Purchase this article with an account.
G. P. Moiseyev, O. Nikolaeva, Y. Takahashi, J. Ma; Mechanism of RPE65 Mutations Leading to Blindness. Invest. Ophthalmol. Vis. Sci. 2009;50(13):1214.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
RPE65 is a membrane-associated protein expressed in the RPE which functions as isomerohydrolase in the visual cycle. Mutations of the RPE65 gene were found to associate with severe retinal dystrophies such as Leber congenital amaurosis (LCA). Mechanism for these mutations to lead to retinal degeneration and blindness is unknown. The purpose of this study is to reveal how some mutations in RPE65 identified previously in patients affects RPE65 structure and function.
RPE65 mutations E102K, E417Q and E417D, were generated by site-directed mutagenesis. RPE65 and the mutants were expressed in 293-LRAT cells using the adenovirus vector. Their expression levels were analyzed by Western blot analysis. The subcellular fractionation was performed using the FractionPREPTM kit. Isomerohydrolase activities of RPE65 and the mutants were determined by in vitro enzymatic assay and quantified by HPLC.
Expression levels of mutants E102K, E417Q and E417D were similar to that of wtRPE65. After subcellular fractionation, wtRPE65 protein was found at a high level in the membrane fraction and a relatively low level in cytosolic fraction. In contrast, E102K, E417Q and E417D predominantly existed in the cytoskeleton/inclusion body fraction suggesting that this mutation disrupt the membrane association of RPE65. Wt RPE65 showed a high stability, with a half-life more than 10 h. Under the same conditions E102K, E417Q and E417D exhibited significantly accelerated degradation with apparent half-life less than 2 h, suggesting that this mutations decrease the stability of the protein. In the isomerohydrolase assay, wtRPE65 generated significant amount of 11-cis retinol while mutants E102K and E417Q did not produce any detectable 11-cis retinol at the similar protein levels. Mutant E417D was found to have isomerohydrolase activity which is 7.5-fold lower than that of wtRPE65.
RPE65 mutations E417Q and E102K identified in patients with retinal dystrophies decrease the stability and membrane association of RPE65 and therefore interrupt the visual cycle. Negative charge of E417 is essential for RPE65 enzymatic activity.
This PDF is available to Subscribers Only