RPE65 is the isomerohydrolase in the retinoid visual cycle

^{Department of Cell Biology, Department of Medicine Endocrinology, University of Oklahoma Health Sciences Center, Oklahoma City, OK 73104; and}^{Department of Biochemistry, Medical University of South Carolina, Charleston, SC 29425}

^{To whom correspondence should be addressed. E-mail:}ude.cshuo@am-gnix-naij.

Edited by Jeremy Nathans, Johns Hopkins University School of Medicine, Baltimore, MD, and approved July 13, 2005

Received 2005 Apr 27

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 pmol/min 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.

Keywords: isomerase, LRAT, retinal dystrophy, retinyl ester, 11-cis retinal

Abstract

In 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).

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Fig. 1.

Scheme of retinoid visual cycle. RDH, retinol dehydrogenase.

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 intensive efforts of several groups over the past two decades. This missing enzyme represents a major gap in the elucidation of the visual cycle (Fig. 1).

RPE65 is an abundant protein in the RPE and is associated with the microsomal membrane (10, 11). A number of mutations of the RPE65 gene are associated with inherited retinal dystrophies in humans (12, 13) and in dogs (14, 15), indicating that RPE65 is essential for normal vision. Although physiologically significant, the exact function of RPE65 has remained elusive. The RPE65 gene knockout in mice resulted in a lack of 11-cis retinoids in the retina and RPE and an overaccumulation of all-trans retinyl ester in the RPE (16), suggesting an interrupted isomerization process. Recent studies demonstrated that purified RPE65 specifically binds all-trans retinyl palmitate, and these workers suggest that RPE65 is a retinyl ester-binding protein that is required for the isomerization reaction (17, 18).

RPE65 shares significant sequence homology only with the β-carotene monooxygenases, which cleaves β-carotene to generate all-trans retinal (19, 20). However, RPE65 itself does not cleave β-carotene (19), and no other enzymatic activities have ever been reported for RPE65.

Here, we show that RPE65, when coexpressed with LRAT in QBI-293A or COS-1 cells, efficiently generates 11-cis retinol from all-trans retinyl ester, suggesting that it is an enzyme responsible for the isomerohydrolase activity.

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Acknowledgments

We thank Drs. Rosalie K. Crouch and Thomas Ebrey for critical review of the manuscript and helpful comments, Dr. Mas Kono for COS-1 cells, and Dr. John Crabb (Cleveland Clinic Foundation, Cleveland) for the cellular retinaldehyde-binding protein expression system. This study was supported by National Institutes of Health Grants EY12231 and ET015650, a research award from the Juvenile Diabetes Research Foundation, a grant from the American Diabetes Association, and a research grant from Oklahoma Center for the Advancement of Science and Technology and Vision Center of Biomedical Research Excellence (to the Oklahoma University Health Sciences Center).

Acknowledgments

Notes

Author contributions: G.M. and J.-x.M. designed research; G.M., Y.C., Y.T., and B.X.W. performed research; Y.C., Y.T., and B.X.W. contributed new reagents/analytic tools; G.M., Y.C., and Y.T. analyzed data; and G.M. and J.-x.M. wrote the paper.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: LRAT, lecithin retinol acyltransferase; RPE, retinal pigment epithelium; Ad-RPE65, adenovirus expressing RPE65; Ad-GFP, adenovirus expressing GFP.

Notes

This paper was submitted directly (Track II) to the PNAS office.

Abbreviations: LRAT, lecithin retinol acyltransferase; RPE, retinal pigment epithelium; Ad-RPE65, adenovirus expressing RPE65; Ad-GFP, adenovirus expressing GFP.

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