RPE65 is the isomerohydrolase in the retinoid visual cycle

Abstract

RPE65 is an abundant protein in the retinal pigment epithelium. Mutations in RPE65 are associated with inherited retinal dystrophies. Although it is known that RPE65 is critical for regeneration of 11-cis retinol in the visual cycle, the function of RPE65 is elusive. Here we show that recombinant RPE65, when expressed in QBI-293A and COS-1 cells, has robust enzymatic activity of the previous unidentified isomerohydrolase, an enzyme converting all-trans retinyl ester to 11-cis retinol in the visual cycle. The initial rate for the reaction is 2.9 pmol͞min per mg of RPE65 expressed in 293A cells. The isomerohydrolase activity of RPE65 requires coexpression of lecithin retinol acyltransferase in the same cell to provide its substrate. This enzymatic activity is linearly dependent on the expression levels of RPE65. This study demonstrates that RPE65 is the long-sought isomerohydrolase and fills a major gap in our understanding of the visual cycle. Identification of the function of RPE65 will contribute to the understanding of the pathogenesis for retinal dystrophies associated with RPE65 mutations.isomerase ͉ LRAT ͉ retinal dystrophy ͉ retinyl ester ͉ 11-cis retinal I n vertebrates, vision is initiated in rod and cone photoreceptors.The photosensitive entities in these cells are the visual pigments which consist of an apoprotein, opsin, and a chromophore, 11-cis retinal, which is attached to the opsin by a Schiff's base bond (1). Upon absorption of light by the pigments, 11-cis retinal is isomerized to all-trans retinal, which leads to the conformational changes of opsin and subsequently activates G protein transducin, initiating vision (1, 2). Efficient regeneration of 11-cis retinal, referred to as the visual retinoid cycle (see Fig. 1) is critical for the regeneration of the visual pigments (for reviews, see refs. 3-5) and maintenance of visual function. Disruption of the visual cycle by mutation or dysfunction of one of the numerous enzymes involved in this process leads to a number of blinding disorders (6).The enzyme in the visual cycle that has eluded identification is the isomerohydrolase, which is responsible for the isomerization and hydrolysis of all-trans retinyl ester to 11-cis retinol. Rando and colleagues (7) first proposed that all-trans retinol is first acylated to retinyl esters by lecithin retinol acyltransferase (LRAT). The generated all-trans retinyl ester is then directly isomerized and hydrolized into 11-cis retinol in the retinal pigment epithelium (RPE). This hydrolysis-isomerization process has been proposed to be catalyzed by a single enzyme, referred to as isomerohydrolase, which is associated with the membrane in RPE microsomes (8).Although isomerohydrolase activity had been demonstrated in the RPE almost 20 years ago, identification of the enzyme catalyzing this reaction has been difficult, because this activity is associated with the membrane and is abolished by solubilization in all of the detergents investigated (9). As a result, the isomerohydrolase has not been identified despite in...

RPE65 from Cone-dominant Chicken Is a More Efficient Isomerohydrolase Compared with That from Rod-dominant Species

“…ments (5)(6)(7). In the present study, we have demonstrated that the RPE65 in chicken eyes has significantly higher enzymatic activity, when compared with that in human and bovine eyes.…”

“…Confirmed human and chicken RPE65 cDNAs were cloned between the NotI and HindIII sites of the pShuttle-CMV vector (Qbiogene, Montreal, Canada) for construction of adenoviruses. Preparations, amplification, and titration of the recombinant adenoviruses were performed as described previously (5).…”

Topology and Membrane Association of Lecithin: Retinol Acyltransferase

“…It has been reported that RPE65 is involved in the isomerization reaction that produces 11-cis-retinol and that retinyl esters are the preferred substrates for this reaction (51)(52)(53). It has also been proposed that palmitoylation of RPE65 by LRAT regulates its subcellular localization and its ligand binding selectivity versus retinol or retinyl esters (54).…”

Parallel visual cycles in the zebrafish retina

“…The resulting all-trans RAL is first reduced to all-trans ROL by an NADPH-dependent dehydrogenase (Cideciyan et al, 2000;Lion et al, 1975;Rattner et al, 2000;Suzuki et al, 1993), and then transported from the photoreceptor to the second compartment involved in visual pigment recycling, the RPE. Upon arrival in RPE cells, all-trans ROL is esterified into retinylesters (REs) by the lecithin:retinol acyltransferase LRAT (Mondal et al, 2000;Ruiz et al, 1999;Saari and Bredberg, 1989;Saari et al, 1993), and then isomerized to 11-cis ROL by the isomerase RPE65 (or Isomerase I) (Gollapalli and Rando, 2003;Jin et al, 2005;Moiseyev et al, 2005;Redmond et al, 2005). Finally, 11-cis ROL is oxidized to 11-cis RAL by a retinol dehydrogenase (Cideciyan et al, 2000;Driessen et al, 1997;Gamble et al, 2000;Haeseleer et al, 1998;Lion et al, 1975;Simon et al, 1999;Suzuki et al, 1993).…”