Proteome profiling of wild type and lumican-deficient mouse corneas
Graphical abstract
Deep coverage of mouse corneal proteome using quantitative proteomics provides insight into functional classes of proteins in the mouse cornea and the main effects of lumican deficiency on corneal homeostasis.
Research Highlights
► Lumican is a major corneal proteoglycan that affects corneal homeostasis. ► An iTRAQ-based quantitative proteomic tool was used to study the Lum–/– mouse cornea. ► Major changes epithelial and stromal proteins were observed.
Introduction
The healthy cornea is an avascular connective tissue-rich barrier of the eye that is both transparent and refractive for normal vision. The stroma, underlying the stratified epithelium, is a key regulator of corneal transparency and refraction, and it is comprised of specialized mesenchymal cells, the keratocytes and a collagen-rich extracellular matrix (ECM) they produce [1]. Injury, infection and corneal diseases can alter the exquisitely balanced cellular and the ECM content of the cornea and compromise its transparency, refractive power and barrier properties. The primary ECM constituents of the corneal stroma are fibrillar collagen types I, III and V and the small leucine-rich repeat proteoglycans (SLRPs), lumican, decorin, biglycan, keratocan and osteoglycin/mimecan [1]. Another SLRP, fibromodulin, is present in the developing cornea but restricted to the corneal periphery at maturity [2]. While the major protein components of the cornea have been identified, little is known of the corneal proteome, or how it is regulated by the major ECM components. The proteomics field has made significant advances in quantitative protein profiling by using isobaric tags for relative and absolute quantification (iTRAQ) that allow simultaneous analysis of four (iTRAQ 4 plex) or eight (iTRAQ 8 plex) different samples [3]. Thus, recent studies of the ocular surface in health and disease are beginning to incorporate these proteomic approaches [4], [5].
Lumican is present in the interstitial ECM of the cornea, skin, intestinal submucosa, cartilage and bone [6]. Lumican is normally expressed by the mesenchymal fibroblasts, and transiently by the injured epithelia [7], [8]. In vitro collagen fibrillogenesis assays and subsequent in vivo studies show that lumican and the other SLRP members of the cornea bind collagen and regulate collagen fibril growth [9], [10], [11], [12]. The lumican-deficient (Lum−/−) mice have cloudy corneas that are 40% thinner than those of the wild type mouse [9], [13]. Our previous studies showed that the loss of corneal transparency in the Lum−/− mice was linked to abnormal collagen fibril architecture and increased light scattering [11], [14], [15]. We further found that wound healing in the Lum−/− corneas was delayed, and the stromal cells showed reduced apoptosis and increased proliferation, suggesting lumican-deficiency to have a broad pleiotropic effect in the cornea [16], [17]. By contrast, deficiencies of the other corneal SLRPs, decorin [18], biglycan [19], fibromodulin [2], [20] and keratocan [21] present a milder corneal phenotype. Therefore, we selected the lumican-null corneas for an in-depth proteomic analysis, and compared the corneal proteomes of lumican-expressing (Lum+/+, Lum+/−) and lumican-deficient (Lum−/−) mice. We performed a multiplexed relative quantification of proteins by LC–MS/MS mass spectrometry using three different iTRAQ tags for Lum+/+, Lum+/− and Lum−/− corneal protein extracts. About 2108 proteins were identified and quantified in the corneal extracts. These included several known cellular proteins, extracellular matrix collagens and proteoglycans, as well as proteins not previously linked to the cornea. Absence of one or both Lum alleles was associated with increases in several oxidative stress-related proteins, increased collagen type I, VI and decreases in the lumican-related SLRPs, decorin, biglycan and keratocan.
Section snippets
Materials
The enzyme chondroitinase ABC was purchased from Associates of CAPE Cod incorporated. TCEP (Tris (2-carboxyethyl) phosphine), cysteine blocking agent (methyl methanethiosulfonate) and SYBR Green master mix were purchased from Applied Biosystems. Sequencing grade trypsin was from Promega. The oligonucleotide primers for PCR analyses were purchased from Eurofins Mwg Operon. KH2PO4, acetonitrile, KCl, formic acid, guanidine-HCl, sodium acetate, Tris–HCl, trifluoroacetic acid (TFA) and NaCl were
MS analysis of iTRAQ-labeled peptides
Analysis of the 24 iTRAQ labeled peptide fractions from each replicate on the LTQ-Orbitrap Velos generated ~ 1.2 million MS/MS spectral search inputs resulting in ~ 130,000 peptide spectral matches. A total of 2173 unique proteins were identified from two replicate analyses at the 1% FDR level. The proteins with shared peptides were ranked based on the score and the peptides were grouped to remove redundancy. Further, the proteins with equal scores were ranked based on their sequence coverage.
Discussion
The proteomes of the heterozygous and the lumican-deficient mouse corneas compared to the wild type show a broad range of changes in cellular and extracellular matrix proteins. The results suggest that lumican deficiency disrupts normal cellular metabolic functions possibly involving the epithelial and the stromal cells. In addition, significant changes are seen in proteins of the basement membranes and interstitial ECM.
Increases in several basement membrane proteins, perlecan [24],
Acknowledgments
Funding support: EY11654 (National Eye Institute, USA) to SC, EY05129 (National Eye Institute, USA) to DB and the Department of Biotechnology (DBT), Government of India research support to the Institute of Bioinformatics, Bangalore, India.
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Authors contributed equally.