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Proteomic and ultrastructural analyses of human lipofuscin [Abstract]

Gugiu, B. G., Rozanowska, Malgorzata, Rozanowski, Bartosz, Rayborn, M. E., Bonilha, V. L.., Gu, X., Salomon, R. G., Hollyfield, J. G., Boulton, Michael Edwin and Crabb, J. W. 2005. Proteomic and ultrastructural analyses of human lipofuscin [Abstract]. Investigative Ophthalmology and Visual Science 46 , E-1739.

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Abstract

Purpose: The progressive accumulation of lipofuscin in the retinal pigment epithelium (RPE), correlates with the pathogenesis of age–related macular degeneration (AMD). We seek a better molecular understanding of the sources and consequences of lipofuscin accumulation, including the protein content of lipofuscin. Methods: Human RPE lipofuscin was purified by conventional sucrose density gradient centrifugation methods. Lipofuscin granule purity was evaluated by light, fluorescence, confocal, and electron microscopy. Lipofuscin preparations were extracted with chloroform/methanol then the chloroform insoluble material was extracted with SDS and subjected to SDS–PAGE, gel bands excised and proteins identified by LC MS/MS. Western analysis was used to probe for oxidative protein modifications. Results: Ultrastructural analyses of lipofuscin purified by conventional methods revealed a heterogeneous core structure composed of lipofuscin granules surrounded by substantial extra–granular material. The chloroform insoluble lipofuscin fraction of the conventional preparation exhibited many fuzzy Coomassie blue stained SDS–PAGE bands, suggesting post–translational modifications. Western blot analysis confirmed the presence of abundant carboxyethylpyrrole adducts. Over 160 proteins were identified, ~33% of which exhibited apparent mass additions. Essentially "pure" lipofuscin granules, free of extra–granular material, were obtained by proteolytic digestion of the conventional preparation. Boiling the purified granules in SDS has so far failed to yield SDS–PAGE detectable bands with Coomassie or silver staining. Conclusions: Lipofuscin granules appear to be embedded in a protein "matrix" similar in content to drusen. Proteomic characterization of purified lipofuscin granules is underway.

Item Type: Article
Date Type: Publication
Status: Published
Schools: Optometry and Vision Sciences
Subjects: R Medicine > RE Ophthalmology
Uncontrolled Keywords: macular pigment, age-related macular degeneration, aging
Publisher: Association for Research in Vision and Ophthalmology
ISSN: 0146-0404
Last Modified: 04 Jun 2017 04:45
URI: http://orca-mwe.cf.ac.uk/id/eprint/44162

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