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.Visual pigments of rods and cones consist of protein opsins and the chromophore, 11-cis-retinal covalently bound to opsins via a Schiff base (1). Upon light absorption, the 11-cis-retinal is photoisomerized to all-trans-retinal, which subsequently activates opsin and triggers the phototransduction cascade (1, 2). The 11-cis-retinal chromophore regenerates through a series of reactions of the retinoid visual cycle (3, 4). The visual cycle has been intensively studied in rod-dominant species such as bovine and mouse. The twocell (photoreceptor and RPE) 2 system of 11-cis-retinal recycling has been proposed for the visual cycle. After reduction of all-trans-retinal by retinol dehydrogenase, the generated all-trans-retinol is transported from the photoreceptor to the RPE and esterified to all-trans-retinyl esters by lecithin retinol acyltransferase (LRAT). The resulting all-trans-retinyl ester can either be stored in the RPE or directly converted to 11-cis-retinol by the isomerohydrolase, which was recently identified as the microsomal protein RPE65 (5-7). RPE65 has been shown to be an essential enzyme in the retinoid visual cycle for 11-cis-retinal regeneration (8). After oxidation of 11-cis-retinol by retinol dehydrogenase-5, the generated 11-cis-retinal is transported back to the photoreceptors to regenerate the visual pigments.It is known that cone...

Identification of Key Residues Determining Isomerohydrolase Activity of Human RPE65

“…2, B-D). cRPE65 produced substantially higher levels of 11cROL than that of hRPE65 after the normalization to its RPE65 protein level, indicating a higher enzymatic activity of cRPE65, consistent with our previous report (44).…”

“…Recombinant cRPE65 showed significantly higher specific isomerohydrolase activity than that of hRPE65 when the activities were normalized by RPE65 protein levels (44), suggesting that RPE65 from conedominant chicken is a more efficient isomerohydrolase, in comparison with mammalian RPE65, probably to meet the higher demand for 11-cis-retinal regeneration in the cone-dominant retina. Chicken and human RPE65 proteins share 90% sequence identity (50 different amino acid residues) at the amino acid level (44). We hypothesized that some of the divergent amino acid residues in cRPE65 were responsible for its higher isomerohydrolase activity.…”

“…Site-directed Mutagenesis-WT hRPE65 and cRPE65 cDNAs were subcloned into cloning vectors as described previously (1,44). The candidate key residues in hRPE65 were replaced by their counterparts in cRPE65 using the QuikChange site-directed mutagenesis kit (Stratagene, La Jolla, CA), following the manufacturer's protocol.…”

“…The membrane was blocked with 5% (w/v) nonfat dry milk in TBST (Tris-buffered saline with 0.1% Tween 20) for 30 min and subsequently incubated overnight at 4°C with a 1:1,000 dilution of an anti-RPE65 polyclonal antibody (27) to identify the key residues and a 1:50,000 dilution of an anti-␤-actin monoclonal antibody (Sigma-Aldrich). We used another RPE65 antibody (44) to compare the expression levels of cRPE65 and chimeric mutants of RPE65 to avoid the immunoreactivity difference on fragment 3. After four washes with TBST, the mem- The chromophore, 11-cis-retinal (11cRAL), in visual pigments is isomerized to all-trans-retinal (atRAL) by light, which triggers the phototransduction cascade.…”

“…We have previously reported that RPE65 in cone-dominant chicken has a substantially higher isomerohydrolase activity compared with that in rod-dominant mammals (44). We have cloned chicken RPE65 (cRPE65) and expressed it in the same heterologous system as that for hRPE65.…”