotein: a multidisciplinary protein. Mol Cancer Res. 2008;6:892–906.
    1. Baker GR, Morton M, Rajapaska RS, Bullock M, Gullu S, Mazzi B, Ludgate M. Altered tear composition in smokers and patients with Graves ophthalmopathy. Arch Ophthalmol. 2006;124:1451–6.
    1. Chandler JW. Ocular surface immunology. In: Pepose JS, Holland GN, Wilhelmus KR, editors. Ocular infection and immunity. St Louis: Mosby; 1996. p. 104–11.
    1. Bonini S, Lambiase A, Juhas T, Rama P. Inflammatory immune-associated diseases of the cornea. In: Ben Ezra D, editor. Ocular inflammation: basic and clinical concepts. London: Martin Dunitz; 1999. p. 151–68.
    1. Lema I, Sobrino T, Durán JA, Brea D, Díez-Feijoo E. Subclinical keratoconus and inflammatory molecules from tears. Br J Ophthalmol. 2009;93:820–4.
    1. Srivastava OP, Chandrasekaran D, Pfister RR. Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas. Mol Vis. 2006;12:1615–25.

Source: PubMed

Sponsors et collaborateurs

Les conditions médicales

Interventions en matière de drogue

3
S'abonner