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S.J. Fliesler, B.J. Kapphahn, D.A. Ferrington, B.A. Nagel, M.J. Richards; Rhodopsin Resists Oxidative Modification by PUFA–Derived Aldehydes and Nitrosylation During Light– and Metabolically–Induced Retinal Degenerations . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4565.
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© ARVO (1962-2015); The Authors (2016-present)
Oxidative modification of proteins has been implicated in various neurodegenerative processes, including covalent derivitization with polyunsaturated fatty acid (PUFA)–derived adducts, such as 4–hydroxynonenal (HNE), 4–hydroxyhexenal (HHE), and carboxyethylpyrrole (CEP), as well as nitrosylation of tyrosine (NT). We assessed rod outer segment (ROS) proteins, particularly rhodopsin, for such modifications in rat retinas undergoing retinal degeneration due to intense light exposure ("light damage") and in a model of Smith–Lemli–Opitz syndrome (SLOS).
Adult (3–mo old) normal and AY9944–treated (SLOS model; see Fliesler et al., Arch. Ophthalmol. 122:1190, 2004) Sprague–Dawley rats were exposed to intense green light (24 h,1700 lux, 490–580 nm), while unexposed cohorts were kept in darkness. ROS membranes were prepared from pairs of retinas for each condition; in parallel, eyes from each group were taken for morphological and immunocytochemical analysis. Proteins were resolved by SDS–PAGE, with and without pre–boiling: one gel from each group was silver–stained, while companion gels were blotted and probed with antibodies to opsin, HNE, HHE, CEP, or NT.Correlative immunfluorescence analysis was performed with these same antibodies, using Cy3–conjugated secondary antibodies and DAPI counterstaining.
Rhodopsin was not immunopositive to anti–HNE or –NT under any condition, whereas other ROS proteins were; however, no ROS proteins reacted appreciably with anti–HHE or –CEP. Boiling promoted opsin oligomerization, exposing more intense HNE–immunoreactivity in the Mr 31–45 kDa region (previously dominated by opsin monomer). Anti–NT immunoreactivity increased in non–opsin higher MW proteins with light damage, but not with AY9944 treatment. Very faint or no ROS immunofluorescence was observed with anti–HNE, –HHE, –CEP, or –NT; in contrast, robust ROS anti–opsin immunofluorescence was observed.
Rhodopsin resists HNE, HHE, CEP, and NT modifications under conditions of intense light exposure or that mimic SLOS, whereas other ROS–resident proteins do acquire some of these modifications. Thus, oxidative damage to opsin is not a significant event in the etiology of these retinal degeneration models.
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