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Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing
Institute of Animal Physiology, Department of Veterinary Sciences, Ludwig-Maximilians-University Munich, D-80539 Munich, Germany
Research Unit Protein Science, Helmholtz Center Munich, German Research Center for Environmental Health, D-85764 Neuherberg, Germany
Department of Ophthalmology, Ludwig-Maximilians-University, Mathildenstrasse 8, D-80336 Munich, Germany
Centre of Ophthalmology, Institute for Ophthalmic Research, University of Tübingen, Röntgenweg 11, D-72076 Tübingen, Germany
* Author to whom correspondence should be addressed.
Received: 11 September 2012; in revised form: 23 October 2012 / Accepted: 25 October 2012 / Published: 31 October 2012
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.
Keywords: equine recurrent uveitis; membrane protein; outer blood-retinal barrier; retinal pigment epithelium cells; cell line; proteomics
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Szober, C.M.; Hauck, S.M.; Euler, K.N.; Fröhlich, K.J.H.; Alge-Priglinger, C.; Ueffing, M.; Deeg, C.A. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing. Int. J. Mol. Sci. 2012, 13, 14053-14072.
Szober CM, Hauck SM, Euler KN, Fröhlich KJH, Alge-Priglinger C, Ueffing M, Deeg CA. Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing. International Journal of Molecular Sciences. 2012; 13(11):14053-14072.
Szober, Christoph M.; Hauck, Stefanie M.; Euler, Kerstin N.; Fröhlich, Kristina J.H.; Alge-Priglinger, Claudia; Ueffing, Marius; Deeg, Cornelia A. 2012. "Profound Re-Organization of Cell Surface Proteome in Equine Retinal Pigment Epithelial Cells in Response to In Vitro Culturing." Int. J. Mol. Sci. 13, no. 11: 14053-14072.