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Olga Nikolaeva, Gennadiy Moiseyev, Yusuke Takahashi, Jian-Xing Ma; Mechanism for a Dominant-Acting D477G Mutation in RPE65 Leading to Vision Impairment. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3762.
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© ARVO (1962-2015); The Authors (2016-present)
RPE65 is a key enzyme in the visual cycle. Multiple recessive point mutations in the RPE65 gene have been shown to lead to impaired vision in patients with retinitis pigmentosa (RP) and Leber congenital amaurosis (LCA). Recently, a dominant mutation D477G of RPE65 has been reported to cause RP through the mechanism yet to be identified. The purpose of this study is to reveal how the mutation D477G of RPE65 affects its enzymatic activity.
Recombinant human wt RPE65 (hRPE65) with a His-tag and its mutant D477G with a 1D4 tag were generated by site-directed mutagenesis. Wt hRPE65 and the mutant were expressed in 293-LRAT cells using either adenovirus or plasmid transfection and their expression was analyzed by Western blot analysis. Subcellular fractionation was performed using FractPrepTM kit. Catalytic activities of wt hRPE65 and the mutant were quantified by HPLC. Velocity sedimentation through a sucrose gradient was used to identify oligomer formation between the mutant and wt RPE65.
Expression level of the D477G mutant was found similar to that of wt hRPE65. The wt hRPE65 showed a high stability, with a half-life more than 10 h whereas the mutant has a decreased half-life, suggesting that the mutation decreases the protein stability. The mutant showed a diminished catalytic activity in comparison to that of wt hRPE65. The subcellular fractionation revealed that considerable amount of wt and D477G were located in membrane fractions. However, when wt and D477G were co-expressed, it caused a decrease of wt RPE65 in the membrane fraction while a simultaneous increase in its inclusion body fraction. Moreover, co-expression of the D477G mutant with wt RPE65 enhances the RPE65 aggregation and causes oligomerization of the wt and the mutant RPE65.
The presence of the D477G mutant affects membrane association of wt RPE65. The D477G mutation enhances the formation of RPE65 oligomers rather than dimers or monomers. In turn, the oligomerization affects the isomerase activity of wt RPE65 and leads to impaired vision in patients with the D477G mutation of RPE65.
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