Abstract
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 ( approximately 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 E(2) 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.
Highlights
Lipofuscin accumulates with age in the retinal pigment epithelium (RPE) in discrete granular organelles and may contribute to age-related macular degeneration
Estimates suggest that lipofuscin contains 30 –70% protein depending upon tissue, species, and study [2, 6] and that this protein may contribute to pathogenesis
Purification of Lipofuscin Granules—RPE lipofuscin granules are usually isolated by sucrose density gradient centrifugation [26] and typically contain significant amounts of extragranular debris as shown (Fig. 1, A, C, E, F, I, and K) by light, confocal, and transmission electron microscopy
Summary
RPE Lipofuscin Preparations—Human eyes were obtained from the Bristol Eye Bank, Bristol, UK with permission for research in accordance to local ethical requirements. Equal amounts (by dry weight) of crude or SDSwashed lipofuscin preparations were subjected to overnight tryptic digestion in 30 mM NEM, pH 8.6, 0.05% SDS containing 0.3 g of trypsin, and soluble components were fractionated by strong cation exchange chromatography using a PolySULFOETHYL Aspartamide column (1.0 ϫ 150 mm, 5-m particle size, 200-Å pore size), a flow rate of 50 l/min, and a gradient of 0 – 600 mM KCl in 25% acetonitrile, 10 mM KH2PO4, pH 3, with fractions collected at 1-min intervals. The redissolved extract was analyzed on an Alliance HPLC system (Waters) equipped with a 2695 Separation Module, a 2996 Photodiode Array Detector (with monitoring at 430 and 510 nm), Empowerா software, and an Atlantisா dC18 column (3 m, 4.6 ϫ 150 mm, Waters) using aqueous trifluoroacetic acid/acetonitrile gradients with a flow rate of 0.8 ml/min as described previously [18]. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay [21]
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