Retinal Pigment Epithelium Lipofuscin Proteomics

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 (ϳ2% (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.

Assessing Susceptibility to Age-related Macular Degeneration with Proteomic and Genomic Biomarkers

“…Of course other oxidative protein modifications accumulate in the eye with age and possibly contribute to AMD pathogenesis. Such modifications include advanced glycation end products in the choriocapillaris, Bruch membrane, and CNV membranes (29,30) and nitrotyrosine and iso [4]levuglandin E 2 adducts (as well as CEP adducts) in RPE lipofuscin granules (31). Why ocular tissues in AMD patients are more susceptible to oxidative damage than normal eye tissue remains to be determined, but oxidative markers formed from retina-rich components like DHA lipids offer a potential early warning system for predicting AMD susceptibility.…”

Simultaneous decomposition of multiple hyperspectral data sets: signal recovery of unknown fluorophores in the retinal pigment epithelium

“…The RPE is responsible for generating vitamin A derivatives required for phototransduction, the initial steps of vision, through a series of biochemical reactions called the visual cycle. Byproducts of the visual cycle are thought to aggregate in the lysosomal compartment of the RPE [12] as lipofuscin, which has an intense fluorescent signal [13][14][15]. Because this signal comes from endogenous substances, rather than exogenously introduced fluorescent markers, it is referred to as lipofuscin autofluorescence (AF).…”

Lipofuscin Accumulation into and Clearance from Retinal Pigment Epithelium Lysosomes: Physiopathology and Emerging Therapeutics

“…Surprisingly, only 12 proteins (11%) of Warburton's list were common with the 65 proteins identiied by Schut et al This discrepancy probably results from variations in the purity of sucrose-isolated granules [33] and from the fact that lipofuscin proteins are microheterogeneous in size due to abundant oxidative modiications while contaminant proteins are intact and therefore, run as well-focused spots. In a third study, Ng et al [34] analyzed the composition of highly puriied RPE-lipofuscin granules devoid of Lysosomes -Associated Diseases and Methods to Study Their Functionmembranes and reported that the luminal material was 98% lipids, mostly LBs [35]. Taking all this information into account, the current concept is that RPE lipofuscin originates from LBs in photoreceptors and is transferred to RPE lysosomes during POS phagocytosis.…”