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Originally published In Press as doi:10.1167/iovs.07-1438 on March 31, 2008
 (Investigative Ophthalmology and Visual Science. 2008;49:2829-2837.)
© 2008 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.07-1438
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Proteomic Analysis of Climatic Keratopathy Droplets

Michelle Menegay,1 DeMia Lee,1 Khalid F. Tabbara,2 Thamara A. Cafaro,3 Julio A. Urrets-Zavalía,4 Horacio M. Serra,3 and Sanjoy K. Bhattacharya1

1From the Bascom Palmer Eye Institute, University of Miami, Miami, Florida; 2The Eye Center and The Eye Foundation for Research in Ophthalmology, Riyadh, Saudi Arabia; 3CIBICI (Centro de Investigaciones en Bioquimica Clinica e Inmunologia), Facultad de Ciencias Quimicas, Universidad Nacional de Cordoba, Haya de la Torre esquina Medina Allende, Córdoba, Argentina; and the 4Department of Ophthalmology, University Clinic Reina Fabiola, Universidad Católica de Córdoba, Córdoba, Argentina.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. To identify the proteins in the corneal droplets of climatic droplet keratopathy (CDK), a disease that results in the formation of droplets on the cornea. Progressive accumulation of droplets in CDK leads to visual loss.

METHODS. Proteomic mass spectrometry of the CDK specimens was performed after fractionation of proteins in 4% to 20% SDS-polyacrylamide gels. Droplets were derived from two human donors. Immunohistochemistry with antibodies was performed to confirm the presence of identified proteins on donor tissues from patients with CDK and control subjects.

RESULTS. Proteomic analyses revealed identification of 105 proteins in CDK specimens. Immunohistochemical analyses confirmed localization of annexin A2 and glyceraldehyde 3-dehydrogenase (GAPDH), proteins identified by proteomic analyses in CDK specimens. The proteins were subjected to analyses with the Kyoto Encyclopedia of Genes and Genomes (KEGG) Database which showed that a few biochemical pathways were more frequent for the identified proteins.

CONCLUSIONS. Approximately 105 proteins were identified in CDK specimens, and a subset of them was confirmed by immunohistochemistry. Several of these may play a role in fibril or deposit formation.

Climatic droplet keratopathy (CDK) is an acquired degenerative disease of the cornea that has been found to be highly prevalent in certain rural communities around the world.1 2 3 It is characterized by the haziness and opalescence of the most anterior layers of the cornea. In the initial stages, the cornea is often characterized by multiple tiny and tightly confluent, translucent, subepithelial yellow-gold deposits, localized peripherally close to the temporal and/or nasal limbus.

In many regions of the world where CDK has been reported, such as Patagonia in Argentina,3 4 North Africa, the Red Sea region,2 and the Punjab in India,5 the climate is characterized by high solar irradiation and reflection, aridity, constant winds, and, in some areas, elevated diurnal temperatures. Thus, there is exposure to high solar ultraviolet radiation because of ground reflection and the lack of cloud cover and shadows. Constant intense winds carrying particles of dust, sand, or ice that can provoke subtle microerosion of the corneal epithelium may play a role in CDK. Although the etiology of CDK is unknown, there is strong epidemiologic evidence that risk factors such as lifetime exposure to those climatic conditions may be involved.6 7 Occurrence of CDK in general does not correlate with any known ocular or other systemic disease. It is primarily a corneal disease; previous studies have rarely found it to be associated with conjunctiva.3 A higher prevalence of pinguecula, pterygium, cataract, and pseudoexfoliation has been found in patients with CDK compared with age-matched control subjects.3 Despite the age of patients affected by CDK and the rigorous climatic and environmental features of the region, dry eye is not a common finding among the patients with CDK or control subjects.4 Based on clinical findings, the patients have been divided into three stages or grades depending on the degree of severity.3 7 Grade 1 is characterized by multiple tiny and tightly confluent, translucent, subepithelial deposits, best observed with back-scattered slit illumination and high magnification, with a prelimbal fringe of clear cornea. In grade 2, haziness spreads over the inferior two thirds of the cornea, giving a tarnished appearance. Grade 3 is characterized by the presence of clusters of golden subepithelial droplets of different sizes, some reaching 1 mm in diameter.3 CDK predominantly occurs in adults, principally in men who work as laborers and are exposed to injurious environments including light throughout their lives, compared with women or children.3 4 6 7 8 9 10 The correlation of corneal aesthesia with CDK has not been systematically evaluated. In many patients with CDK, a moderate to severe corneal hypoesthesia has been observed in advanced cases.3 4 There appears to be a correlation between the grade of CDK and corneal sensitivity.

Histopathologic examination of CDK tissue under a light microscope often demonstrates globular deposits of different sizes under the corneal epithelium, within Bowman’s membrane and the anterior stroma. The coalescence and increased volume of these spherules may cause disruption of Bowman’s membrane and elevation and thinning of the corneal epithelium.1 11 12 13 The droplets generally lack positive staining for fat or calcium.1 12 13 Electron microscopy has shown that the globules are round, electron-dense, sharply demarcated structures, always surrounded by basement membrane material and adjacent disorganized collagen fibrils.1 13 The origin and exact composition of the droplets remain unclear; however, proteins appear to be at least some of the components.11 12 13 Analyses of CDK globules in corneas undergoing penetrating keratoplasty (PKP) showed high protein content, with average protein contents of 72 µg/mg of wet tissue. SDS-PAGE has shown that the proteins of molecular mass 20 to 300 kDa are contained in the droplets, with a major fraction appearing to be of molecular mass 67 kDa.11 14 Recent immunohistochemical studies in surgical specimens of CDK with monoclonal antibodies to N-(carboxymethyl)-L-lysine (CML), N-(carboxyethyl)-L-lysine (CEL), pyrraline, pentosidine, and imidazolone have shown these moieties to be immunoreactive for protein modifications in CDK.15 We have described these droplets as superficial, only to indicate that they occur in and just beneath the epithelial layer and do not penetrate the deep stroma and endothelial layers of the cornea. Further investigation and protein identification in the droplets (CDK specimens) is expected to advance our understanding of the pathogenesis of this disease. Given the nature of the disease, of particular interest would be to identify enzymatic proteins or proteins that bear the potential for deposit formation. Toward this goal, the proteomic analyses of superficial corneal specimens obtained during PKP was performed. We expect that targeting pathways most frequently involving the identified proteins early on and simultaneously will be helpful for intervention. Toward this goal, we performed bioinformatic analyses to determine the biochemical pathway of identified proteins.


   Methods
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 Abstract
 Methods
Results
 Discussion
 References
 
Tissue Procurement and Isolation of CDK Specimens
The CDK specimens (predominantly droplet-containing/droplet-rich tissue samples) were obtained from the cornea of a patient undergoing corneal transplantation. The cornea with CDK droplets was procured from a donor after surgery, according to a protocol approved by institutional review process and the tenets of the Declaration of Helsinki. The corneal specimens: cases 1 and 2 were the corneas from left eyes of male patients (outdoor laborers), case 1 is a 67-year-old, and case 2 is a 72-year-old, both of them with typical CDK grade 2 in the right eye and grade 3 in the left eye. Both patients had had a bilateral, painless, and slowly progressive decrease in visual acuity over the past 10 years. Best corrected visual acuity (BCVA) was 20/200 in the right eye and counting fingers in the left eye. Both eyes in both patients presented moderately prominent nasal pingueculae and moderate nuclear sclerosis of the lens. In both eyes of both patients, no abnormalities of the iris were observed biomicroscopically. Ocular tension was normal in both eyes of both patients, and the fully dilated fundus examination was unremarkable in both eyes of both patients. The CDK specimen was collected by using sharp scissors and fine forceps, capturing an area of the droplets for examination by microscope.

Protein Analyses
The proteins were extracted from CDK specimens by using 125 mM Tris-HCl (pH 7.0), 100 mM NaCl, 0.1% Triton X-100, 0.1% Genapol C-100, and 0.1% SDS. Insoluble materials were removed by centrifugation (8000g for 5 minutes), and soluble proteins were quantified by the Bradford assay.16 Proteins were fractionated over 4% to 20% gradient SDS-polyacrylamide gels (Invitrogen Inc., Carlsbad, CA). The proteins (10 µg) fractionated on the gels were stained with blue gel stain (GelCode; Pierce Biotechnology, Inc., CA). The protein bands were excised, destained, reduced with dithiothreitol (DTT), alkylated with iodoacetamide, and subjected to mass spectrometric analysis.

Mass Spectrometry
For protein identification, gel slices were excised and digested in situ with sequencing-grade trypsin (Promega Biosciences, Inc., Madison, WI). Digestion mixtures were loaded onto precolumns (360 mm OD x 100 mm ID fused silica; Polymicro Technologies, Phoenix, AZ), packed with 3-cm irregular C18 (5–15 µm nonspherical; YMC, Inc., Wilmington, NC), and washed with 0.1 M AcOH for 5 minutes before switching in-line to the resolving column (7-cm spherical C18, 360 x 100). Once the columns were in line, the peptides were gradient eluted with a gradient of 0% to 100% A in 30 minutes, where A was 0.1 M AcOH in nanopure H2O, and B was 0.1 M AcOH in 80% MeCN. All samples were analyzed on a mass spectrometer (model LTQ; Thermo Electron Finnigan, San Jose, CA). Electrospray was accomplished with a spray system (TriVersa Nanomate; Advion Biosystems, Ithaca, NY) with a voltage of 1.7 kV and a flow rate of approximately 250 nL/min. The mass spectrometer was operated in a data-dependent mode with the top five most abundant ions in each spectrum being selected for sequential tandem (MS/MS) experiments. The exclusion list was used (1 repeat, 180 seconds return time) to increase the dynamic range. All MS/MS spectra were searched (Sequest, ver. 2.7; Sequest Technologies, Inc., Lisle, IL, and Mascot; Matrix Science, Boston, MA) using NCBI nonredundant, Ensemble17 and Swiss Prot18 databases. Searches of database entries were restricted to Metazoa (animals) and allowed a maximum of two missed cleavages. The protonated molecule ions MH+ and monoisotopic were defined for the peak mass data input. For protonated MH+ peptides, Sequest Sp and Xcorr cutoff scores were 500 and 1.8, respectively. For Mascot searches, the score was greater than 78. All spectra were visually inspected for determination of correct database assignment. The potential chemical modifications of a peptide such as the alkylation of a cysteine, carbamidomethyl (C), and the oxidation of a methionine residue (M) and acetylation of lysine (K) were also considered in the search. For all mass lists, the peptide tolerance/error was at most 50 ppm, and no restriction was applied for both the protein isoelectric point and molecular weight. Database search results were tabulated and visually inspected (Scaffold; Proteome Software, Portland, OR). These methods are routinely used in our laboratory. A combined list of all identified proteins was prepared. The Swiss Prot and GenBank accession numbers for proteins were obtained, and the latter was used for search against the Dragon database (http://pevsnerlab.kennedykrieger.org/annotate.htm) to obtain information from the Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway Database (http://www.genome.jp/kegg/pathway.html). The identified proteins were also subjected to comparison with a normal human soluble corneal proteomic dataset,19 by using suitable modification of an available in-house written macro program (Excel; Microsoft, Redmond, WA).20

Immunohistochemistry
Histologic evaluations were made according to published protocols for human ocular tissues used in our laboratory.21 22 23 The 4% paraformaldehyde in phosphate-buffer-fixed and subsequently paraffin-embedded anterior eye sections (~8 µm) were prepared and stained with commercially procured antibodies to annexin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The presence of annexin and GAPDH proteins was verified by florescence analyses with Alexa 488- and Alexa 594-coupled secondary antibodies (Invitrogen-Molecular Probes, Eugene, OR). To ensure identical processing and uniform exposure, control samples (without antibody and primary antibody treated sections) were examined side by side on the same slide. Fluorescence images were obtained with a laser scanning confocal microscope (model TCS-SP5; Leica Microsystems, Exton, PA). A series of 1-µm x–y (en face) images were collected and stitched together for an image representing a three-dimensional projection of the entire 8-µm section. Confocal microscopic panels were composed with image-analysis software (Photoshop 8.0; Adobe Systems, San Jose, CA).


   Results
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 Abstract
 Methods
 Results
Discussion
 References
 
Isolation and Fractionation of CDK Specimens
Anterior segment images of affected human eyes were taken with the informed consent of the patients (Fig. 1) . The CDK that affects people from the Patagonia region of Argentina3 4 is characterized by the presence of characteristic droplets in the cornea that are more prominent in advanced cases (Figs. 1A 1B) . Characteristic droplet-containing specimens, as indicated by the arrow (Fig. 1A) , were excised and subjected to fractionation on SDS-PAGE. The fractionated CDK specimen proteins were subjected to staining with Coomassie blue (Fig. 2) and subsequently subjected to mass spectrometric analyses.


Figure 1
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  		FIGURE 1. Image of the anterior part of the eye. (A) Image of an eye with grade 3 climatic droplet keratopathy with typical subepithelial golden-yellow confluent vesicles or drops. (B) Same case examined biomicroscopically with higher magnification and retroillumination better delineating the vesicles.

 

Figure 2
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  		FIGURE 2. Fractionation of proteins from human climatic keratopathy droplets. The isolated droplets from cornea removed during transplantation was extracted and separated on a 4% to 20% SDS-polyacrylamide. Samples 1 and 2 are from cases 1 and 2, respectively. The gel bands from top to bottom were excised and subjected to LC MS/MS analysis.

 
Proteomic Identification and Bioinformatic Analyses
Each band in both samples (Fig. 2) was excised, reduced, alkylated, and trypsin digested, and the resultant peptides were subjected to sequence identification by mass spectrometry and to protein identification by database searches. Using this approach, we were able to identify 105 proteins in the CDK specimens (Table 1) . Only the proteins that presented good mass spectra and at least two sequenced peptides were included in the identification list shown in the table. All identified proteins were examined to determine their position in the cellular biochemical pathways by using Dragon annotation tools and the KEGG database. As shown in Figure 3 , this resulted in the determination of the most frequent biochemical pathways to which the identified CDK specimen proteins belong. Most of the proteins identified in CDK specimens belong to cell junctions (Fig. 3) followed by glycolysis, suggesting that cell-junction proteins are likely to be involved in CDK’s etiology. Several identified proteins such as lysozyme, transketolase, pyruvate kinase, aldehyde dehydrogenase, and retinal dehydrogenase 1 (Table 1) are enzymatic. We observed differences in the number of proteins identified between the two samples (Table 1) , and because of the identical outcome in two technical repeats (not shown), we attribute these differences to the nature of these samples. It is likely that the samples posed differences in their digestibility and ionization due to the inherent differences leading to identification in the different number of proteins in them. CDK droplets are often yellow and have not been found to stain positively for fat12 13 ; however, the catalysis of the natural small molecule substrates for several of these enzymes, such as retinal dehydrogenase and aldehyde dehydrogenase, may lead to local accumulation of their products in the droplets, which would cause the yellow color. Several of the proteins (Table 1) such as the isoform of plectin-124 25 , desmoplakin,26 27 tenascin,28 and {alpha}-actinin-429 are either secreted extracellular matrix (ECM) proteins or have the potential to form deposits and, under certain conditions, to initiate fibril formation, leading to formation of isolated deposits. Many of these proteins bind to proteoglycans,28 which may provide the nucleation necessary to form deposits. Several proteoglycans, such as gelsolin and decorin, were also identified in the CDK specimens. Several of the proteins or their mutant variants have also been implicated in disease processes such as keratosis26 27 or glomerulosclerosis,29 which are characterized by deposits, plaques, and lesions. Since surgical techniques are far from perfect, the possibility that samples of droplets in the cornea are contaminated with surrounding cells from normal cornea cannot be ruled out. We have performed a comparison of proteins identified in the current analyses with a previously reported dataset of soluble human cornea proteins that were identified by peptide mass fingerprinting and tandem mass spectrometry.19 In the previous investigation normal cornea, 1-D and 2-D gel fractionation, and several different detergent extractions were used, and the total protein data from these analyses were compared with the current proteomic data from droplets (Supplementary Table S1, online at http://www.iovs.org/cgi/content/full/49/7/2829/DC1). A previous study, due to its utilization of normal cornea, was not limited by tissue availability. Many cell junction proteins, a category that appeared most frequently among the identified proteins in our bioinformatic analysis (Fig. 3) also has high potential to initiate formation of deposits. Epiplakin, which has been uniquely observed in droplets (Supplementary Table S1), and desmoplakin are proteins30 that play roles in the formation of ceramide deposits. Previous proteomic and bioinformatic analyses have also suggested that the importation of plasma proteins into the human cornea31 and the presence of hemoglobin, endoplasmin, and serum albumin in the droplets (Table 1 , Supplementary Table S1) are consistent with this hypothesis. Inactivation of proteolytic activities suggested by the presence of calpain-1 and serpin among the identified proteins and uniquely present in the droplets (Supplementary Table S1) could be a mechanism for formation of deposits. It may be that these proteases are posttranslationally modified due to oxidative modification or to UV cross-linking or both, leading to nonclearance of some initial surface deposition or a submicroscopic nuclear deposition.


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  		TABLE 1. Proteins from Climatic Keratopathy Droplets on Cornea

 

Figure 3
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  		FIGURE 3. Analyses identified proteins using the KEGG Database for biochemical pathways. Total identified proteins were subjected to analyses of pathways. (A) The pathways (pathway numbers are as in the KEGG Database) that had a frequency higher than two in proteins from CDK specimens are presented. Pathways related to infection have been omitted. (B) The names of pathways identified by number in (A).

 
Immunohistochemical Analyses
Annexin and GAPDH are among the proteins that were identified in CDK specimens (Table 1) . The entire length of cornea from a normal donor was subjected to immunohistochemical analyses by using antibodies to annexin and GAPDH and confocal microscopy (Fig. 4) . Nuclear staining was performed to show corneal cellularity (Fig. 4A) . As shown in Figure 4B , the presence of annexin in the normal cornea was rather toward the limbus region at the edge of the cornea, not in the central part. The absence or weak presence of annexin in the central cornea is further shown in Figure 4C , and a relative increase in annexin is observed along the edges of the cornea (Fig. 4 D); however, in multiple control specimens (corneas obtained from different healthy donors) very weak staining was observed for annexin in the cornea, with the exception of the presence in the limbus region.


Figure 4
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  		FIGURE 4. Immunohistochemical analyses of identified proteins. (A) DAPI-stained anterior human cadaveric eye segments. (B) The same sections were stained with anti-annexin A2 secondary antibody coupled with Alexa 488. The boxed regions were imaged for Alexa 488 fluorescence and are shown at higher magnification in (C), the central region of the cornea, and (D), the peripheral region/edge of the cornea.

 
In CDK specimens, annexin was captured in abundance from the central location, prominently associated with droplets, which is in contrast to its presence in normal cornea (Fig. 5A) . Further immunohistochemical analyses of corneas with CDK showed a strong presence of annexin and GAPDH in the droplets and a rather diffused presence in the rest of the cornea. In certain instances, the droplet-like deposits were present in the cornea (Supplementary Fig. S1, online at http://www.iovs.org/cgi/content/full/49/7/2829/DC1). The antibody control (without primary antibody), in identical settings, did not show any fluorescence (Fig. 5C) .


Figure 5
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  		FIGURE 5. Immunohistochemical analyses of identified proteins. (A) Cornea section from a surgical patient with CDK (62-year-old man from Saudi Arabia) and from (B) a human cadaveric eye (67-year-old Asian man) with no known eye disease. The sections were stained with DAPI, mouse monoclonal anti-annexin A2, and rabbit polyclonal GAPDH, as indicated. The secondary antibodies were coupled with Alexa 488 and Alexa 594, respectively. (C) Cornea section from the patient in (A) subjected to staining with DAPI, with mouse monoclonal MOPC-21 antibody, and without any primary antibody, as indicated.

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
In the present analysis, we identified approximately 105 proteins in CDK specimens. As shown in Figure 3 , the most frequent pathways for which the proteins were identified were cell junction, focal adhesion, and regulation of cytoskeleton in addition to energy metabolism-associated proteins.

Annexin A1 and A2, both were identified in our proteomic analyses, and immunohistochemical analysis has shown the presence of annexin A2 in CDK specimens. It is important to note that extensively positive staining for annexin II has been shown in corneal epithelial basal cells in rat cornea32 in contrast to very weak staining in the epithelial cell layer in the present investigation (Fig. 5) . However, similar results were observed with different commercial antibodies to annexin II (rabbit polyclonal [ab19415] and two mouse mAbs [ab8146;Abcam Inc., Cambridge, MA, and H00000302-M02; Novus Biologicals, Littleton, CO]). The observed differences between human and rat corneas are probably due to epitope masking in different immunohistochemical procedures, or human cornea may be inherently slightly different from rat cornea, or the translocation of annexin II from cytoplasm to cell surface is less effective in humans, giving a diffuse, less-intense staining. Annexin A1,33 and to a lesser extent annexin A2,34 as well as GAPDH,35 are known for membrane fusion. If the CDK deposits form due to the formation of fused membrane that eventually becomes lipid-containing fusion of proteins, then the membrane fusion property of annexin A1 and -A2 and GAPDH would help catalyze some of the stages of the fusion process. Annexin A1 has been implicated in the formation of fibrotic deposits in the lungs, in cultured lung cells after irradiation,36 and in the aggregation and fusion of complex liposomes.37 It has been found to be associated with granular material in the skin as well.38 Annexins bind to lipids, are key mediators of cellular dynamic membrane-cytoskeleton interactions,39 and have been implicated in a variety of inflammatory pathways, including the regulation of cell death signaling and in phagocytic clearance of apoptotic cells.39 40 41 42 These processes that are likely to be altered in corneas predisposed to form CDK droplets and annexins as well GAPDH overexpression have the potential to mediate some of the early or intermediate steps. It is also important to note that GAPDH, which is a housekeeping protein has been noted in the normal cornea (Fig. 5) , however, the staining is rather diffuse compared with that in the droplets. The possibility that the droplets have substances that release spontaneous fluorescence that may augment secondary antibody-mediated fluorescence cannot be ruled out. A consecutive section without antibody or stained with a nonspecific mouse monoclonal MOPC-21 antibody,43 44 however, did not show such fluorescence (Fig. 5C) . Proteomic identification and immunohistochemical observation of the droplets only suggest an elevated presence of GAPDH and annexin in the droplets and not a complete lack of them in the normal cornea. It is likely that these observations suggest increased accumulation of GAPDH and annexin in CDK compared with normal cornea. The proteomic identification and immunohistochemical confirmation presented herein will pave the way for further mechanistic investigation into the role of these proteins in the initial stages of change in the cornea leading to droplet formation.

Research on CDK is limited by the lack of a suitable animal model, but perturbation of these pathways in organ or tissue culture models as well as in animal models may provide some clue about the initiation of protein deposition. Analysis of the presence of polymorphisms of identified enzymatic proteins will also provide clues as to whether these activities are responsible for the initiation of reactions that lead to deposit formation. Continued investigation will also provide further details about the proteins, particularly secreted ECM proteins with the potential to form deposits.


   Acknowledgements
 
The authors thank Gabriel S. Gaidosh for assistance with confocal microscopy and Victor Perez and Valery Shestopalov for advice and comments on the manuscript.


   Footnotes
 
Supported in part by a career development award from Research to Prevent Blindness (SKB), National Institutes of Health Grant S10 RR019382 and National Eye Institute Grant P30 EY014801 (BPEI center core grants), an unrestricted grant to the University of Miami from Research to Prevent Blindness, and the HHMI (Howard Hughes Medical Institute) internship program (MM, DL).

Submitted for publication November 7, 2007; revised February 6, 2008; accepted May 14, 2008.

Disclosure: M. Menegay, None; D. Lee, None; K.F. Tabbara, None; T.A. Cafaro, None; J.A. Urrets-Zavalía, None; H.M. Serra, None; S.K. Bhattacharya, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Sanjoy K. Bhattacharya, McKnight Vision Research Building, Bascom Palmer Eye Institute, University of Miami, 1638 NW 10th Avenue, Room 706A, Miami, FL 33136; sbhattacharya{at}med.miami.edu.


   References
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 Discussion
 References
 

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