Rpe65 Is a Retinyl Ester Binding Protein That Presents Insoluble Substrate to the Isomerase in Retinal Pigment Epithelial Cells

Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of β-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65. Photon capture by a rhodopsin pigment molecule induces 11-cis to all-trans isomerization of its retinaldehyde chromophore. To restore light sensitivity, the all-trans-retinaldehyde must be chemically re-isomerized by an enzyme pathway called the visual cycle. Rpe65, an abundant protein in retinal pigment epithelial (RPE) cells and a homolog of β-carotene dioxygenase, appears to play a role in this pathway. Rpe65-/- knockout mice massively accumulate all-trans-retinyl esters but lack 11-cis-retinoids and rhodopsin visual pigment in their retinas. Mutations in the human RPE65 gene cause a severe recessive blinding disease called Leber's congenital amaurosis. The function of Rpe65, however, is unknown. Here we show that Rpe65 specifically binds all-trans-retinyl palmitate but not 11-cis-retinyl palmitate by a spectral-shift assay, by co-elution during gel filtration, and by co-immunoprecipitation. Using a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, we demonstrate that Rpe65 extracts all-trans-retinyl esters from phospholipid membranes. Assays of isomerase activity reveal that Rpe65 strongly stimulates the enzymatic conversion of all-trans-retinyl palmitate to 11-cis-retinol in microsomes from bovine RPE cells. Moreover, we show that addition of Rpe65 to membranes from rpe65-/- mice, which possess no detectable isomerase activity, restores isomerase activity to wild-type levels. Rpe65 by itself, however, has no intrinsic isomerase activity. These observations suggest that Rpe65 presents retinyl esters as substrate to the isomerase for synthesis of visual chromophore. This proposed function explains the phenotype in mice and humans lacking Rpe65. Light perception in vertebrates is mediated by a group of G protein-coupled receptors called the opsins. Most opsin pigments contain 11-cis-retinaldehyde (11cRAL) 1The abbreviations used are: 11cRAL, 11-cis-retinaldehyde; IMH, isomerohydrolase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; Ab, antibody; FRET, fluorescent resonance energy transfer; HPLC, high performance liquid chromatography; ER, endoplasmic reticulum; atROL, all-trans-retinol; atRAL, all-trans-retinaldehyde; atRE, all-trans-retinyl ester; RPE, retinal pigment epithelium; 11cRP, 11-cis-retinyl palmitate; CRALBP, cellular retinaldehyde-binding protein; LCA, Leber's congenital amaurosis. as the light-absorbing chromophore. Absorption of a photon induces 11-cis to all-trans isomerization of the chromophore, resulting in the activated species, metarhodopsin II. After a brief period, metarhodopsin II decays to yield apo-rhodopsin and free all-trans-retinaldehyde (atRAL). Before light sensitivity of the pigment can be restored, the atRAL must be chemically re-isomerized to 11cRAL by a metabolic pathway called the visual cycle. Most steps in this pathway take place within cells of the retinal pigment epithelium (RPE) adjacent to the photoreceptors. The key step in this pathway is all-trans to 11-cis re-isomerization of the retinoid, which is catalyzed by an enzyme activity called isomerohydrolase (IMH). IMH has been shown to use fatty acyl esters of retinol as a substrate (1.Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus (106) Google Scholar, 2.Gollapalli D.R. Rando R.R. Biochemistry. 2003; 42: 5809-5818Crossref PubMed Scopus (59) Google Scholar), harnessing the energy of ester hydrolysis [ΔG = -5 kcal/mol (3.Deigner P.S. Law W.C. Canada F.J. Rando R.R. Science. 1989; 244: 968-971Crossref PubMed Scopus (157) Google Scholar)] for the endothermic conversion of all-trans-retinol (atROL) to 11-cis-retinol (11cROL) (+4.1 kcal/mol, Ref. 4.Rando R.R. Chang A. J. Am. Chem. Soc. 1983; 105: 2879-2882Crossref Scopus (74) Google Scholar). IMH has never been purified or cloned. Leber's congenital amaurosis (LCA) is a severe and relatively common autosomal recessive disease that results in blindness at birth. LCA is frequently caused by mutations in the RPE65 gene (5.Gu S.M. Thompson D.A. Srikumari C.R.S. Lorenz B. Finckh U. Nicoletti A. Murthy K.R. Rathmann M. Kumaramanickavel G. Denton M.J. Gal A. Nat. Genet. 1997; 17: 194-197Crossref PubMed Scopus (550) Google Scholar, 6.Marlhens F. Bareil C. Griffoin J.M. Zrenner E. Amalric P. Eliaou C. Liu S.Y. Harris E. Redmond T.M. Arnaud B. Claustres M. Hamel C.P. Nat. Genet. 1997; 17: 139-141Crossref PubMed Scopus (520) Google Scholar). Rpe65, the product of this gene, is an abundant protein of unknown function in cells of the RPE (7.Bavik C.O. Eriksson U. Allen R.A. Peterson P.A. J. Biol. Chem. 1991; 266: 14978-14985Abstract Full Text PDF PubMed Google Scholar). Rpe65 has high affinity for phospholipid membranes but contains no membrane-spanning domains (8.Bavik C.O. Busch C. Eriksson U. J. Biol. Chem. 1992; 267: 23035-23042Abstract Full Text PDF PubMed Google Scholar, 9.Tsilou E. Hamel C.P. Yu S. Redmond T.M. Arch. Biochem. Biophys. 1997; 346: 21-27Crossref PubMed Scopus (41) Google Scholar). Mice with a knockout mutation in rpe65 massively accumulate all-trans-retinyl esters (atRE) in their RPE and have no detectable 11-cis-retinoids (10.Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (815) Google Scholar). Photoreceptors in rpe65-/- mice are morphologically normal but contain only apo-rhodopsin instead of rhodopsin pigment. These observations led to speculation that Rpe65 may be IMH. In the current study, we sought to define the function of Rpe65 and its relationship to IMH. We show by several approaches that Rpe65 specifically binds all-trans-retinyl palmitate (atRP), the most abundant retinyl ester in RPE cells (11.Futterman S. Andrews J.S. J. Biol. Chem. 1964; 239: 81-84Abstract Full Text PDF PubMed Google Scholar). Further, we present data suggesting that atRP bound to Rpe65 is the substrate for IMH. Expression of Bovine Rpe65 in Baculovirus-infected Sf9 Cells—We cloned the complete coding sequence of bovine Rpe65 into pFastBac bacmid (Invitrogen) and used it to transfect Spodoptera frugiperda (Sf9) cells following the manufacturer's procedure. Following infection, adherent cells were lysed in phosphate-buffered saline, pH 7.0, 1% CHAPS, and protease inhibitor mixture (Roche Diagnostics) and centrifuged at 100,000 × g for 30 min. The detergent-soluble fractions were analyzed by gel filtration (Zorbax GF-250) on an Agilent 1100 series liquid chromatograph (mobile phase = 20 mm Na2HPO4, 130 mm NaCl, flow = 1 ml/min, 280 nm detection). Proteins eluting at Mr 158-43 kDa were collected and concentrated using Centricon 30,000 molecular weight cut-off (MWCO) ultrafiltration devices (Millipore). The retained proteins were separated on an ÄKTA FPLC (Amersham Biosciences) using a Mono Q anion exchange column equilibrated with 25 mm Tris-OAc, pH 7.4, 1 mm EDTA, 0.1 mm dithiothreitol, 10 mm NaOAc, flow = 1 ml/min. A gradient to 0.5 m NaOAc over 60 min was initiated 10 min after sample injection. Collected gradient fractions were analyzed by SDS-PAGE and Western blotting to confirm the presence of Rpe65. For purified Rpe65 or proteins from Sf9 cells were into mm pH 7.4, mm NaCl, 1 mm using 30,000 ultrafiltration In the detergent-soluble fractions were used Bovine Rpe65 from bovine Rpe65, was to and as an with complete (Invitrogen) into were at using was purified from on a protein A The was on 1 of 10 to the manufacturer's SDS-PAGE and Western were by SDS-PAGE on a gradient gel and to membranes in a to the manufacturer's After in were with the at were using to the manufacturer's and were from atROL, atRAL, and or were from 11cRAL and to R.A. PubMed Scopus Google Scholar). were purified by to of sample to and by using PubMed Google Scholar). were in at to of separated by a were used in to was in which a protein sample of Rpe65 in 10 mm pH mm NaCl, in and the atROL, 11cRP, atRAL, or at in the in the adjacent The sample was and were at the using the as a = 30 were in an using protein or bovine and was at to proteins from Sf9 cells or Sf9 cells Rpe65. were at and centrifuged at × 30 min to from the binding were analyzed by gel filtration with and and in of Rpe65 10 were with 30 of sample of in phosphate-buffered at were on and protein Following the were centrifuged at × 30 and the fractions were The were with of phosphate-buffered and in of of of were for and for Western in was used to for was used to confirm of Rpe65. of and were to Biochemistry. 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Full Text Full Text PDF PubMed Scopus Google Scholar). membranes were with light min on to and were in mm pH 1 mm dithiothreitol, 1 mm EDTA, 1 The sample was for 1 at by at × min. in the was for as protein = = The protein sample was to an of in 10 mm to a protein of and a of A. Chen S. J. Bok D. 1998; PubMed Scopus Google was to a of 10 and the sample was at for 1 The sample was with to the and D. A. M. Biophys. PubMed Scopus Google Scholar, S. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). were and as To and of protein from Sf9 cells and atRP or Sf9 cells and atRP were and at for 1 were by the to an of the by and as for bovine RPE were that the binding were to a gel filtration column to the Rpe65 The was to an of the and were as only atRP were by of atRP in with a mixture and in To sample in enzyme of protein atRP were and of or to as substrate for in bovine RPE protein was by of the with J. Lee J. Biochemistry. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). A of the substrate was to of RPE and the were for min at was to and was for at were with of and of were into × and analyzed by as N. G. Full Text Full Text PDF PubMed Scopus Google Scholar). of Rpe65 on IMH in and rpe65-/- knockout mice (10.Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (815) Google were in at for Mice = were by of and and by were and the and were The were in 10 mm pH 1 mm dithiothreitol, 1 mm using a and centrifuged at × g for min. The resulting were in and the step was The which of the was and the protein was in 1% by brief × 20 and min at The were centrifuged at × g for 30 min. The fractions were and to gel filtration to as = The protein was concentrated and for IMH activity or Rpe65 from Sf9 cells. of protein sample were to a mixture 25 in and of Rpe65 or of 1 of a of atRP in was to the atRP = the were as and at for 1 with to activity of Rpe65 were as that the RPE proteins were with or were used as were with of and the into of was a of and the sample were in were analyzed by as on an Agilent column × and a phase of flow = 1 ml/min. of of the used in and in were by light were on a with a and a were after min of These data were with data from of Rpe65 and atRP a in the Absorption an and its light-absorbing induces a in the of this is the presence of or in from and after of the with the protein R.K. Harris D.A. and A Scholar). To for binding of to Rpe65, we of atRP and Rpe65 to the of a In this and we used Sf9 cells as a of Rpe65. These cells no detectable the Rpe65 protein was in the or were at after sample in the of atRP with Rpe65 caused a in at nm and an at 280 resulting in an at nm The of atRP in binding was with Rpe65 the of free We this the 11-cis-retinyl palmitate for no in was during with Rpe65 suggesting that the with atRP is for the all-trans We used the assay to for Rpe65 and atRAL or to 11cRP, we no in the of the with Rpe65 We for an Rpe65 and with Rpe65 in no in the of Rpe65 with a in with of an at nm a we on and cellular retinaldehyde-binding protein which specifically binds and 11cRAL J. Biol. Chem. Full Text PDF PubMed Google Scholar). we an in the results suggest that Rpe65 with atRP and atROL, but not with 11cRP, atRAL, 11cRAL, or of Rpe65 and atRP by and Rpe65 binds atRE, atRP with Rpe65 during gel filtration We a of Sf9 cells with an of a we a of Sf9 cells with We separated by gel filtration and atRP by min with an We collected fractions for by The in The was in the column to the in was or with a of Sf9 cells The of was in fractions of the sample and the of Rpe65 by protein gel of was in the sample from Sf9 cells. We at the of in the Rpe65 The from the a of nm of atRP in The from the a of 280 of with at nm These data suggest that atRP binds to Rpe65. To confirm that atRP binds specifically to Rpe65, we an from Rpe65. We extracts of and Sf9 cells with an of were with the We analyzed the and fractions for We of the and fractions with the Rpe65 Most were in the to the of This Rpe65 to binding to the were present in the were detectable in the of the sample We of the by following gel filtration and results Rpe65 as the protein binding for for an with Rpe65 we the gel filtration for we no co-elution of with Rpe65 This that the of with Rpe65 may be to Rpe65 atRP and from esters are in and not exchange J. Biol. Chem. 1989; Full Text PDF PubMed Google Scholar, R.R. Biochem. Biophys. PubMed Scopus Google Scholar). A function for Rpe65 may be to and to their from membranes. To this we a novel fluorescent resonance energy transfer (FRET) binding assay in liposomes, using the fluorescent Biochemistry. PubMed Scopus Google and A. M. G. Biochemistry. PubMed Scopus Google Scholar). the and for and for atRP in The of and with the of the the of atRP with the of or To of we or We of at the for and the were from or A and to of with The fluorescent were from or A and The fluorescent in the presence of atRP were to of in in a we used the assay to of atRP into the and of We of purified Rpe65 from or fractions from Sf9 and or of protein were used in the and We free atRP in to of for and fluorescent at or nm with After a the at nm was in Rpe65 with A was on the was in Rpe65 with These results show of atRP into the with The is to a in and is to the is a to the of the with is to from atRP in the of the is from atRP in the The of was in the in the of and The the with an of These data that free atRP in the must the to the To that the fluorescent in Rpe65 proteins were to of or we at the of and fluorescent at or For the were in Rpe65 and the in the and were not caused by of the is that of the to the Rpe65 and atRP bound to Rpe65, and atRP within the The of to is this is in the Rpe65 and To confirm of we analyzed the and by after addition of We with light at nm and using a only atRP is The a of suggesting that the atRP was to the membranes. We fluorescent in the the The of was in we atRP in the and the fluorescent were never in Rpe65 the results in show that Rpe65 not only binds but can from membranes into the of These suggest a function for Rpe65. Rpe65 for of atRP but role for Rpe65 as an may be to present this substrate to IMH for To this we RPE membranes from wild-type and rpe65-/- knockout mice (10.Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (815) Google Scholar). After to the membranes were in and for of from atRP substrate or Rpe65. of during are shown in in we of the by in and with the of the assay of were by the RPE membranes from wild-type of Rpe65 to membranes synthesis of to was by RPE membranes from rpe65-/- addition of Rpe65 to rpe65-/- membranes the IMH activity of We the of Rpe65 on IMH activity in from bovine we of with a of that only of were A of was the atRP were with that atRP Sf9 of were the bovine RPE were with that atRP Rpe65 The of in the assay with atRP or atRP Sf9 proteins are to Rpe65 during of the bovine RPE A of was in assay was and In a we to RPE Following a of was suggesting that the in the is a product of The results in that IMH activity is the presence of Rpe65. To the that Rpe65 is IMH, we Rpe65 atRP or and analyzed for of by was in We membranes from wild-type RPE with atRP and Rpe65. Here no was These data that Rpe65 has no intrinsic isomerase activity. In this we that Rpe65 binds We that and atRP to yield a molecular We that atRP with Rpe65 during gel filtration and we that Rpe65 take free atRP from the liposomes, and that a of the atRP in is within the we that the conversion of atRP to by IMH is on Rpe65, but that Rpe65 not this isomerization Rpe65 is to which the conversion of β-carotene to atRAL T.M. S. Yu S. B. E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, F. N. Chang J. M. M. P. K. PubMed Scopus Google Scholar). this of the of Rpe65 and the of may be the molecular for and the of we no β-carotene with Rpe65, suggesting that Rpe65 not Rpe65 not possess activity J. K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The and Rpe65 that proteins with To the of atRP binding by Rpe65, we analyzed the of atRP into proteins from and Sf9 cells. the Sf9 proteins were with Rpe65 was present on and of the We atRP to the in a of which in of atRP as a We used a fluorescent of and a fluorescent to the presence of atRP within the and of liposomes, We the Rpe65 of atRP by the of fluorescent from Rpe65 a of Sf9 The in the with was with and the These the of Rpe65 on atRP into liposomes, the and to the or A and in Rpe65 and Sf9 Rpe65 strongly the of atRP into the membranes and its transfer to the We the fluorescent of atRP to it in by In the of atRP were in the the in RPE cells of rpe65-/- mice (10.Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (815) Google and K. M. A. Redmond T.M. 2003; PubMed Scopus Google Scholar). We never in the of Rpe65 on the of may with to Rpe65 may from the membranes to atRP from is that a of from the and to Rpe65 the of a phospholipid at its of atRP in explains the of atRP in membranes from of rpe65-/- with normal Rpe65 caused of the from the K. M. A. Redmond T.M. 2003; PubMed Scopus Google Scholar). Rpe65 can from in The presence of atRP in the of in the current and is that Rpe65 extracts atRP from membranes. proteins have been that of retinol and retinaldehyde J. Biol. Chem. Full Text PDF PubMed Google Scholar), cellular protein D. F. Google Scholar), protein Biophys. PubMed Scopus Google Scholar), and protein T.M. B. Lee Biochemistry. PubMed Scopus Google Scholar). no binding proteins for retinyl esters have been The binding proteins are to their and to from with cellular however, are relatively is the for an protein as esters the of A (11.Futterman S. Andrews J.S. J. Biol. Chem. 1964; 239: 81-84Abstract Full Text PDF PubMed Google Scholar, PubMed Scopus Google Scholar), and the substrate for synthesis of visual in the RPE (1.Moiseyev G. Crouch R.K. Goletz P. Oatis Jr., J. Redmond T.M. Ma J.X. Biochemistry. 2003; 42: 2229-2238Crossref PubMed Scopus (106) Google Scholar, 2.Gollapalli D.R. Rando R.R. Biochemistry. 2003; 42: 5809-5818Crossref PubMed Scopus (59) Google Scholar). The of in the for a binding protein to from the and in a to IMH. RPE membranes from rpe65-/- mice no IMH activity and we complete of this phenotype with addition of Rpe65 to the assay mixture and we of IMH activity in bovine RPE with addition of Rpe65 The lack of in the that the with Rpe65 is not caused by Sf9 A of the data in is that Rpe65 and IMH are the no was Rpe65 was with atRP or or with atRP RPE proteins from wild-type These observations the that Rpe65 and IMH are the a that Rpe65 is a of a IMH The of Rpe65 in RPE cells this A for the of isomerase activity on the presence of Rpe65 is that this protein is for substrate by IMH. for the function of Rpe65 is shown in We suggest that the role of Rpe65 is to from and present to IMH. This explains the phenotype in rpe65-/- mice of by is normal in Redmond T.M. D.R. 2003; PubMed Scopus Google Scholar). Rpe65 to atRE, IMH is for substrate and no is accumulate at to the in wild-type RPE to K. U. S. A. PubMed Scopus Google Scholar). The the and as which the following K. U. S. A. PubMed Scopus Google and are in rpe65-/- RPE by (10.Redmond T.M. Yu S. Lee E. Bok D. Hamasaki D. Chen N. Goletz P. Ma J.X. Crouch R.K. Pfeifer K. Nat. Genet. 1998; 20: 344-351Crossref PubMed Scopus (815) Google Scholar). Rpe65 is present in at in RPE cells J. Redmond T.M. Crouch R.K. Biophys. 1998; PubMed Scopus Google Scholar, Crouch R.K. G. Ma J. Google Scholar). and have been shown to contain and in in addition to RPE N. G. Full Text Full Text PDF PubMed Scopus Google Scholar, Am. J. 1989; Google Scholar, G. A. A. Biochem. PubMed Scopus Google Scholar). The cellular of retinyl esters within the is not a pathway that was in and N. G. Full Text Full Text PDF PubMed Scopus Google Scholar). in that that esters accumulate in cells. it is that contain of atRE, which may be by Rpe65. The presence of Rpe65 in may the of function in rpe65-/- mice C. A. F. Yu S. Redmond T.M. Nat. Genet. PubMed Scopus Google Scholar, B. P. PubMed Scopus Google Scholar). Rpe65 was shown to with a of all-trans-retinyl C. N. K. Rando R.R. Biochemistry. 2003; 42: PubMed Scopus Google Scholar). This was by addition of and all-trans-retinyl with fatty acyl esters of as atRP was not data with the results In we have shown that Rpe65 is a binding protein for This binding function to atRE, which are to membranes and We have shown that Rpe65 is for IMH activity but has no intrinsic isomerase activity, suggesting that Rpe65 substrate to IMH. We for Redmond for rpe65-/- knockout mice, and Crouch for of

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