Comparative study of cathepsins D and S in rat IPE and RPE cells

Abstract

To investigate differences between activities related to phagocytosis in iris pigment epithelial (IPE) and retinal pigment epithelial (RPE) cells, an aspartic protease, cathepsin D (cat D), and a cysteine protease, cathepsin S (cat S), of IPE and RPE were studied. IPE and RPE cells were isolated from Long Evans rat eyes. The origin of the isolated cells was determined by pigmentation and cytokeratin labelling. The mRNA expressions of cat D and cat S in cultured IPE or RPE cells were investigated by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Enzyme activities of cat D and cat S in IPE or RPE cells were measured by using specific fluorogenic substrates, MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Phe-Arg-Leu-Lys-(Dnp)D-Arg-NH2 and Z-Val-Val-Arg-MCA, respectively. Western blot analysis of both proteins was also performed. The cultured cells, both of IPE and RPE cells were pigmented and showed positive labelling with an anti-cytokeratin monoclonal antibody. The cat D activity in RPE cells was 37 times that in IPE cells. The cat S activity in RPE cells was four times that in IPE cells. On the other hand, mRNA expression levels of cat D in RPE cells were at the same level with IPE cells, cat S mRNA expression in RPE cells were 10 times that in IPE cells. These results were also correlated with the Western blot analysis. In this study, we measured the characteristic expressions of cat D and S in IPE and RPE cells for the first time to compare their lysosomal activities. IPE cells have the lysosomal activities like RPE cells, however, the function of lysosomal activity in IPE cells is beneath RPE's. These results indicated that the ability of ROS digestion in IPE cells was not same as RPE cells.