Negative charge of the glutamic acid 417 residue is crucial for isomerohydrolase activity of RPE65

Abstract

RPE65 is the isomerohydrolase essential for regeneration of 11-cis retinal, the chromophore of visual pigments. Here we compared the impacts of two mutations in RPE65, E417Q identified in patients with Leber congenital amaurosis (LCA), and E417D on isomerohydrolase activity. Although both mutations decreased the stability of RPE65 and altered its sub-cellular localization, E417Q abolished isomerohydrolase activity whereas the E417D mutant retained partial enzymatic activity suggesting that the negative charge of E417 is important for RPE65 catalytic activity. Loss of charge at this position may represent a mechanism by which the E417Q mutation causes blindness in LCA patients.

Fig. 1

Mutations in RPE65 alter its subcellular fractionation. The 293A-LRAT cells infected with Ad-wtRPE65, Ad-E417Q and Ad-E417D at MOI of 100 were harvested 18 h after infection, homogenized and fractionated. A, total cell lysate (32 μg), and equal amount of protein (8 μg) from the cytosolic, membrane, nuclear and cytoskeletal/including inclusion body fractions were analyzed by Western blot using the anti-RPE65 antibody. B, protein levels of wtRPE65 and the mutants were quantified by densitometry and presented as % of total protein levels of wtRPE65 or its mutants correspondingly (mean±S.D., n =3).

Fig. 2

RPE65 mutations E417Q and E417D cause deterioration of the protein stability. A, The adenoviruses infected 293A-LRAT cells were harvested at consecutive time intervals after blockage by CHX and RPE65 levels were determined by Western blot. B, RPE65 protein levels were semi-quantified by densitometry and expressed as % of that before the addition of CHX (mean±S.D., n=4).

Fig. 3

Expression level and isomerohydrolase activities of E417O and E417D. The 293A-LRAT cells were infected with Ad-wtRPE65, Ad-E417D and Ad-E417Q at MOI of 100. A, the cells were harvested 24 hr after the infection, and 25 μg of total proteins from each sample along with positive and negative controls (1 μg of bovine microsomes (BMF) and 25 μg of uninfected cells) were immunoblotted with the antibodies for RPE65 (top) and β-actin (bottom). B, protein levels of wtRPE65 and its mutants from the cells infected with MOI of 100 (mean±S.D., n=3). C–E, Cell lysates from (A) (250 μg of protein) were used for isomerohydrolase activity assays. C, cells infected with Ad-wtRPE65; D and E, cells infected with Ad-E417D and Ad-E417Q, respectively. Peak 1, retinyl esters; peak 2, all-trans retinal; peak 3, 11-cis retinol. F, Dependence of isomerohydrolase activities of wtRPE65 protein and E417D mutant on MOIs.

Fig. 4

RPE65 molecular model showing the vicinity of Fe²⁺ binding site, A, Predicted structure of RPE65, where the conserved Fe²⁺–binding site marked with red dashed line. B, a close-up view of predicted structure of wt RPE65 in proximity of E417 residue and the iron binding site; C-D, close-up views of predicted structures of RPE65 with a mutation E417D (C) and the patient mutant E417Q (D). The colors are coded as a carbon (white), a nitrogen (blue), an oxygen (red); and the Fe²⁺ ion is presented as a gray sphere.