November 1994
Volume 35, Issue 12
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Articles  |   November 1994
Detection and possible functions of a cysteine protease involved in digestion of rod outer segments by retinal pigment epithelial cells.
Author Affiliations
  • P E Rakoczy
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
  • K Mann
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
  • D M Cavaney
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
  • T Robertson
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
  • J Papadimitreou
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
  • I J Constable
    Department of Molecular Biology, Lions Eye Institute, University of Western Australia, Nedlands.
Investigative Ophthalmology & Visual Science November 1994, Vol.35, 4100-4108. doi:
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      P E Rakoczy, K Mann, D M Cavaney, T Robertson, J Papadimitreou, I J Constable; Detection and possible functions of a cysteine protease involved in digestion of rod outer segments by retinal pigment epithelial cells.. Invest. Ophthalmol. Vis. Sci. 1994;35(12):4100-4108.

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

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Abstract

PURPOSE: To investigate the presence and role of a recently cloned cysteine protease (cathepsin S) in the digestion of rod outer segments (ROS) by cultured retinal pigment epithelial (RPE) cells. METHODS: RPE cell cultures were established from eye bank donor eyes. Total RNA was extracted from freshly harvested cultures, and after reverse transcription, the cDNA was subjected to polymerase chain reaction (PCR). Cathepsin S (Cat S) mRNA translation was inhibited by antisense oligonucleotides, and the effect of inhibition on the accumulation of fluorescent debris was examined. The activity of cysteine and aspartic proteases in ROS-challenged RPE cell cultures was inhibited by leupeptin and pepstatin, respectively. The accumulation of autofluorescent debris within RPE cells was measured by a fluorophotometric flow cytometer. The presence of phagosomes in antisense DNA-inhibited and control cultures was demonstrated by electron microscopy. RESULTS: The expression of Cat S in RPE cells was demonstrated by RNA-PCR. Using antisense oligonucleotide-mediated-specific inhibition of Cat S, a significant ROS-derived increase in autofluorescence was detected within the RPE cells when they were compared with the unchallenged control cultures and cultures in the presence of ROS and sense oligonucleotides. Electron microscopy demonstrated the presence of a large number of phagosomes that enveloped structures similar to ROS. The accumulation of autofluorescent debris was also demonstrated in cysteine protease-inhibited, ROS-challenged RPE cultures, but it was not detected with aspartic protease inhibition. CONCLUSIONS: The expression of Cat S in RPE cells and the accumulation of an autofluorescent debris in cultures in which cysteine proteases or Cat S activity is inhibited suggest a key role for this enzyme, either in the ROS digestion process or in the activation of cathepsin D, the major lysosomal enzyme present in RPE cells.

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