Global and Comparative Proteome Signatures in the Lens Capsule, Trabecular Meshwork, and Iris of Patients With Pseudoexfoliation Glaucoma

Abstract

Pseudoexfoliation (PXF) is characterized by the accumulation of the exfoliative material in the eye and high rates of blindness if left untreated. Pseudoexfoliation glaucoma (PXG) is generally diagnosed too late due to its asymptomatic nature, necessitating the development of new effective screening tools for the early diagnosis of the disease. Thus, the increasing prevalence of this disease due to an aging population has demanded the identification of suitable biomarkers for the early detection of the disease or detection of the onset of glaucoma in the eyes with PXF. We applied a proteomics strategy based on a high-throughput screening method for the determination of proteins involving PXF and PXG pathogenesis. The lens capsule (LC), iris, and trabecular meshwork (TM) samples with PXF and PXG were taken by surgical trabeculectomy, and control samples were taken from the donor corneal buttons obtained from the institutional eye bank to characterize the proteome profile. Peptides from the LC were analyzed using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The protein of interest and cytokine/chemokine profiles were verified using immunohistochemistry and the bio-plex kit assay, respectively. There were a total of 1433 proteins identified in the human LC, of which 27 proteins were overexpressed and eight proteins were underexpressed in PXG compared with PXF. Overexpressed proteins such as fibromodulin, decorin, lysyl oxidase homolog 1, collagen alpha-1(I) chain, collagen alpha-3(VI) chain, and biglycan were the major components of the extracellular matrix (ECM) proteins involved in cell-matrix interactions or ECM proteoglycans and the assembly and cross-linking of collagen fibrils. The ECM composition and homeostasis are altered in glaucoma. Thus, quantitative proteomics is a method to discover molecular markers in the eye. Monitoring these events can help evaluate disease progression in future studies.

Keywords: extracellular matrix; iris; lens capsule; lysyl oxidase; mass spectroscopy; pseudoexfoliation; pseudoexfoliation glaucoma; trabecular meshwork.

FIGURE 1

Protein profiles in LC with PXG compared to PXF. (A) Box plot of the protein expressions in each sample of PXF and PXG (n = 3), showing very little variability among the triplicate samples. (B) Abundance ratios (log2) of proteins in PXF vs. PXG with overexpressed or underexpressed proteins indicated in the graph. (C) Venn list was derived of all 1,433 proteins in PXF vs. control, PXG vs. control, and PXF vs. PXG for the identification of common and unique proteins in PXF and PXG. (D) Network generated of protein–protein interactomes with the top 27 proteins identified in PXG (> 2.5 folds) involved in extracellular homeostasis and protein binding using STRING analysis. (E) Fold change of cytokines/chemokines using a bioplex kit assay with PXG compared to PXF. Means ± SEM shown, *p < 0.05, **p < 0.01, one-way ANOVA post hoc t-test with Tukey correction, PXF-pseudoexfoliation, and PXG-pseudoexfoliation glaucoma.

FIGURE 2

Functional enrichment protein analysis of overexpressed/underexpressed PXG proteins compared to PXF by the FunRich database tool. A total of 27 overexpressed and eight underexpressed proteins were subjected to this analysis. (A) Percentage of the cellular component showing the collagen type-VI trimer more enriched in PXG. (B) Percentage of molecular functions showing glycosaminoglycan binding, ferrous iron binding, and ECM binding more enriched in PXG. (C) Percentage of biological process classification showing peptide cross-linking, collagen fibril organization, and response to cadmium ions more enriched in PXG. (D) Protein interaction of neighboring proteins from the UniProt dataset involved in four major pathways showing maximum protein counts including “ECM proteoglycans,” “assembly of collagen fibrils and other multimeric structures,” “collagen biosynthesis and modifying enzymes,” and “cross-linking of collagen fibrils” by six important proteins (Col1A1, Col6A3, LOXL1, FMOD, DCN, and BGN). The red node represents the target protein, and the green node represents the neighboring protein. Col1A1-collagen alpha-1(I) chain, Col6A3-collagen alpha-3(VI) chain, LOXL1-lysyl oxidase homolog 1, FMOD-, DCN-decorin, and BGN-biglycan.

FIGURE 3

Evaluation of the protein of interest involved in ECM aggregation in LCs by immunohistochemistry. Bright-field microscopic images at ×40 magnifications of PCOLCE, TGFβ1, a-SMA, fibulin V, and fibronectin in the LC. Black arrows indicate immunopositivity of the highlighted/target proteins in the LC. TGFβ1, a-SMA, fibulin V, and fibronectin were highly expressed in PXG compared to PXF/control. Scale bar-10 um, PXF-pseudoexfoliation, PXG-pseudoexfoliation glaucoma, PCOLCE-procollagen C-endopeptidase enhancer 1, TGFβ1-transforming growth factor, a-SMA (anti-alpha smooth muscle actin).

FIGURE 4

Protein profile in LC with PXG compared to the control. (A) Abundance ratios (log2) of proteins overexpressed or underexpressed are indicated in the graph. (B) Fold change of cytokines/chemokines using a bioplex kit assay with PXG compared to the control. (C) Functional enrichment protein analysis by the FunRich database tool using 43 proteins subjected to this analysis. The percentage of the cellular component highlights chylomicron more enriched in PXG. (D) Molecular functions such as heparan sulfate proteoglycan binding and protein-membrane adaptor activity are more enriched in PXG. (E) Biological process classifications such as peptide cross-linking are more enriched in PXG. Means ± SEM shown, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, one-way ANOVA post hoc t-test with Tukey correction, PXG-pseudoexfoliation glaucoma.