RPE65 from cone-dominant chicken is a more efficient isomerohydrolase compared with that from rod-dominant species

Abstract

Cones recover their photosensitivity faster than rods after bleaching. It has been suggested that a higher rate regeneration of 11-cis-retinal, the chromophore for visual pigments, is required for cones to continuously function under bright light conditions. RPE65 is the isomerohydrolase catalyzing a key step in regeneration of 11-cis-retinal. The present study investigated whether RPE65 in a cone-dominant species is more efficient in its enzymatic activity than that from roddominant species. In vitro isomerohydrolase activity assay showed that isomerohydrolase activity in the chicken retinal pigment epithelium (RPE) was 11.7-fold higher than in the bovine RPE, after normalization by RPE65 protein levels. Similar to that of human and bovine, the isomerohydrolase activity in chicken RPE was blocked by two specific inhibitors of lecithin retinal acyltransferase, indicating that chicken RPE65 also uses all-trans-retinyl ester as the direct substrate. To exclude the possibility that the higher isomerohydrolase activity in the chicken RPE could arise from another unknown isomerohydrolase, we expressed chicken and human RPE65 using the adenovirus system in a stable cell line expressing lecithin retinal acyltransferase. Under the same conditions, isomerohydrolase activity of recombinant chicken RPE65 was 7.7-fold higher than that of recombinant human RPE65, after normalization by RPE65 levels. This study demonstrates that RPE65 from the cone-dominant chicken RPE possesses significantly higher specific retinol isomerohydrolase activity, when compared with RPE65 from rod-dominant species, consistent with the faster regeneration rates of visual pigments in cone-dominant retinas.

FIGURE 1.

Alignment of the deduced amino acid sequences of human, bovine, mouse, and chicken RPE65. The identical residues are indicated by dots. The four conserved histidine residues in the catalytic center are marked by arrows. The potential palmitoylation site Cys-330 conserved in mammalian RPE65 is boxed.

FIGURE 2.

Abundance of RPE65 in chicken RPE microsomes. A, to obtain purified chicken RPE65 as the standard for quantifying RPE65 concentrations in the microsomes from chicken RPE, 293A cells were infected by Ad-chRPE65 and harvested 24 h following the infection. Microsomes were prepared, and RPE65 was purified to homogeneity as described under “Experimental Procedures.” The purified RPE65 was examined by SDS-PAGE followed by Coomassie Blue staining. Lane 1, molecular markers; lane 2, microsomal fraction from the 293A cells infected by Ad-chRPE65 (50 μg); lane 3, purified recombinant chicken RPE65 (5 μg). B, RPE65 levels were measured in microsomes isolated from chicken and bovine RPE by Western blot analysis and by comparing with the purified RPE65 standards. RPE65 levels were expressed as relative abundance in microsomes (μg/mg of total microsomal proteins, mean ± S.D., n = 3). For comparison, RPE65 levels in microsomes from bovine RPE were measured at the same time using the purified bovine RPE65 as a standard.

FIGURE 3.

Inhibition of retinyl ester and 11-cis-retinol formation by apo-CRBP and AcDCMK. All-trans-[³H]retinol (0.2 μm) was incubated with 3 μg of chicken (A–C) or 10 μg of bovine (D–F) RPE microsomes for 2 h, and the generated retinoids were analyzed by HPLC. A and D, no inhibitor was added; B and E, 13 μm of apo-CRBP in the reaction; C and F, 20 μm of AcDCMK in the reaction. Peak 1, retinyl ester; peak 2, all-trans-retinal; peak 3, 11-cis-retinol; peak 4, all-trans-retinol.

FIGURE 4.

Comparison of the isomerohydrolase activities in chicken and bovine RPE. A and B, microsomal protein (14.5 μg) from chicken (A) and 29 μg from bovine RPE (B) were separately incubated with all-trans-[³H]retinol (0.2 μm) for various time intervals as indicated. The generated retinoids at each time point were analyzed by HPLC. Amounts of 11-cis-retinol generated were quantified based on the standard and averaged (mean ± S.D., n = 3). C and D, RPE65 levels were measured in the microsomes from chicken and bovine RPE by Western blot analysis. The amounts of 11-cis-retinol generated at each time point from chicken (C) and bovine (D) RPE microsomes were normalized by RPE65 concentrations in the samples (mean ± S.D., n = 3). E, the specific isomerohydrolase activities of chicken and bovine were measured from the slopes of the linear time courses of 11-cis-retinol generation normalized by RPE65 levels as shown in C and D (mean ± S.D., n = 3).

FIGURE 5.

Comparison of the isomerohydrolase activities of recombinant human and chicken RPE65. A and B, the same amount of microsomal proteins (32 μg) from 293A-LRAT cells expressing chicken (A) and human (B) RPE65 was incubated with all-trans-[³H]retinol (0.2 μm) for various time intervals as indicated. The generated retinoids at each time point were analyzed by HPLC, and 11-cis-[³H]retinol generated was quantified and averaged (mean ± S.D., n = 3). C and D, RPE65 levels were measured in the same microsomes from 293A-LRAT cells expressing chicken (C) and human (D) RPE65 as those used in A and B by Western blot analysis. The amounts of 11-cis-retinol generated at each time point from recombinant chicken (C) and human (D) RPE65 were normalized by RPE65 concentrations in the samples (mean ± S.D., n = 3). E, the specific isomerohydrolase activities of the chicken and human RPE65 were measured from initial slopes of the time course of 11-cis-retinol generation as shown in C and D (mean ± S.D., n = 3).

FIGURE 6.

LRAT activity measured in bovine and chicken RPE microsomes. All-trans-[³H]retinol (0.2 μm) was separately incubated for indicated times with 29 μg of bovine (A) or 14.5 μg of chicken (B) RPE microsomes, which were used for the isomerohydrolase assay, and the generated retinyl ester was quantified by HPLC (mean ± S.D., n = 3). C, the specific LRAT activities of chicken and bovine microsomes were measured from the slopes of the linear time courses of retinyl ester generation and normalized by total microsomal protein level (mean ± S.D., n = 3).