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N.V. Gordiyenko, J.W. Lee, M. Marchetti, N. Tserentsoodol, R.N. Fariss, H. Weissbach, M. Kantorow, I.R. Rodriguez; Expression and Localization of Methionine Sulfoxide Reductase a in the Retina . Invest. Ophthalmol. Vis. Sci. 2005;46(13):5144.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: To localize msrA in the monkey retina and cultured RPE cells and measure the effect of siRNA knockdown on the susceptibility of cultured RPE cells to oxidative stress. Methods: MsrA protein and mRNA expression were measured in monkey retina and cultured RPE cells by Northern and Western blot analyses. The msrA peptide was detected using a rabbit polyclonal anti–msrA antibody. Localization of msrA in monkey retina and cultured RPE cells was performed by fluorescent confocal microscopy using a Cy5 conjugated secondary antibody. MsrA–GFP fusion constructs were transfected into ARPE19 cells. SiRNA mediated gene silencing was conducted with separate siRNA sequences and RPE viability monitored by MTT assays in the presence or absence of increasing TBHP concentrations. Results: Northern blot analyses indicate msrA is expressed mainly in RPE with some expression in neural retina. In the RPE/Choroid msrA immunoreactivity was observed in bands at 28 kDa (actual size) and 150 kDa. In the neural retina and ARPE19 cells a ∼50 kDa peptide was observed. Immunohistochemical analysis of the monkey retinal sections localized msrA in the apical side of the RPE as well as in the outer plexiform and inner nuclear layers. In cultured RPE cells endogenous msrA was localized in the mitochondria and cytosol. Transfection of the RPE cells with msrA–GFP fusion constructs corresponding to two different isoforms of msrA showed that full length of msrA localizing to the mitochondria, while the shorter transcript missing of the N–terminal sequence localized to the cytosol. SiRNA–mediated gene silencing of msrA resulted in loss of RPE viability with decreased resistance to oxidative stress. Conclusions:MsrA is localized mainly to the apical side of the RPE but is also present in the outer plexiform and inner nuclear layers of the retina. In the cultured RPE cells msrA is localized to the mitochondria and cytosol depending on the presence or absence of an alternatively spliced leader sequence. The large molecular weight sizes observed on Western blots suggest that msrA is forming covalently bound complexes with itself and/or other proteins. Increased sensitivity to oxidative stress shown in cultured RPE cells after siRNA knockdown suggests msrA plays an important role in RPE survival and retinal function.
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