Cloning of a major human retinal pigment epithelial lysosomal aspartic protease and mapping its transcriptional start sites.
OBJECTIVE
It has been proposed that the major proteolytic enzyme responsible for the proteolysis of photoreceptor outer segments is an aspartic protease similar or identical to cathepsin D. The aim of this study was to determine if the major retinal pigment epithelial (RPE) aspartic protease was cathepsin D, to study its distribution and investigate its transcription start sites (TSS).


METHODS
An RPE cDNA library was screened at low stringency for the presence of aspartic proteases. DNA sequencing was used to analyze the identified clones. The expression of cathepsin D mRNA was analyzed by Northern blot while immunohistochemistry was used to demonstrate cathepsin D related immunoreactivity in an eye section. RNase protection assay was used to map the Cathepsin D TSS in RPE cells.


RESULTS
The aspartic protease identified in the RPE cDNA library was identical to the DNA sequence of cathepsin D found in other tissues. Northern blot analysis of a wide range of cell types showed that the highest level of cathepsin D mRNA expression was found in RPE cells which was similar only to cathepsin D expression in MCF7 breast cancer cells. Immunohistochemical staining of a human retina confirmed the inherent nature of high cathepsin D expression in RPE cells. An RNase protection assay demonstrated two major cathepsin D TSS in RPE cells. One of them was identical to the TSS responsible for the constitutive expression of cathepsin D and the other, a TATA box-controlled TSS, was identical to a TSS found in MCF7 cells. Cathepsin D expression was not enhanced by the phagocytosis and digestion of rod outer segments (ROS) and ROS phagocytosis did not induce the use of other cathepsin D TSS in the RPE cells.


CONCLUSIONS
The major aspartic protease identified in RPE cells was cathepsin D. In addition, it was demonstrated that the high level of cathepsin D expression in RPE cells is the intrinsic nature of these cells and that it is linked to a TATA box-controlled TSS. This TSS has previously been described as an estrogen regulated TSS and further studies are required to identify the RPE-specific inducer or indirect factors that may be responsible for the enhanced expression of cathepsin D in the RPE cells.
Cavaney-Brooker
D M
DM
Centre for Ophthalmology and Visual Science, Lions Eye Institute, University of Western Australia, Nedlands, Australia.
Rakoczy
P E
PE
D015152:Blotting, Northern; D002402:Cathepsin D / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D006801:Humans; D007150:Immunohistochemistry; D008247:Lysosomes / Q000201:enzymology*; D008875:Middle Aged; D010587:Phagocytosis / Q000502:physiology; D010857:Pigment Epithelium of Eye / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism; D012374:Rod Cell Outer Segment / Q000502:physiology; D015533:Transcriptional Activation / Q000502:physiology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D002402:Cathepsin D