Abstract
Abstract: :
Purpose: The retinal pigment epitherium (RPE) cells fulfill many functions of vital importance for the retina, and disruption of RPE fuction is related to retinal degeneration. Rigorous studies related to the function of the RPE are required. The porpose of this study is the identification and characterization of dominant RPE proteins. Methods:Monoclonal antibodies against the RPE were established, and the antigen was identified by MS/MS analysis. The roles of identified microsomal glutathione S–transferase 1 (MGST1) were assessed with primary RPE and cultured RPE cells. Anti–oxidative effects were examined by gene silencing technique with culture cell systems under H2O2 challenging. MGST1’s functions and expression in the RPE was compared in young and old mice. Results: MGST1 is shown to be a dominant, highly expressed enzyme in RPE microsomes that displays significant reduction activity towards synthetic peroxides, oxidized RPE lipids, and oxidized retinoids. MGST1–transfected HEK293 cells exhibited greater viability following oxidative challenge and oxidized docosahexaenoic acid (70 ± 4% survival) compared with untransfected control cells (46 ± 4% survival) challenged with 20 µM H2O2. Cultured ARPE19 cells transfected with MGST1 siRNAs exhibited lower expression of MGST1 (12% of the controls) and significantly lower GPx activity (44 ± 13%), and thus were more susceptible to oxidative damage. Immunoblotting revealed that in vivo expression of MGST1 in mouse RPE decreases 3–4–fold with age, to trace levels in 18–month–old mice. GPx activity in the RPE was also found to be reduced in 12–month–old mice to ∼67%. Conclusions: We demonstrate that MGST1 is one of the most active detoxification enzymes of the RPE. In addition, a gradual decline in the expression of MGST1 in mouse RPE with age is observed, suggesting a possible correlation with progressive age–related retinal diseases associated with oxidative injury such as age–related macular degeneration.
Keywords: antioxidants • oxidation/oxidative or free radical damage • age-related macular degeneration