May 2003
Volume 44, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2003
Proteome Analysis of Lipofuscin and Detection of Posttranslational Protein Modifications in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • F. Schutt
    Department of Ophthalmology, University of Heidelberg, Heidelberg, Germany
  • B. Ueberle
    Protein Analysis Facility, German Cancer Research Center, Heidelberg, Germany
  • M. Schnoelzer
    Protein Analysis Facility, German Cancer Research Center, Heidelberg, Germany
  • J. Kopitz
    Department of Pathobiochemistry, University of Heidelberg, Heidelberg, Germany
  • F.G. Holz
    Department of Pathobiochemistry, University of Heidelberg, Heidelberg, Germany
  • Footnotes
    Commercial Relationships  F. Schutt, None; B. Ueberle, None; M. Schnoelzer, None; J. Kopitz, None; F.G. Holz, None.
  • Footnotes
    Support  DFG Grant Ho 1926/2-2, Schu 1388/2-1, DFG Priority Research Program AMD (1088)
Investigative Ophthalmology & Visual Science May 2003, Vol.44, 3141. doi:
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      F. Schutt, B. Ueberle, M. Schnoelzer, J. Kopitz, F.G. Holz; Proteome Analysis of Lipofuscin and Detection of Posttranslational Protein Modifications in Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):3141.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Abstract: : Purpose: Lipofuscin accumulation in postmitotic RPE cells is a common downstream pathogenetic pathway in various retinal diseases including age-related macular degeneration. To better understand lipofuscinogenesis and possible adverse effects of its constituents we analyzed the proteome of isolated human lipofuscin granules from human RPE cells and screened for posttranslational modifications. Methods: After homogenization and fractionation by gradient ultracentrifugation of RPE/choroid complex from 10 pairs of human donors, protein compounds were separated by 2D gel electrophoresis and analyzed using matrix-assisted laser desorption/ionization mass spectrometry and HPLC-coupled electrospray tandem mass spectrometry. Lipofuscin proteins were screened for posttranslational modifications including malondialdehyde (MDA) , 4-hydroxynonenal (HNE) and advanced glycation endproducts (AGE) to identify toxic compounds. Results: A total of 76 proteins were identified in lipofuscin granules including cytosceleton proteins, proteins of visual cycle, enzymes of metabolism, proteins of mitochondrial respiratory chain, ion channel proteins and chaperones. A broad spectrum of lipofuscin proteins showed specific posttranslational modifications. Conclusions: This is the first description of protein components of human ocular lipofuscin isolated from RPE cells. The presence of photoreceptor segment (POS) specific proteins such as recoverin underscores the contribution of phagocytosed POS to lipofuscinogenesis, while a range of proteins originate from autophagic processes within the RPE cell. Some of the detected posttranslational alterations indicate the significance of oxidative damage. MDA- and HNE-modifications triggered lysosomal enzyme inhibition may contribute to impairment in degradative capacity and further accumulation of lipofuscin material. Such mechanisms may be operative in retinal diseases associated with excessive lipofuscin accumulation.

Keywords: retinal pigment epithelium • protein purification and characterization • protein modifications-post translational 
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