Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle

Abstract

RPE65 is essential for isomerization of vitamin A to the visual chromophore. Mutations in RPE65 cause early-onset blindness, and Rpe65-deficient mice lack 11-cis-retinal but overaccumulate alltrans-retinyl esters in the retinal pigment epithelium (RPE). RPE65 is proposed to be a substrate chaperone but may have an enzymatic role because it is closely related to carotenoid oxygenases. We hypothesize that, by analogy with other carotenoid oxygenases, the predicted iron-coordinating residues of RPE65 are essential for retinoid isomerization. To clarify RPE65's role in isomerization, we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cis-retinoids, but coexpression with lecithin:retinol acyltransferase was needed for high-level production. Accumulation was significant, amounting to >2 nmol of 11-cis-retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolish this isomerization. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect on isomerization activity. Mutations associated with Leber congenital amaurosis/early-onset blindness cause partial to total loss of isomerization activity in direct relation to their clinical effects. These findings establish a catalytic role, in conjunction with lecithin:retinol acyltransferase, for RPE65 in synthesis of 11-cis-retinol, and its identity as the isomerohydrolase.

Fig. 1.

Specific transfection of RPE65 and LRAT induces 11-cis-retinol synthesis. (A) 11-cis-retinol synthesis by 293-F cells transfected with RPE65. Synthesis of 11-cis-retinol is seen only in 293-F cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 (red trace), and not when pVitro3/LRAT+RDH5 alone is transfected (blue trace). Traces are representative of multiple analyses. Peak identification: 1, 11-cis-retinol; 2, 13-cis-retinol; 3, all-trans-retinol. (Inset) Absorption spectra of peaks 1, 2, and 3 confirm the identity of the isomers (29). (B) Isomerization of all-trans- to 11-cis-retinol occurs optimally with coexpression of RPE65 and LRAT. Cultures are transfected with six different combinations of pVitro2/RPE65+CRALBP, pVitro2/RPE65, pVitro2/CRALBP, and/or pVitro3/LRAT+RDH5, or no DNA, and incubated with 2.5 μM all-trans-retinol, and retinols are analyzed after 6 h. Data represent means and SDs of three independent sets of experiments.

Fig. 2.

RPE65, CRALBP, LRAT, and RDH5 are expressed only in transfected 293-F cells. (A) Immunoblot analysis of whole-cell lysates probed with antisera to, from top, RPE65, CRALBP, RDH5, and LRAT. Lane 1, untransfected 293-F cells; lane 2, 293-F cells transfected with an irrelevant plasmid; lane 3, 293-F cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5. (B) Single-channel and merged confocal images of 293-F cells transfected with pVitro2/RPE65+CRALBP and labeled to demonstrate coexpression of RPE65 and CRALBP. Immunolabeling: (a) rabbit antibody to RPE565 (green); (b) mouse monoclonal antibody to CRALBP (red); (c) merged triple-labeled image; (d) merged image of untransfected 293 cells labeled with RPE65 and CRALBP antibodies. DAPI labels cell nuclei (blue) in all images.

Fig. 3.

Isomerization activity is inhibited by the iron chelator 2,2-DP. Extracts of 293-F cell cultures transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 vectors were treated with 2.5 μM all-trans-retinol for 3.5 h in the presence of 0-2,000 μM 2,2-DP. Retinoids were extracted, saponified, and analyzed by HPLC. Values for 11-cis-retinol synthesis in the presence of chelator are normalized to activity in the presence of ethanol vehicle alone, taken as 100% activity. Results are means and SDs of three independent experiments.

Fig. 4.

Effect of mutation of iron-coordinating residues on RPE65 activity and expression. (A) Mutation of iron-coordinating residues abolishes isomerase activity. Synthesis of 11-cis-retinol is normalized to RPE65 immunoreactivity quantified by densitometry. Relative activities of mutants are compared with wild type, expressed as the mean and SD of three determinations. (B) Effect of histidine and glutamate mutations on RPE65 expression. Equivalent volumes of whole-cell lysates of transfections with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 constructs analyzed by immunoblot using RPE65 (Upper) and CRALBP (Lower) antibodies. The expression level of all mutants except H180A was lower than wild type. Lane 1, untransfected control; lane 2, wild-type RPE65; lane 3, H180A; lane 4, H241A; lane 5, H313A; lane 6, H527A; lane 7, E417A. The lower band in Upper is a nonspecific reactant present in both untransfected and transfected 293-F cells.

Fig. 5.

Effect of mutation of “palmitoylation switch” cysteines. (A) Individual conservative changes do not affect 11-cis-retinol synthesis by RPE65, but nonconservative (C330Y) or double (CC329/330SS) mutations abolish activity. Differences between wild type, C231S, C329S, and C330T are not significant. Synthesis of 11-cis-retinol is normalized to RPE65 immunoreactivity quantified by densitometry. Relative activities of mutants are compared with wild type, expressed as the mean and SD of three independent determinations. (B) Effect of cysteine mutations on RPE65 expression. Equivalent volumes of whole-cell lysates of cells transfected with pVitro2/RPE65+CRALBP and pVitro3/LRAT+RDH5 constructs analyzed by immunoblot with primary antibodies to RPE65 (Upper) and CRALBP (Lower). Lane 1, wild-type RPE65; lane 2, C231S; lane 3, C329S; lane 4, C330T; lane 5, C330Y; lane 6, CC329/330SS. The lower band in Upper is a nonspecific reactant present in both untransfected and transfected 293-F cells (see Fig. 4).

Fig. 6.

Comparison of RPE65 with ACO. (A) Selected residues of RPE65 are plotted to equivalent positions on the carbon backbone of the ACO crystal structure (22) by using the visual molecular dynamics program (Version 1.8.3; www.ks.uiuc.edu/Research/vmd) (42). Residues are iron-coordinating (blue), conserved residues associated with disease (pink), conserved/nonconserved cysteines (yellow), and weak pathogenic/nonpathogenic mutations (red). The iron-coordinating residues are clustered in the proposed substrate binding tunnel. Residues more sensitive to mutation tend to occur in the core. (B) Sequence alignment of RPE65 and ACO showing key iron-coordinating, cysteine, and other residues. The original alignment obtained with DeepView/Swiss-PdbViewer (Version 3.7; http://ca.expasy.org/spdbv) was manually adjusted to maximize gap suppression and alignment with predicted structural features (β-sheets and α-helices). Residues are color-coded as above. ^*, identity; ·, similarity.