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Molecular & Cellular Proteomics 7:1397-1405, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Retinal Pigment Epithelium Lipofuscin Proteomics^*,S

Kwok-Peng Ng, Bogdan Gugiu^,, Kutralanathan Renganathan^,¶,||, Matthew W. Davies^**, Xiaorong Gu, John S. Crabb, So Ra Kim, Malgorzata B. Róanowska^**, Vera L. Bonilha, Mary E. Rayborn, Robert G. Salomon^¶, Janet R. Sparrow, Michael E. Boulton^**,, Joe G. Hollyfield^,¶¶ and John W. Crabb^{,¶,¶¶,||||}

From the Cole Eye Institute and Lerner Research Institute, Cleveland Clinic Foundation and ^¶ Department of Chemistry, Case Western Reserve University, Cleveland, Ohio 44195, ^** School of Optometry and Vision Sciences, Cardiff University, CF103XQ Wales, United Kingdom, Department of Ophthalmology, Columbia University, New York, New York 10032, Ophthalmology and Visual Sciences, University of Texas Medical Branch, Galveston, Texas 77555, and ^¶¶ Department of Ophthalmology, Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Cleveland, Ohio 44106

Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration. Because previous studies suggest that lipofuscin contains protein that may impact pathogenic mechanisms, we pursued proteomics analysis of lipofuscin. The composition of RPE lipofuscin and its mechanisms of pathogenesis are poorly understood in part because of the heterogeneity of isolated preparations. We purified RPE lipofuscin granules by treatment with proteinase K or SDS and showed by light, confocal, and transmission electron microscopy that the purified granules are free of extragranular material and associated membranes. Crude and purified lipofuscin preparations were quantitatively compared by (i) LC MS/MS proteomics analyses, (ii) immunoanalyses of oxidative protein modifications, (iii) amino acid analysis, (iv) HPLC of bisretinoids, and (v) assaying phototoxicity to RPE cells. From crude lipofuscin preparations 186 proteins were identified, many of which appeared to be modified. In contrast, very little protein (2% (w/w) by amino acid analysis) and no identifiable protein were found in the purified granules, which retained full phototoxicity to cultured RPE cells. Our analyses showed that granules in purified and crude lipofuscin preparations exhibit no statistically significant differences in diameter or circularity or in the content of the bisretinoids A2E, isoA2E, and all-trans-retinal dimer-phosphatidylethanolamine. The finding that the purified granules contain minimal protein yet retain phototoxic activity suggests that RPE lipofuscin pathogenesis is largely independent of associated protein. The purified granules also exhibited oxidative protein modifications, including nitrotyrosine generated from reactive nitrogen oxide species and carboxyethylpyrrole and iso[4]levuglandin E₂ adducts generated from reactive lipid fragments. This finding is consistent with previous studies demonstrating RPE lipofuscin to be a potent generator of reactive oxygen species and supports the hypothesis that such species, including reactive fragments from lipids and retinoids, contribute to the mechanisms of RPE lipofuscin pathogenesis.

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