Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing

Christoph Szober,Stefanie Hauck,Kristina Fröhlich,Claudia Alge-Priglinger,Kerstin Euler,Cornelia Deeg,Marius Ueffing

doi:10.3390/ijms131114053

Abstract

The purpose of this study was to characterize the cell surface proteome of native compared to cultured equine retinal pigment epithelium (RPE) cells. The RPE plays an essential role in visual function and represents the outer blood-retinal barrier. We are investigating immunopathomechanisms of equine recurrent uveitis, an autoimmune inflammatory disease in horses leading to breakdown of the outer blood-retinal barrier and influx of autoreactive T-cells into affected horses’ vitrei. Cell surface proteins of native and cultured RPE cells from eye-healthy horses were captured by biotinylation, analyzed by high resolution mass spectrometry coupled to liquid chromatography (LC MS/MS), and the most interesting candidates were validated by PCR, immunoblotting and immunocytochemistry. A total of 112 proteins were identified, of which 84% were cell surface membrane proteins. Twenty-three of these proteins were concurrently expressed by both cell states, 28 proteins exclusively by native RPE cells. Among the latter were two RPE markers with highly specialized RPE functions: cellular retinaldehyde-binding protein (CRALBP) and retinal pigment epithelium-specific protein 65kDa (RPE65). Furthermore, 61 proteins were only expressed by cultured RPE cells and absent in native cells. As we believe that initiating events, leading to the breakdown of the outer blood-retinal barrier, take place at the cell surface of RPE cells as a particularly exposed barrier structure, this differential characterization of cell surface proteomes of native and cultured equine RPE cells is a prerequisite for future studies.

Highlights

Protein expression analysis with mass spectrometry has become an important tool to understand participating proteins in physiological networks and on cellular levels [1]
We showed that the cell surface proteome of equine retinal pigment epithelium (RPE) cells shifted from proteins with RPE specialized functions (CRALBP, retinal pigment epithelium-specific protein 65 kDa (RPE65) and retinol dehydrogenase 5 (RDH5)) in native cells (Table 1, proteins 29, 37 and 38; Figures 2–4) to proteins that are associated with cell adhesion and extracellular matrix (ECM) formation in cultured cells
The results of this study indicate a profound re-organization of the cell surface proteome of passage-4 equine RPE cells compared to native cells

Summary

Introduction

Protein expression analysis with mass spectrometry has become an important tool to understand participating proteins in physiological networks and on cellular levels [1]. Analysis technologies continually improved in the field of proteomics in the last years, especially regarding sensitivity of mass spectrometric analysis [3]. This enables increasingly better investigations of complex and widely dynamic protein concentrations [3]. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) allowed resolution of thousands of proteins [4], but it has major disadvantages, mainly inadequate resolution of cell surface membrane proteins. Several different approaches were applied to enhance cell surface membrane protein identification in tissues through variations in sample preparation [5,6]. Mass spectrometry is rapidly advancing, and liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) has emerged as a highly precise and sensitive high-throughput technique for protein identification and characterization and facilitates discovery of proteins [3,6]

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