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B. M. Tam, S. M. Noorwez, S. Kaushal, O. L. Moritz; The Light Activated Conformations of Rhodopsin Glycosylation Mutants T4K and T17M Are Unstable and Toxic to Photoreceptors. Invest. Ophthalmol. Vis. Sci. 2010;51(13):4073.
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We have previously demonstrated that the RP-associated rhodopsin mutations T4K and T17M cause light-dependent retinal degeneration in transgenic X. laevis. We hypothesize that this is due to decreased stability of the light activated conformation of the mutant rhodopsins. Therefore, we investigated whether inactivating the mutant proteins or increasing their thermal stability would protect against light-induced retinal degeneration. Furthermore, we investigated whether constitutively active forms of these mutants would exacerbate retinal degeneration.
T4K and T17M mutations were individually engineered into the human rhodopsin cDNA. Identical mutants were also constructed in the context of cDNAs containing the mutations N2C/N282C, K296R, and K296E. WT and N2C/N282C rhodopsins were expressed in HEK293 cells, regenerated with 11-cis retinal, and purified. We assessed the stability of the purified rhodopsins at 59C by measuring the absorbance at 500nm at two minute intervals over a 30 minute period. We generated transgenic X. laevis expressing wild-type and mutant rhodopsins in rod photoreceptors. Retinas of transgenic X. laevis were analyzed by dot blot for total and transgenic rhodopsin, and by confocal microscopy to assess the severity of retinal degeneration and subcellular protein localization.
Similar to previous results for bovine rhodopsin, we found that engineering two novel cysteines at residues 2 and 282 in human rhodopsin resulted in a protein that was more resistant to thermal denaturation than the wild type molecule. Moreover, retinal degeneration induced by the T17M mutation was less severe in the context of the stabilized N2C/N282C rhodopsin. Constitutively inactivated T4K and T17M rhodopsins (i.e. via introduction of the K296R mutation) were also much less toxic to photoreceptors. However, constitutively active T4K and T17M rhodopsins (i.e. via introduction of the K296E mutation) caused rod death even when the animals were reared in complete darkness.
Our results indicate that the light-activated conformations of human T4K and T17M rhodopsins are unstable. Maintaining these mutants in their inactive conformation or stabilization of the molecules significantly reduces their light induced toxicity, whereas constitutively active variants cause rod death which can no longer be rescued by dark rearing.
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