May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Detection of HNE– and MDA–Modifications on Lysosomal Cathepsins in Human Retinal Pigment Epithelial Cells
Author Affiliations & Notes
  • T.U. Krohne
    Ophthalmology, University of Bonn, Bonn, Germany
  • E. Kammerer
    Molecular Pathology, University of Heidelberg, Heidelberg, Germany
  • J. Kopitz
    Molecular Pathology, University of Heidelberg, Heidelberg, Germany
  • F.G. Holz
    Ophthalmology, University of Bonn, Bonn, Germany
  • Footnotes
    Commercial Relationships  T.U. Krohne, None; E. Kammerer, None; J. Kopitz, None; F.G. Holz, None.
  • Footnotes
    Support  Gerok Scholarship, Bonn University Bonfor Foundation, to TUK
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 1374. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to authenticated users only.
      Sign In or Create an Account ×
    • Get Citation

      T.U. Krohne, E. Kammerer, J. Kopitz, F.G. Holz; Detection of HNE– and MDA–Modifications on Lysosomal Cathepsins in Human Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):1374.

      Download citation file:


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

      ×
  • Supplements
Abstract

Purpose: : Progressive formation of lipofuscin granules and drusen in early–stage age–related macular degeneration (AMD) is thought to result from accumulation of incompletely digested material due to impaired lysosomal function of the retinal pigment epithelium (RPE). Products of lipidperoxidation, in particular 4–hydroxynonenal (HNE) and malondialdehyde (MDA) were previously shown to inactivate lysosomal proteolytic activities in RPE cells. Besides substrate stabilization, covalent binding of MDA and HNE to lysosomal cathepsins is considered a major cause. We tested this hypothesis by directly detecting the corresponding adducts on cathepsin B and L in MDA– and HNE–treated RPE cells.

Methods: : Cultured human RPE cells were treated with HNE and MDA. Subsequently lysosomes were isolated from the cells and lysosomal proteins separated by 2D–gelelectrophoresis. Protein spots were visualized by fluorescent protein staining. Cathepsin B and L as well as HNE– and MDA–modified proteins were also detected by Western blotting. Matching of the Western Blots to the protein gel finally resulted in the identification of spots representing HNE– and MDA–modified cathepsin B and L, which were further characterized by mass spectrometry.

Results: : In HNE– and MDA–treated RPE cells, modifications by these lipidperoxidation products were detected on cathepsin B and L. Mass spectrometric analysis of tryptic peptides obtained from the cathepsins indicated modifications of critical active site residues in both proteases.

Conclusions: : Products of lipidperoxidation like HNE and MDA cause covalent modifications in the active center of RPE lysosomal cathepsins. This may result in the observed inhibitory effect of lipidperoxidation products on lysosomal proteolytic activities and may contribute to RPE lysosomal dysfuntion in early–stage AMD.

Keywords: age-related macular degeneration • retinal pigment epithelium • oxidation/oxidative or free radical damage 
×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×