|Molecular Vision 2006;
Received 21 September 2005 | Accepted 14 December 2006 | Published 20 December 2006
Molecular changes in selected epithelial proteins in human keratoconus corneas compared to normal corneas
Om P. Srivastava,1
Roswell R. Pfister2
1Department of Vision Sciences, University of Alabama at Birmingham and 2Eye Research Laboratory, Birmingham, AL
Correspondence to: Om P. Srivastava, Department of Vision Sciences, Worrell Building, 924-South 18th Street, University of Alabama at Birmingham, Birmingham, AL, 35294; Phone: (205) 975-7630; FAX: (205) 934-5725; email: email@example.com
Purpose: The purpose of the study was to determine molecular changes in selected epithelial proteins in human keratoconus (KC) corneas compared to normal corneas.
Methods: 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, α-enolase and β-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.
Results: 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 α-enolase (48 kDa) and β-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 α-enolase and β-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.
Conclusions: The results showed relatively low or negligible levels of α-enolase and β-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.
The keratoconic cornea assumes a conical shape as a result of noninflammatory thinning of corneal stroma. It is a gradually progressive disease with an unknown cause, inducing corneal thinning, irregular astigmatism, myopia, and central or paracentral conical protrusion. As the disease progresses, glasses, contact lenses or, ultimately, keratoplasty might be required. The incidence of keratoconus (KC) is 1 per 2000 in the general population . The classical histopathological features include stromal thinning, iron deposits in the epithelial basement membrane, and breaks in Bowman's layer. Several reports describe an association of keratoconus with Down's syndrome , Lebers congenital amaurosis, and mitral valve prolapse . Despite intensive investigations into the pathogenesis of keratoconus, the biochemical mechanism of the disease is poorly understood. It has been suggested that thinning of the cornea may be due to defective formation or destruction of extracellular matrix because altered or abnormal levels of fibronectin and type VI collagen were observed . In the early stages of KC, the cell membrane breakage along with disappearance of the basal cells occur , and also a deposition of particulate materials between the surface of basal epithelial cells and Bowman's layer was observed . As the basal cells degenerate, they might release proteolytic enzymes that destroy the underlying tissue. Indeed, the abnormality in corneal collagenase activity has been suggested as a cause of corneal thinning [6,7]. Molecular studies have shown an increased expression of leukocyte common antigen-related protein (LAR) in cells of KC corneas . LAR is a transmembrane protein belonging to a family of proteins called phosphotyrosine phosphatases, which play a role in cell-cell interactions, cell-matrix interactions, and cell differentiation and proliferation. The appearance of LAR in KC corneas might cause apoptosis because LAR expression plays a role in apoptosis .
Biochemical and immunohistological studies of KC corneas have suggested that the loss of corneal stroma could be caused by increased levels of proteases and other catabolic enzymes or by decreased levels of protease inhibitors. Decreased levels of proteinase inhibitors, i.e., α1-proteinase inhibitor and α2-macroglobulin were also observed . An increased level of the proteoglycans and changes in their location with respect to collagen fibrils were also observed in KC corneas [10,11]. The interaction between collagen fibrils and proteoglycans might be important in maintaining normal corneal strength; therefore, these abnormalities could result in stretching and thinning of the stroma [11,12]. Wilson and Kim  proposed a role for an interleukin (IL)-1 system in the cornea in the pathogenesis of KC because KC keratocytes had four fold greater number of IL-1 receptors than normal corneas. In spite of these biochemical changes in the KC versus normal corneas, no clear pathology of KC has emerged from these studies.
Rabinowitz  has suggested that genetics play a role in the development of KC based on results from the Genetics Program at Cedars-Sinai Medical Center. KC is thought to be an autosomal inherited disease because of its bilaterality [15-17], its occurrence more often in certain families [18,19], and its development in homozygous twins . To date, genetic analyses have identified a KC gene at several chromosomal locations, i.e., 6.5 cM at chromosome 21 , association at 20q12 in seven related Tasmanian patients , third locus to chromosome 16q in autosomal keratoconus in a small (20 individuals) Finnish pedigree , two distinct heterozygous mutations in the VSXI homeobox gene , and at chromosome 3p14-q13 in an Italian family with autosomal dominant pure keratoconus . The apparent association of markers on chromosome 20 (identified in the aforementioned Tasmanian study ) to MMP-9 (a nearby candidate gene) was excluded. Similarly, the COL8A1 gene (maps at 3q14-q13) that encodes for human alpha (VIII) chain of type VIII collagen (expressed in different layers of the cornea) was also excluded. Another genome-wide linkage study indicated that a major gene responsible for 50-60% familial KC was localized within 1.69 MB region at 2p24 . Additionally, a causative gene of a family with autosomal dominant inheritance of keratoconus in association with cataract mapped to the long arm of chromosome 15 . Therefore, based on these reports, no markers for clinical diagnosis of KC are known. A recent microarray analysis to identify differentially expressed genes in keratoconus epithelium  showed that during keratoconus, massive changes in cytoskeleton caused reduced extracellular matrix remodeling, altered transmembrane signaling and modified cell to cell and cell-matrix interactions. Although this microarray analysis provided vast information but no valuable markers were evident for the diagnosis of KC.
The focus of the current study was to determine comparative up- or downregulation in selected epithelial proteins with molecular weights (MW) between 40 kDa and 60 kDa in the normal and KC corneas. Using the proteomic approach, we identified two proteins (α-enolase and β-actin) that showed downregulation in the epithelium of KC corneas compared with normal corneas.
Normal human corneas were obtained within 6-12 h of death from the Alabama Eye Bank through the Shared Ocular Tissue Module of the University of Alabama at Birmingham. Diseased corneas were obtained from a local corneal surgeon, Dr. Roswell Pfister, within 4-6 h following surgery. The sizes of the normal corneas were 10-13 mm in diameter and the diseased corneal buttons were 7-8 mm in diameter. Corneas were stored in Optisol-GS (Chiron Ophthalmic, Irvine, CA) at 4 °C before use. Prestained and unstained protein molecular weight markers were obtained from Life Technologies (Carlsbad, CA). Chemicals used in this study were obtained from either Fisher Scientific (Atlanta, GA) or Sigma (St. Louis, MO) unless stated otherwise.
Preparation of protein extracts from corneal epithelial cells
The corneal epithelium was scraped with a corneal gill knife (Strotz, St. Louis, MO), collected in ice-cold phosphate buffer saline (PBS), and stored at -20 °C until utilized. The cells were centrifuged at 5000x g for 10 min in a microcentrifuge (kept at 5 °C), and the pellet was suspended in resolubilization buffer (5 M urea, 2 M thiourea, 2% 3-[C3-cholamidoproyl] dimethyl-ammonio-1-propansulfonat (CHAPS), 2% caprylyl sulfobetaine 3-10, 2 mM tri-butyl phosphine, 40 mM Tris, pH 8.0)  for isoelectric focusing (IEF). The preparation was vortexed for 5 min to solubilized proteins, and these were either used immediately or the aliquots were stored at -20 °C for later use.
Two-dimensional gel electrophoretic analysis
Samples were analyzed by IEF in the first dimension followed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the second dimension. IEF was carried out according to the instructions of the manufacturer (GE Healthcare, Piscataway, NJ) using 11 cm IEF strips with pH range of 3-10. SDS-PAGE analysis was performed using 15% polyacrylamide gel by the Laemmli method . Two proteins preparations, one from five normal corneas (mean age: 55 years) and the other from five KC corneas (mean age: 50 years) were used for 2-D gel electrophoretic analysis to develop proteomic maps. The KC corneas showed no scarring. Identical amounts of proteins (900 μg) from keratoconus or normal corneas, and the identical IEF strips and SDS-polyacrylamide gels (15% polyacrylamide) were used. The first dimensional IEF and second dimensional SDS-PAGE electrophoresis of proteins from normal and keratoconus corneas were conducted under identical conditions to normalize the electrophoretic process.
MALDI-TOF and micromass QTOF mass spectrometric analyses
For mass spectrometric analyses, the individual protein spots were excised from a polyacrylamide gel, washed with doubly deionized water, and destained after treating with ammonium bicarbonate and acetonitrile. Trypsin solution (12 ng/μl) was added, and the preparation was resuspended in 25 mM ammonium bicarbonate, pH 7.8. The samples were digested by trypsin at 37 °C overnight, and the next day, they were analyzed by the MALDI-TOF method (Model Voyager-DE2 PRO; Perspective Biosciences, Forest City, CA). The MALDI-analysis and ES-MS/MS sequencing (micromass QTOF-2) were performed at the Comprehensive Cancer Center Mass Spectrometric Shared Facility of the University of Alabama at Birmingham. The MALDI-TOF-identity of proteins was established by using the NCBInr database of MatrixScience. During the ES-MS/MS analysis of the tryptic fragments, the data base of "Proteinlynx Global Server" was used along with manual interpretations as needed.
Immunohistochemical analysis of normal and KC corneal sections was done with a confocal microscope with commercially available antibodies raised against α-enolase (Biogenesis, Biogenesis, A division of MorphoSys US Inc., Kingston, NH, no clone number available from the company) and to β-actin (Clone AC-15; Accurate Chemical and Scientific, Westbury, NY). Three normal corneas (mean age: 55 years) and three KC corneas (mean age: 47 years) were used for immunohistochemical analysis. The KC corneas showed no scarring. Serial microtome sections (10 μM thick) of normal and KC corneas were cut starting from the periphery to the center.
The two primary antibodies were either a polyclonal anti-human α-enolase antibody or a fluorescein isothiocyanate (FITC)-conjugated monoclonal anti-human β-actin antibody. The anti-α-enolase antibody was raised against the whole protein molecule, whereas the anti-β-actin was raised against the NH2-terminus of the protein molecule. The secondary antibodies used were Alexafluor 488 goat anti-mouse IgG or Alexafluor 488 goat anti-rabbit IgG (Molecular Probes). Some of the corneal sections were stained for cell nuclei with 4,6-diamidino-2-phenylindole dihydrochloride (DAPI).
Corneas were fixed by using a procedure described by Takacs et al. . Tissue sections were washed 3X in 0.1 M PBS. The sections were then blocked using 2% bovine serum albumin in 0.1 M PBS for 1 h and washed again in 0.1 M PBS. Later, the sections were incubated in the primary antibodies in a solution of 0.3% Triton X-100 in 0.1 M PBS. These sections were kept at 4 °C overnight. The next day, the sections were washed in 0.1 M PBS and then incubated in the secondary antibodies for 1 h. After three to four washes in 0.1 M PBS, the sections were incubated in DAPI nuclear stain for 2 to 3 min. The tissue sections were again washed in 0.1 M PBS several times after the incubation. This procedure was conducted in minimal light to avoid damage to the fluorophores. The tissues were later washed in double-distilled water and covered with coverslip Permafluor. The sections were stored at 4 °C in dark and were later analyzed by a confocal microscope (Leica confocal imaging spectrophotometer TCS SP unit). In the control experiments, the individual primary antibodies were omitted but the secondary antibody was used as described above.
Isolation of RNA from corneal epithelial cells and Reverse-Transcription Polymerase Chain Reaction (RT-PCR)
Total RNA from the epithelial cells of normal or KC corneas was isolated using the TRIZOL reagent (Life Technologies, Carlsbad, CA). Published sequences of α-enolase and β-actin were used to design the primers for RT-PCR. A forward primer and a reverse primer were synthesized (Sigma, St. Louis, MO) with the sequences as shown: (a) forward primer of α-enolase: CGT GAC CGA GTC TCT TCA; reverse primer of α-enolase: ACG AGG CTC ACA TGA CTC (GenBank M14328, ). The reverse primer was intron-spanning. (b) forward primer of β-actin: CGT GAC ATT AAG GAG AAG; reverse primer of β-actin: CCA TGC CAA TCT CAT CTT (GenBank NM_001101).
Titan one-tube RT-PCR kit (Boehringer Mannheim, Indianapolis, IN) was used according to supplier instructions. PCR amplification using the Gene Amp PCR system 2400 (Perkin-Elmer, Foster City, CA) was carried out using the following temperature profile: 30 min at 50 °C, 2 min 94 °C, 10 cycles for 10 s at 94 °C, 30 s at 50 °C, 2 min at 68 °C, 11-25 cycles for 10 s at 94 °C, 30 s at 50 °C, 2 min at 68 °C, 1 cycle for 7 min at 68 °C. Agarose gel electrophoretic analysis was used to verify that the amplified product corresponded to the predicted size of the PCR products of α-enolase and β-actin.
The protein concentration in the corneal extracts was determined by the bicinchoninic acid method (Pierce, Rockford, IL) by using the resolubilization buffer (the buffer used to solubilize the epithelial cell proteins prior to 2-D gel electrophoresis) as a control blank. Epithelial protein yield in the extract ranged from 500 to 900 μg per cornea.
Western blot analysis
Western blot analysis was performed by the method of Towbin et al. . Protein preparations from pooled five normal corneas (mean age: 65 years) or five KC corneas (mean age: 55 years) were used for western blot analysis. The immunoreaction was visualized by peroxidase staining for α-enolase and β-actin.
Proteomic analysis of epithelial proteins from normal human corneas
Proteomic analyses of epithelial proteins from normal corneas were carried out by 2-D gel electrophoretic separation followed by mass spectrometric analyses. We used a resolubilization buffer to solubilize both cytosolic and membrane proteins of corneal epithelial cells. As shown in Figure 1, a total of 81 major spots were identified on a 2-D gel, and these were numbered starting with one with the lowest molecular weight. Following excision of individual spots and trypsin digestion, mainly MALDI-TOF, and in some cases, MALDI-TOF followed by ES-MS/MS method were used to determine the identity of the protein spots. As shown in Table 1, the MALDI-TOF method identified all but seven of the 81 epithelial proteins. The identified epithelial proteins by mass spectrometry showed a probability-based mowse (probability-based molecular weight search engine) score greater than 71, which is significant for the identification of a particular protein (www.matrixscience.com).
Comparative proteomic maps of epithelial proteins from human KC and normal corneas
The epithelial cell proteins from human KC and normal corneas were analyzed by 2-D gel electrophoresis to determine whether certain proteins were up- or downregulated during the disease. As stated in the methods, two proteins preparations, one from five normal corneas (mean age: 55 years) and the other from five KC corneas (mean age: 50 years) were used for 2-D gel electrophoretic analysis to develop proteomic maps. Almost all the spots were reproducibly seen in 2-D gel protein profiles, and this was further confirmed by analyzing 2-D gel protein profiles of four individual keratoconus and four individual normal corneas. After overlapping of the representative 2-D gel protein profiles, few spots were found only in the normal corneas and were absent in KC corneas and vice versa. The spots present in normal corneas but were either absent or present at low levels in KC corneas were numbers 21 and 29 (Figure 2A,B). After tryptic digestion and MALDI-TOF mass spectrometric analysis, spot number 21 was identified to be of β-actin, and spot number 29 as α-enolase. Because additional spots that showed up- or downregulation were present in insufficient quantities for mass spectrometric analysis, they could not be identified.
The MALDI-TOF mass spectrometric profile of tryptic fragments of spot number 29 (α-enolase) and the mass of tryptic fragments are shown in Figure 3 and Table 2, respectively. The identification score of α-enolase was 61, which were less than the optimal 71 (the score considered significant for identification of protein by MALDI-TOF). Therefore, tandem mass spectrometric analysis of the precursor ions of α-enolase was done, and two fragments of mass 1804 (fragment 1) and 1426 (fragment 2) were identified. The ES-MS/MS profiles of fragments 1 and 2 are shown in Figure 4A,B, respectively. Fragment 1 (mass 1804) showed a sequence of AAVPSGASTGIYEALELR (Figure 4A), and the second fragment with mass of 1426 showed a sequence of YISPDQLADLYK (Figure 4B). On overlapping sequences of the two fragments with the α-enolase sequence from the literature, the first fragment contained residue number 32-49 and the second fragment, residue number 269-280 of the protein (Figure 5). The mass of tryptic fragments of β-actin (spot number 21) during MALDI-TOF are shown in Table 3, which clearly showed that an observed and predicted mass of tryptic fragments of β-actin matched.
Immunohistochemical analysis of α-enolase and β-actin in KC and normal corneal sections
During the confocal miscroscopic immunohistochemical analysis of the normal corneas using polyclonal anti-α-enolase antibody, we saw a uniform immunofluorescence in the superficial, wing, and basal columnar cells (Figure 6A). The nuclei of the cells were seen in blue after staining with DAPI. In contrast, the KC tissue sections showed relatively weak immunofluorescence of the wing and superficial cells compared to the cells from normal cornea (Figure 6B). The control sections did not show any fluorescence (results not shown). A similar confocal microscopic immunohistochemical analysis with monoclonal FITC-conjugated anti-β-actin antibody showed a uniform immunofluorescence in the superficial, wing, and basal columnar cells from a normal cornea (Figure 6C). In contrast, the KC section revealed relatively weak immunofluorescence of the wing and basal cells (Figure 6D). The control sections lacked any fluorescence (Results not shown). Together, the data showed that the wing and superficial cells of KC corneas exhibited decreased levels of both α-enolase and β-actin compared to these cells from normal corneas. Next, the question to consider was whether the reduced levels of α-enolase and β-actin were due to their reduced expression or because of their degradation. To determine this, western blot analysis, was performed.
Western blot analysis using anti-α-enolase- and anti-β-actin antibodies
Western blot analysis using anti-α-enolase antibody on normal corneas revealed spots having molecular weights between 28 kDa and 50 kDa (Figure 7A). The KC corneas showed relatively greater number of spots having molecular weights between 28 and 48 kDa compared to normal corneas (Figure 7B).The molecular weight of native α-enolase is 48 kDa, so it apparently was degraded in both normal and KC corneas but more so in the latter.
Western blot analysis with anti-β-actin antibody showed two major spots of 42 kDa in the normal corneas (Figure 7C), and two minor spots of 40 kDa in the KC corneas (Figure 7D). Because the MW of the native β-actin is 42 kDa, this protein was degraded in the KC corneas.
RT-PCR to detect transcripts of α-enolase and β-actin
RT-PCR with primers of α-enolase and β-actin showed products of about 550 bp (Figure 8). The expected sizes of PCR products of α-enolase and β-actin were 538 and 579 bp, respectively. The data suggested that mRNA of the two proteins exist in epithelial cells of normal corneas of a 60-year-old donor.
Presence of α-enolase in the limbal and central regions of normal corneas
Previous publications have shown that α-enolase is a marker for stem cells, yet it was only present in the limbal region and not in the central cornea (see Discussion); therefore we investigated its localization in both limbal and central corneal regions. The limbal and central corneal regions of two normal corneas were carefully scraped and their proteins were separated by 2-D gel electrophoresis. Selected protein spots were analyzed by MALDI-TOF mass spectrometric methods as previously described. The 2-D gel protein profiles of the limbal region and the central cornea are shown in Figure 9A,B, respectively. The protein spots, identified by arrows in the limbal (Figure 9A) and the central corneal (Figure 9B) regions on tryptic digestion and MALDI-TOF analysis, were identified as α-enolase (Results not shown).
The purpose of the study was to identify proteins that undergo up- or downregulation in KC corneas compared to normal corneas so that their potential roles either as disease markers as well as their involvement in the molecular mechanism of the disease could be determined.
The major finding of our proteomic investigation was that α-enolase and β-actin showed downregulation in the KC corneas compared to the normal corneas. Immunohistochemical/confocal microscopic analyses further showed that both proteins were present at relatively lower levels in the epithelial wing and superficial cells of KC corneas compared to those from normal corneas. These reduced levels could be due to their degradation as suggested by the results of western blot analyses.
In this study, proteins from 8 mm corneal epithelial rings from the keratoconus corneas were compared with 12 mm rings from normal corneas. It was possible some of the observed decrease in the two protein levels could be due to comparison of different corneal regions. Because the 2-D gel electrophoretic protein profiles were supported by our additional immunohistochemical analyses of α-enolase and β-actin in normal versus keratoconus corneas, a downregulation of the two proteins isolated from two sets of rings might indeed be true. Furthermore, additional comparative immunohistochemical analysis of successive corneal sections from periphery to the center of the two types of corneas also showed a downregulation of the two proteins as shown in representative tissue sections of Figure 6. Additionally, our finding about α-enolase was supported by a recent report .
The hallmark of our investigation was that we observed reproducible 2-D gel protein profiles following silver staining even from a single KC or normal corneal button. The 2-D gel protein profile showed that the majority of the epithelial proteins showed MW between 40-60 kDa in normal and KC corneas. Therefore, these proteins were the focus of our study. To prevent degradation of corneal proteins prior to the 2-D gel analysis, they were solubilized in protein-denaturating resolubilization buffer  that contained urea, thiourea, CHAPS, caprylyl sulfobetaine 3-10,tri-butyl phosphine, and Tris (see composition in Methods). The chaotropic agents, such as urea, allow proteins to unfold and thus expose their hydrophobic residues to a solution. The use of thiourea in combination with urea has been shown to enhance solubility of hydrophobic proteins and lead to visualization of yet more proteins . Among available surfactants, sulfobetaine and CHAPS are preferred for their solubility in a high concentration of urea . An additional surfactant, N-decyl-N-dimethyl-3-ammonio-1-propane sulfonate, has been used along with CHAPS to solubilize proteins that require a stronger surfactant for solubility . Dithiothreitol, a sulfhydryl reducing agent, has been replaced with an uncharged reducing agent, tributyl phosphine, which greatly enhances protein solubility during IEF . Because of the use of denaturing conditions for corneal epithelial protein solubilization, the proteinase activity was expected to be inactivated, and therefore proteinase inhibitors were not included in the resolubilization buffer.
Immunohistochemical-confocal analyses with commercially available antibodies raised against α-enolase or β-actin revealed a similar pattern of immunoreactivity in the central cone region of KC (i.e., where the epithelium appeared thin in the sections when compared with the corneal periphery [results not shown]). Immunohistochemical analysis further showed that although the two proteins were present in both normal and KC corneas, their distribution in the three epithelial layers differed, i.e., a relatively intense and uniform immunostaining of the two proteins in the epithelial basal, wing, and superficial cells of the normal corneas but in the KC cornea, a maximum staining of the basal cells, which greatly diminished in the wing and superficial cells. A strong immunofluorescence was also observed at the junction of the basal cell layer and Bowman's layer in normal corneas. Together, the data suggested the presence of relatively lower levels of the two proteins in the wing and superficial cells of the KC corneas compared to similar cells from normal corneas. As stated, a lower level of α-enolase in the keratoconus corneas compared to normal corneas was shown in a recent report . Because we observed a decreased level of β-actin in the epithelium of keratoconus corneas compared to normal corneas, the protein ought to be precluded to normalize the standard 1-D western blots of epithelial proteins.
Our results of the localization of the α-enolase in the central corneal region were in contrast to the previous reports of Zeiske et al. [36-39]. These researchers localized α-enolase in the basal cells of the limbal region using a monoclonal antibody raised against an immunogen, which was a 0.1 M ammonium acetate extract of limbus-to-limbus scrap of rat corneal epithelium that was harvested 18 h after a debridement wound of 3 mm in diameter. Zieske further showed that the antibody specifically immunoreacted with a 50 kDa protein in the basal cells in the limbus of rat, rabbit, and human corneas and the antigen was identified as α-enolase . Zeiske et al.  also reported that α-enolase was expressed on cell migration during wound closure, and immunohistochemically localized α-enolase in cultured rabbit epithelial cells under variety of environmental conditions. The protein was present in all epithelial cell layers but was restricted to the basal cells under certain conditions . Our results of MALDI-TOF mass spectrometric analyses of 2-D gel separated proteins from cells of limbal, peripheral and central regions of a normal cornea also showed the presence of α-enolase in all three regions of the human cornea (Figure 9). This was further supported by a report by Piatigorsky et al. , that showed α-enolase as a major soluble protein during examination of taxon-specific recruitment of proteins in the corneal epithelium of three mammals, chicken, and squid. A further support for the α-enolase localization was provided by Nishida et al. , who identified α-enolase among the 62 active genes in human corneal epithelium. Nishida et al.  found that the abundant expression of the α-enolase gene was intriguing because except for this enzyme, no other enzyme of the glycolytic pathway was correspondingly active in the cornea. The presence of the two proteins in the cells of normal and KC corneas was further confirmed by the RT-PCR analysis (Figure 8). Together, our results and present literature strongly suggest the presence of α-enolase in the human corneal epithelium.
Nishida et al.  speculated that the unusually high expression of α-enolase in human corneal epithelium might due to its novel role other than energy production. α-Enolase acts as a cell surface receptor for plasminogen, which has binding interactions with several adhesion proteins of the pericellular matrix [42,43]. Indeed, the plasminogen synthesis by corneal epithelial cells has been demonstrated [44,45]. Therefore, the degradation or a relatively lower level of expression of α-enolase might cause a loss of interactions with the proteins of the cell matrix. This loss of interactions may lead to disassembly of intact structures in the matrix, which might result in the thinning of the cornea, a hallmark of KC. Also, α-enolase has been shown to bind to actin and tubulin, which are cytoskeletal proteins . An association of α-enolase with the centrosome also indicate that α-enolase, like other glycolytic enzymes, is involved in the bundling of microtubules . Therefore, the reduced expression or degradation of α-enolase could cause unstable cytoskeletal assembly, leading to changes associated with the disease. However, whether changes in α-enolase levels have such effects in KC need further investigation.
An identification of differentially expressed genes in keratoconus epithelium by microarray method revealed massive changes of cytoskeleton, reduced extracellular matrix remodeling, altered transmembrane signaling, and modified cell to cell and cell-matrix interactions . The major changes in cytosketon included up-regulation of keratins and also microfilaments, intermediate filaments and microtubules suggesting internal cell structures are heavily reinforced in KC corneas. This could be due to ECM degradation in KC corneas because during KC, elevated levels of cathepsins V/L2, -B, and -G were observed . Further, the involvement of metalloproteinases in the development of keratoconus has also been suspected .
Altered expression of cytoplasmic proteins like α-enolase and β-actin might be contributing factors in KC. These proteins may be useful as markers and also in the elucidation of biochemical abnormalities in KC-corneal epithelial cells. Future research in our laboratory will address these important questions.
The authors thank the Alabama Eye Bank for providing the normal corneas. The research was supported by a grant from Fight for Sight Inc. of Prevent Blindness America.
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