Molecular changes in selected epithelial proteins in human kertoconus corneas compared to normal corneas

Department of Vision Sciences, University of Alabama at Birmingham, Birmingham, AL 35294, USA.
Molecular vision (Impact Factor: 1.99). 02/2006; 12(182-86):1615-25.
Source: PubMed


The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas.
Two-dimensional (2-D) gel electrophoretic profiles of epithelial cell proteins from normal and keratoconus corneas were compared, and the selected protein spots that showed either up- or downregulation were identified. The desired spots were identified after trypsin digestion and mass spectrometric analysis. Based on the results, two proteins, alpha-enolase and beta-actin, were further analyzed by immunohistochemical and western blot methods, using respective antibodies. To determine the presence of mRNA of the two proteins in the epithelial cells, RT-PCR studies were performed.
On comparison of the 2-D gel electrophoretic protein profiles, two protein spots were identified in normal corneas that were either absent or present at lower levels in keratoconus corneas. The two spots were determined to be alpha-enolase (48 kDa) and beta-actin (42 kDa) by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF), and ES-MS/MS mass spectrometric methods. Immunohistochemical analysis revealed that alpha-enolase and beta-actin were present at extremely low levels in the epithelial superficial and wing cells of the keratoconus corneas compared to these cells of normal corneas. 2-D gel electrophoresis followed by western blot analysis revealed relatively greater degradation of the two proteins in the keratoconus corneas compared to normal corneas. RT-PCR analysis showed the mRNA expression of the two proteins in the epithelial cells of both normal and keratoconus corneas.
The results showed relatively low or negligible levels of alpha-enolase and beta-actin in the wing and superficial epithelial cells of keratoconus corneas compared to normal corneas. This was attributed to relatively greater degradation of the two proteins in keratoconus corneas compared to normal corneas.

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Available from: Deepa Chandrasekaran, May 01, 2015
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    • "Strangely, our study did not identify changes in expression levels of either degradative enzymes or protease inhibitors, but it might be indicated by the suppressed metabolic activity of enzymes as observed in KC corneas. A stress-related heat shock 71 kDa protein exhibited up-regulation in epithelium, which is supported by a previous report that showed altered expression of several wound healing or stress-related proteins (vimentin, tenascin, transforming growth factor-beta, interleukin-1, heat shock protein 27, and ubiquitin 9) in KC corneas (Srivastava et al., 2006). Nuclear lamins are intermediate filaments that are involved in maintenance of shape and stability of mammalian cells. "
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    ABSTRACT: The purpose of the study was to identify epithelial and stromal proteins that exhibit up- or down-regulation in keratoconus (KC) vs. normal human corneas. Because previous proteomic studies utilized whole human corneas or epithelium alone, thereby diluted the specificity of the proteome of each tissue, we selectively analyzed the epithelium and stromal proteins. Individual preparations of epithelial and stromal proteins from KC and age-matched normal corneas were analyzed by two independent methods, i.e., a shotgun proteomic using a Nano-Electrospray Ionization Liquid Chromatography Tandem Mass Spectrometry [Nano-ESI-LC-MS (MS)(2)] and two-dimensional-difference gel electrophoresis (2D-DIGE) coupled with mass spectrometric methods. The label-free Nano-ESI-LC-MS (MS)(2) method identified 104 epithelial and 44 stromal proteins from both normal and KC corneas, and also quantified relative changes in levels of selected proteins, in both the tissues using spectral counts in a proteomic dataset. Relative to normal corneal epithelial proteins, six KC epithelial proteins (lamin-A/C, keratin type I cytoskeletal 14, tubulin beta chain, heat shock cognate 71 kDa protein, keratin type I cytoskeletal 16 and protein S100-A4) exhibited up-regulation and five proteins (transketolase, pyruvate kinase, 14-3-3 sigma isoform, phosphoglycerate kinase 1, and NADPH dehydrogenase (quinone) 1) showed down-regulation. A similar relative analysis showed that three KC stromal proteins (decorin, vimentin and keratocan) were up-regulated and five stromal proteins (TGF-betaig h3 (Bigh3), serotransferrin, MAM domain-containing protein 2 and isoforms 2C2A of collagen alpha-2[VI] chain) were down-regulated. The 2D-DIGE-mass spectrometry followed by Decyder software analysis showed that relative to normal corneas, the KC corneal epithelium exhibited up-regulation of four proteins (serum albumin, keratin 5, L-lactate dehydrogenase and annexin A8) and down-regulation of four proteins (FTH1 [Ferritin heavy chain protein 1], calpain small subunit 1, heat shock protein beta 1 and annexin A2). A similar relative analysis of stroma by this method also showed up-regulation of aldehyde dehydrogenase 3A1 (ALDH3A1), keratin 12, apolipoprotein A-IV precursor, haptoglobin precursor, prolipoprotein and lipoprotein Gln in KC corneas. Together, the results suggested that the Nano-ESI-LC-MS(MS)(2) method was superior than the 2D-DIGE method as it identified a greater number of proteins with altered levels in KC corneas. Further, the epithelial and stromal structural proteins of KC corneas exhibited altered levels compared to normal corneas, suggesting that they are affected due to structural remodeling during KC development and progression. Additionally, because several epithelial and stromal enzymes exhibited up- or down-regulation in the KC corneas relative to normal corneas, the two layers of KC corneas were under metabolic stress to adjust their remodeling.
    Full-text · Article · Apr 2011 · Experimental Eye Research
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    • "However, the robustness of the results gives support to the need to explore the role of these molecular markers as a new therapeutic or diagnostic tool in the KC. Although sample size is not so large, similar proteomic studies have used similar sample sizes [32]. Another limitation is that proteomics based on two-dimensional gels has a sensitivity in the order of magnitude of nanograms. "
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    ABSTRACT: 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. 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. 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. 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.
    Full-text · Article · Oct 2010 · Molecular vision
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    • "Proteomics technology provides a powerful high throughput screening tool to identify protein expression differences that may underlie age-related changes at the functional level. Protocols similar to those used in the current study have been used to conduct proteomic analyses of other ocular cells [25,31,36,37]. The experimental approach used for these studies involved collection of protein extracted from five individual donors within each age group. "
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    ABSTRACT: To establish a baseline protein fingerprint of cultured human corneal endothelial cells (HCEC), to determine whether the protein profiles exhibit age-related differences, and to identify proteins differentially expressed in HCEC cultured from young and older donors. Corneas were obtained from five young (<30 years old) and five older donors (>50 years old). HCEC were cultured, and protein was extracted from confluent passage 3 cells. Extracts from each age group were pooled to form two samples. Proteins were separated on two-dimensional (2-D) gels and stained with SyproRuby. Resultant images were compared to identify protein spots that were either similarly expressed or differentially expressed by at least twofold. Protein spots were then identified by matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry. Protein spots were well resolved, and patterns were reproducible on 2-D gels using either pH 3-10 or pH 4-7 IPG strips. Two-dimensional gels prepared with pH 4-7 IPG strips were used for differential display analysis, which was reproduced on three separate pairs of gels. MALDI-TOF identified 58 proteins with similar expression; 30 proteins were expressed twofold higher in HCEC from young donors; five proteins were expressed twofold higher in cells from older donors; and 10 proteins were identified in gels from young donors that did not match in gels from older donors. Several proteins expressed at higher levels in younger donors support metabolic activity, protect against oxidative damage, or mediate protein folding or degradation. This is the first proteomic comparison of proteins expressed in HCEC cultured from young and older donors. Although restricted to proteins with isoelectric points between pH 4.0 and pH 7.0, the data obtained represent an initial step in the investigation of molecular mechanisms that underlie physiologically important age-related differences in cultured HCEC, including differences that may affect proliferative capacity. Results indicate that HCEC from older donors exhibit reduced expression of proteins that support important cellular functions such as metabolism, antioxidant protection, protein folding, and protein degradation. These differences may affect the ability to consistently obtain a sufficient number of healthy cultured HCEC for use in preparing bioengineered endothelium as an alternative method for the treatment of endothelial dysfunction.
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