COMMENTARY COMMENTARY

RPE65 takes on another role in the vertebrate retina

T. Michael Redmonda,1

The high-acuity central vision of humans, and other loci (e.g., complement/immune-modulatory, lipid me- primates, depends on a region of the central retina tabolism, extracellular matrix genes) contributing to called the macula lutea (Latin: yellow spot), containing risk and disease pathogenesis (6). A multifactorial dis- not only a high concentration of both cone and rod ease, AMD not only involves the macular and foveal photoreceptors but also a high concentration of neural retina but also the underlying retinal pigment xanthophyll carotenoids; hence, its name (1). At the epithelium (RPE) and the vascular choriocapillaris. center of the macula is the fovea (Latin: pit) centralis, While some surgical and monoclonal antibody thera- a region of especially high-acuity vision, containing pies are available to treat severe complications (neo- only cone photoreceptors and Muller glia cells, where, vascularization), treatment options are limited and in the human fovea, the density of cones is estimated cure is currently unavailable. to reach 1–3 × 105 per square millimeter. In the fovea, Epidemiological studies, such as the Age-Related there are no overlying interneurons or retinal ganglion Eye Disease Studies (AREDS and AREDS 2) have cells, as in the rest of the retina. Instead, the axons of found that higher levels of macular xanthophyll intake the foveal cones are elongated and connect to bipolar are correlated with lower risk of developing severe cells located on the rim of the foveal pit. Anatomically, AMD (5) and higher macular pigment optical density these elongated axonal processes form a layer called (MPOD) is an important phenotypic parameter associ- the fibers of Henle that overlies the cone photorecep- ated with macular health (1, 3). Accordingly, AREDS tors like a thin cap. It is here that the macular xantho- 2 proposed preventative measures to mitigate risk of phylls accumulate (2). developing AMD, including supplementation with lu- It is thought that the protective function of the tein and zeaxanthin, as well as other antioxidants, such xanthophylls is twofold: serving as a blue-light filter as copper and zinc, and ascorbic acid. Xanthophylls and as scavengers of reactive oxygen species (ROS), are taken up by the gut and transported in the blood both potentially damaging to cone photoreceptors (via HDL) to the RPE and taken up into the pri- (3). The electron-rich polyene chains of carotenoids mate retina by scavenger receptor class B member efficiently quench ROS, thereby preventing mem- 1(SCARB1) and CD36 (3). While significant levels of brane damage. Three xanthophylls are found in the lutein and zeaxanthin, neither of which can be synthe- macula and fovea: two, lutein and zeaxanthin, are sized by animals, are both widely available from di- abundant in nature, while the third, meso-zeaxanthin, etary sources (e.g., green leafy vegetables, fruits, is uncommon elsewhere outside primate and bird corn, egg yolks), meso-zeaxanthin, rare in commonly retinas (although synthetic meso-zeaxanthin is now consumed dietary sources, is not (3). Hence, the origin commercially available). Both zeaxanthins have a of macular meso-zeaxanthin has been controversial. better singlet oxygen-quenching ability than lutein Some investigators favor a dietary source for meso- due to an additional conjugated double bond, while zeaxanthin, where trace levels are found in fish skin meso-zeaxanthin may have the greatest antioxidant and egg yolks (7), suggesting that metabolic pro- ability of the three (4). cesses are not necessary to explain its occurrence in Threats to the health of the macula, and vision the retina, while others hold that it is metabolically itself, include various inherited simple Mendelian transformed in the retina from some other xantho- gene disorders, all relatively rare, but also the far phyll, probably lutein (3). While accumulation of meso- more common condition of age-related macular de- zeaxanthin has been shown to occur in a developmentally generation (AMD) (5). AMD is a major cause of blind- regulated manner, first in the RPE/choroid of chicken ness in older adults, with the risk increasing with age. It embryos followed by its appearance in the retina, and is a complex disease, with environmental factors (e.g., nowhere else in the embryo (8), the underlying meta- smoking) and common and rare variants in >30 genetic bolic mechanism was unknown.

aLaboratory of Retinal Cell & Molecular Biology, National Eye Institute/NIH, Bethesda, MD 20892 Author contributions: T.M.R. wrote the paper. The author declares no conflict of interest. See companion article on page 10882. 1Email: [email protected].

10818–10820 | PNAS | October 10, 2017 | vol. 114 | no. 41 www.pnas.org/cgi/doi/10.1073/pnas.1715064114 Downloaded by guest on September 28, 2021 Fig. 1. Dual roles of RPE65 retinol isomerase in the primate and avian RPE. (A) In its canonical role, RPE65 catalyzes the concerted ester cleavage of all-trans retinyl esters and the all-trans– to 11-cis–isomerization of the retinyl moiety, as the critical retinol isomerase in the retinoid visual cycle. (B) In its newly described role, RPE65 catalyzes the isomerization of lutein to meso-zeaxanthin by migrating the 4′-5′ double bond (highlighted in red) of lutein to the 5′-6′ position (highlighted in red) of meso-zeaxanthin, thereby converting an e-ionone ring to a β-ionone ring. The meso-zeaxanthin generated in the RPE is transported by several binding proteins (SR-B1, CD36, and IRBP) to the cone photoreceptor axons, where it is sequestered (indicated by yellow) by GSTP1 to form the macula lutea. CD36, platelet glycoprotein 4; GI, gastrointestinal; GSTP1, glutathione S-transferase (GST P); IRBP, interphotoreceptor retinoid-binding protein (retinol-binding protein 3); LRAT, lecithin/retinol acyltransferase; SR-B1, scavenger receptor class B member 1; Vit A, vitamin A.

In PNAS, Shyam et al. (9) now reveal that RPE65, a protein the Rpe65 gene in the mouse (17) gives rise to a phenotype in highly preferentially expressed at high levels in the RPE and the which 11-cis retinoids are completely absent in the retinae of ho- well-known retinol isomerase of the vertebrate visual cycle (10–12) mozygous knockout mice, while all-trans retinyl esters accumulate; (Fig. 1A), is also the lutein to meso-zeaxanthin isomerase (Fig. 1B). concomitantly, these mice are extremely insensitive to all but the Although a remarkable and exciting finding that explains an im- very highest light intensities. portant phenomenon, the concept that RPE65 is the responsible In earlier work, Bernstein and coworkers (8) had shown that enzyme should not be entirely surprising, given its identity as a meso-zeaxanthin first appears in the RPE/choroid of dark- divergent member of a superfamily of carotenoid-cleaving oxy- incubated chicken embryos at embryonic day (E) 17, increasing genases (CCOs). CCOs, widespread in all kingdoms of life, include as the embryo developed further and appearing in the retina at apocarotenoid oxygenases (ACOs) in bacteria, epoxycarotenoid- E19, but nowhere else in the embryo. In PNAS, Shyam et al. show cleaving enzymes required for abscisic acid biosynthesis in plants (9) that this period of development correlates with the significant [NCEDs (viviparous14s [VP14s])], and carotenoid-cleaving enzymes up-regulation of RPE65 mRNA and protein, also required for pro- in plants (CCDs) and animals. Vertebrates express three members duction of 11-cis retinal chromophore for the developing photore- ′ β ′ ′ of the superfamily: 15,15 - -carotene oxygenase (BCO1), 9 ,10 - ceptors (Fig. 1A). Supply of HPLC-purified lutein to HEK293T cells β -carotene oxygenase (BCO2), and RPE65. All CCOs are nonheme heterologously expressing RPE65 revealed accumulation of meso- iron (II) oxygenases, with ferrous iron coordinated by four histidines zeaxanthin, in a dose- and time-dependent manner, while control in a seven-bladed beta-propeller chain fold. Crystal structures of the cells or cells exposed to isomerically pure zeaxanthin do not. Fur- cyanobacterial Synechocystis ACO, vertebrate RPE65 from Bos tau- thermore, primary cultures of chicken RPE, which (unlike mamma- rus (13), plant VP14 from Zea mays, and bacterial resveratrol- lian RPE cells) retain robust expression of RPE65, also accumulated cleaving dioxygenase NOV1 from Novosphingobium confirm the meso-zeaxanthin when incubated with lutein, also in a dose- and conservation of overall fold and active site arrangement in all CCO time-dependent manner. To test the hypothesis in an in vivo system, superfamily members. All CCOs, except for RPE65, oxidatively pharmacological inhibition of RPE65 by ACU-5200-HCl, a compet- cleave conjugated polyene substrates. In contrast, the major sub- itive inhibitor of RPE65 and an analog of emixustat (18), injected into strate of RPE65 is all-trans retinyl ester. RPE65 performs a concerted theyolksacatE17andatE19blockedmeso-zeaxanthin accumula- O-alkyl cleavage of the all-trans retinyl ester into retinol and fatty tion in injected embryos in a dose-dependent manner. acid, concomitant with carbocation-mediated (or possibly radical Docking of lutein into a homology model for chicken RPE65 cation-mediated) trans-tocis-isomerization of the retinyl moiety indicates that the e-ionone ring lies in proximity to the histidines (13, 14), a novel function acquired by an ancestral carotenoid oxy- coordinating the iron center, with the C4′/C5′/C6′ segment, genase. Mutations in RPE65 are associated with a rare autosomal where the relevant double-bond migration occurs, close to the recessive blinding disorder called Leber congenital amaurosis, or histidines. Binding of the e-ionone ring in the pocket may be early onset severe retinal dystrophy (15, 16). Targeted disruption of facilitated by hydrogen bonding with Glu417 and Trp331 present

Redmond PNAS | October 10, 2017 | vol. 114 | no. 41 | 10819 Downloaded by guest on September 28, 2021 in the cavity and/or aromatic ring stacking with phenylalanine res- not inconceivable that it should have a secondary role as the lutein idues also present. Double-bond migration from the 4′-5′ position to meso-zeaxanthin isomerase. Although double-bond migration of to the 5′-6′ position is proposed to be mediated by coordinated lutein to meso-zeaxanthin is different from trans-tocis-isomerization acid-base catalysis. This double-bond migration confers the addi- of retinol, the actual bond broken by RPE65’s iron center is the O- tional conjugated double bond (11 instead of 10 in lutein) to the alkyl linkage of the retinyl ester. So, consistent with the divergent resultant meso-zeaxanthin. Given what we know about the cata- functional path that RPE65 has taken from its fellow BCOs and CCOs, lytic potential of the nonheme iron center, catalyzing double-bond lutein to meso-zeaxanthin isomerization may be yet another string cleavage of CCOs or O-alkyl cleavage of the ester bond of all-trans to the bow of this versatile nonheme iron center. retinyl esters (in the case of RPE65) (13, 14, 18), this is a reasonable Finally, these exciting findings will provoke much further conjecture. Alternatively, a carbocation mechanism, as in the trans- study in this important area of vision research with implications in to cis-isomerization of retinoids (13, 14, 18), may be responsible. AMD, a disease of major impact on quality of life of older adults. The catalytic efficiency, as indicated by the rate of accumulation of Other important questions remaining include the following: (i)the meso-zeaxanthin in the experiments presented, is low and likely precise cellular localization(s) of the meso-zeaxanthin (photorecep- much slower than that for the trans-tocis-isomerization of reti- tor cell bodies, photoreceptor axons, muller glia, other cells?), noids, already considered to exhibit low turnover. A direct com- (ii) trafficking mechanisms of the meso-zeaxanthin from the RPE parison of substrates, between all-trans retinyl ester and lutein, to its final destination, and (iii) whether there is reduced accu- would be informative. mulation of meso-zeaxanthin in the maculae of individuals with Despite the low turnover, resultant meso-zeaxanthin is trans- RPE65-associated retinal dystrophies and/or unaffected carriers ported across the interphotoreceptor space to the cone photore- of a mutant RPE65 allele. Interestingly, with respect to the last ceptors (Fig. 1B), where it is thought to be specifically sequestered by point, an SNP in RPE65, along with SNPs in many other genes in glutathione S-transferase [GST P1 (GSTP1)], the zeaxanthin-binding carotenoid and lipid metabolism and transport, is associated protein of the human macula (19). Although RPE65 is expressed with MPOD variability in the Carotenoids in Age-Related Eye throughout the RPE, and cone photoreceptors are dispersed Disease Study (20). throughout the peripheral retina, the concentration of/exclusive pres- ence of cones in the fovea may serve to increase the relative prom- Acknowledgments inence of meso-zeaxanthin in this region. In conclusion, while the This work was supported by the Intramural Research Program of the National primary role of RPE65 is as retinol isomerase in the visual cycle, it is Eye Institute/NIH.

1 Krinsky NI, Landrum JT, Bone RA (2003) Biologic mechanisms of the protective role of lutein and zeaxanthin in the eye. Annu Rev Nutr 23:171–201. 2 Meyer zu Westrup V, Dietzel M, Pauleikhoff D, Hense HW (2014) The association of retinal structure and macular pigment distribution. Invest Ophthalmol Vis Sci 55:1169–1175. 3 Bernstein PS, et al. (2016) Lutein, zeaxanthin, and meso-zeaxanthin: The basic and clinical science underlying carotenoid-based nutritional interventions against ocular disease. Prog Retin Eye Res 50:34–66. 4 Li B, Ahmed F, Bernstein PS (2010) Studies on the singlet oxygen scavenging mechanism of human macular pigment. Arch Biochem Biophys 504:56–60. 5 Aronow ME, Chew EY (2014) Age-related eye disease study 2: Perspectives, recommendations, and unanswered questions. Curr Opin Ophthalmol 25:186–190. 6 Fritsche LG, et al. (2016) A large genome-wide association study of age-related macular degeneration highlights contributions of rare and common variants. Nat Genet 48:134–143. 7 Nolan JM, Meagher K, Kashani S, Beatty S (2013) What is meso-zeaxanthin, and where does it come from? Eye (Lond) 27:899–905. 8 Gorusupudi A, et al. (2016) Developmentally regulated production of meso-zeaxanthin in chicken retinal pigment epithelium/choroid and retina. Invest Ophthalmol Vis Sci 57:1853–1861. 9 Shyam R, Gorusupudi A, Nelson K, Horvath MP, Bernstein PS (2017) RPE65 has an additional function as the lutein to meso-zeaxanthin isomerase in the vertebrate eye. Proc Natl Acad Sci USA 114:10882–10887. 10 Jin M, Li S, Moghrabi WN, Sun H, Travis GH (2005) Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium. Cell 122:449–459. 11 Moiseyev G, Chen Y, Takahashi Y, Wu BX, Ma JX (2005) RPE65 is the isomerohydrolase in the retinoid visual cycle. Proc Natl Acad Sci USA 102:12413–12418. 12 Redmond TM, et al. (2005) Mutation of key residues of RPE65 abolishes its enzymatic role as isomerohydrolase in the visual cycle. Proc Natl Acad Sci USA 102:13658–13663. 13 Kiser PD, Golczak M, Lodowski DT, Chance MR, Palczewski K (2009) Crystal structure of native RPE65, the retinoid isomerase of the visual cycle. Proc Natl Acad Sci USA 106:17325–17330. 14 Redmond TM, Poliakov E, Kuo S, Chander P, Gentleman S (2010) RPE65, visual cycle retinol isomerase, is not inherently 11-cis-specific: Support for a carbocation mechanism of retinol isomerization. J Biol Chem 285:1919–1927. 15 Gu SM, et al. (1997) Mutations in RPE65 cause autosomal recessive childhood-onset severe retinal dystrophy. Nat Genet 17:194–197. 16 Marlhens F, et al. (1997) Mutations in RPE65 cause Leber’s congenital amaurosis. Nat Genet 17:139–141. 17 Redmond TM, et al. (1998) Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet 20:344–351. 18 Kiser PD, et al. (2015) Catalytic mechanism of a retinoid isomerase essential for vertebrate vision. Nat Chem Biol 11:409–415. 19 Bhosale P, et al. (2004) Identification and characterization of a Pi isoform of glutathione S-transferase (GSTP1) as a zeaxanthin-binding protein in the macula of the human eye. J Biol Chem 279:49447–49454. 20 Meyers KJ, et al. (2013) Genetic determinants of macular pigments in women of the Carotenoids in Age-Related Eye Disease Study. Invest Ophthalmol Vis Sci 54:2333–2345.

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