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(Investigative Ophthalmology and Visual Science. 2002;43:1604-1609.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Identification of the RPE65 Protein in Mammalian Cone Photoreceptors

Sergey L. Znoiko, Rosalie K. Crouch, Gennadiy Moiseyev and Jian-xing Ma

From the Storm Eye Institute, Medical University of South Carolina, Charleston, South Carolina.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians.

METHODS. The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections.

RESULTS. RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested.

CONCLUSIONS. RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
RPE65 is a protein that is expressed abundantly in the retinal pigment epithelium (RPE).1 2 We have previously reported that RPE65 is expressed in salamander cone photoreceptors. The RPE65 mRNA was detected by single-cell RT-PCR in isolated salamander cones, but not in rods.3 The protein has also been reported to be expressed in the iris,4 ARPE-19 cells,5 transformed kidney cells, and some malignant kidney tumors.6

The physiological function of RPE65 is presently uncertain, but evidence has suggested that it has an important role in retinoid metabolism.7 8 In the RPE65-knockout mouse, disturbed retinoid profiles have been observed in the RPE and retina. The 11-cis retinoids are absent in the retina, and thus rhodopsin regeneration is impaired, although free opsin is available.8 In contrast, retinyl ester is accumulated in the RPE of the knockout mouse. Ester saponification has shown that all the retinyl ester is in the all-trans form, whereas the 11-cis ester is absent.8 These results indicate that in the knockout mouse, the regeneration of 11-cis retinal is blocked at the isomerization–hydrolysis step, supporting the hypothesis that this protein is essential for isomerohydrolase activity.8 Recently, it has been demonstrated that supplementation of 9-cis retinal can partially reverse the functional phenotypes of the knockout retina,9 indicating that the absence of 11-cis retinal is responsible for the functional abnormalities of the knockout retina. These findings support the role of RPE65 in the visual cycle of retinoids.

Mutations in the RPE65 gene are associated with several forms of inherited retinal dystrophies, including retinitis pigmentosa, Leber congenital amaurosis, autosomal recessive childhood-onset severe retinal dystrophy and early-onset severe rod–cone dystrophies.10 11 12 13 The homozygous RPE65-knockout mouse (RPE65-/-) has shown a diminished rod response in the ERG and photoreceptor degeneration.8 These observations suggest that an intact RPE65 protein is essential for maintaining normal vision. Recent results regarding the rescue of the RPE65-/- dog using a recombinant adeno-associated virus carrying wild-type RPE6514 provide further evidence for the essential role of this protein.

Rod photoreceptors are known to rely on the RPE to recycle 11-cis retinal for rhodopsin regeneration.15 However, physiological evidence has shown that cones are distinct from rods in retinoid transporting and processing.16 17 Cones have been suggested to have an alternative retinoid–metabolic pathway independent of the RPE. Our previous studies have identified the RPE65 mRNA in salamander cones, but not in rods photoreceptors.3 The purpose of this study was to determine whether RPE65 is expressed at the protein level in cones of mammals and other amphibians.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Animals and Tissues
Three animals of each species were used per analysis. BALB/c, 129Sv, and C57/Bl6 mice (4 months old) were purchased from Harlan-Sprague Dawley (Indianapolis, IN). Homozygous RPE65-knockout (RPE65-/-) mice (1 and 4 months old) were obtained from Michael Redmond of the National Eye Institute (Bethesda, MD). Bovine eyes were purchased from a local abattoir and processed within 12 hours of death. Eyes of New Zealand White rabbits (5 months old) were obtained from the Antibody Center at the Medical University of South Carolina (Charleston, SC). Xenopus laevis (5–7 cm long) were purchased from the Charles Sullivan Company, Inc. (Nashville, TN).

All animals were kept in a 12-hour light–dark cycle. Care, use, and treatment of all animals in this study were in strict agreement with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, as well as the guidelines set forth in the Care and Use of Laboratory Animals by the Medical University of South Carolina.

RPE65 Antibody
A polyclonal antibody was raised using a synthetic peptide of bovine RPE65, NFITKINPETLETIK (residues 150-162 of RPE65) synthesized in the Biotechnology Resource Laboratory at the Medical University of South Carolina. The peptide was conjugated to keyhole limpet hemocyanin (KLH) protein. Rabbits were subcutaneously injected with an emulsion of 0.3 mg of the peptide-KLH and complete Freund’s adjuvant (Gibco-BRL, Gaithersburg, MD) and intramuscularly boosted with 0.3 mg of the same emulsion at 3-week intervals. After significant immune responses had developed (4 months after the first injection), the rabbits were killed, and the whole serum was collected. Specific antibody to the RPE65 peptide was purified by passing the serum through a column of the epitope peptide coupled to agarose beads (AminoLink; Pierce, Rockford, IL) according to a protocol recommended by the manufacturer. The antibody was eluted and stored at -70°C.

Western Blot Analysis
BALB/c and RPE65-/- mouse eyes were dissected to remove the anterior part of the eye and the retina. The remaining eyecup was homogenized by sonication in PBS (pH 7.4) three times for 30 seconds each, on ice, and protein concentration was determined by protein assay (Bio-Rad, Hercules, CA). Samples (50 µg total protein) were resolved with SDS-PAGE (8%–16% Tris-glycine gel) and electrotransferred to a nitrocellulose membrane (Hybond-ECL; Amersham, Piscataway, NJ), according to the manufacturer’s instructions. The membrane was blocked with 5% (wt/vol) blocking reagent (Blotto; Santa Cruz Biotechnology, Santa Cruz, CA) in TBST (20 mM Tris-HCl, [pH 7.6], 137 mM NaCl, 0.1% Tween-20) for 2 hours at room temperature and subsequently incubated overnight at 4°C with a 1:1000 dilution of the anti-RPE65 peptide antibody. After three 15-minute washes in the blocking reagent, the membrane was incubated with a horseradish peroxidase–conjugated donkey anti-rabbit IgG (Amersham) at a 1:7500 dilution in blocking reagent for 3 hours. The membrane was washed four times in TBST to remove any unbound antibody, and bands were detected using the enhanced chemiluminescence (ECL) Western blot analysis kit (Amersham) according to manufacturer’s instructions.

Immunohistochemistry
Fixation.
The eyes were dissected and placed in cold PBS (pH 7.2). The cornea and sclera were separated from the choroid-RPE-retina-lens complex. The RPE–choroid layer was detached from the retina–lens complex, which was fixed in cold 5% formaldehyde solution in PBS for 4 hours at 4°C. After washing with PBS (30 minutes at 4°C, three times), the retina was separated from the lens and vitreous body before labeling.

Labeling of Flatmounted Retina.
All the following steps were performed on ice or at 4°C. The retina was blocked with blocking solution (1% BSA and 0.05% saponin in PBS) for 40 minutes. The anti-RPE65 antibody and peanut agglutinin (PNA) lectin (FITC-conjugated peanut lectin 0.2 mg/mL Arachis hypogaea; Sigma, St. Louis, MO) were dissolved in the blocking solution at 1:100 dilution and added to the retina. In the negative control, no anti-RPE65 antibody was added. After 12 hours of incubation, the retina was washed three times in PBS (20 minutes each), and a secondary (Cy-3 conjugated donkey anti-rabbit IgG) antibody in blocking solution (1:200) was added and incubated for another 12 hours. The retina was washed three times in PBS and covered by a coverslip after the application of several drops of an antifade solution (ProLong; Molecular Probes, Eugene, OR).

Retina Section Preparation.
The labeled retina was transferred into an embedding medium (Tissue-Tek; Sakura Finetek USA, Inc., Torrance, CA) and frozen. Frozen sections (10 µm) were cut, counterstained with nuclear staining 4',6'-diamino-2-phenylindole (DAPI, dilactate; Molecular Probes), and covered by a coverslip for microscopic analysis.

Microscopic Analysis.
The samples were analyzed by fluorescence microscope (Axioplan II; Carl Zeiss Inc., Jena, Germany) equipped with differential interference contrast (DIC) in incident light components, 100-W mercury light source, FITC and rhodamine filters and 20x/0.50 and 100x/1.3 objective lens (Plan-Neofluar; Carl Zeiss Inc.). Images were captured with a digital camera, (Spot RT; Diagnostic Instruments, Inc., Sterling Heights, MI) attached to the microscope. The image capturing and processing were performed using the software provided (Spot software, ver. 3.0; Diagnostic Instruments, Inc.).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Anti-RPE65 Antibody Specificity
A polyclonal antibody against RPE65 was raised in rabbit as described in the Methods section. The specificity of the antibody was examined by Western blot analysis. The antibody recognized a single band with an apparent molecular weight of 65 kDa in the eyecup homogenate of the BALB/c mouse, but not in that of the RPE65-/- mouse (Fig. 1) , demonstrating the specificity of the antibody.



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  		Figure 1. Western blot analysis. Eyecup homogenates (50 µg total protein each) from BALB/c and RPE65-/- mice were blotted with the anti-RPE65 antibody. The antibody recognized a single band in the eyecup homogenate from the BALB/c but not in the eyecup from the RPE65-/- mice.

 
Identification of RPE65 in Mammalian Cones at the Protein Level
The anti-RPE65 antibody labeled a type of cell with cone-shaped outer segments in the photoreceptor layer in flatmounted retinas of all species examined, including mouse, cow, rabbit, and Xenopus laevis. The density and morphology of the stained cells suggest that they are cone photoreceptors (Fig. 2) . Staining was observed in the outer and inner segments and cell body of the cones. No cells were stained in these species when the primary antibody was absent (negative control, data not shown), demonstrating the specificity of the antibody labeling.



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  		Figure 2. Staining of cones by the anti-RPE65 antibody in multiple species. The flatmounted retinas of the (A) mouse, (B) cow, (C) rabbit, and (D) Xenopus laevis were stained with the anti-RPE65 antibody (dilution 1:100) followed by a Cy-3–conjugated anti-rabbit IgG antibody (dilution 1:200). Staining was observed in the outer segments (OS), the myoid area of the inner segment (ISm), which is known to contain the rough endoplasmic reticulum and Golgi complex, and the ellipsoid region of the inner segment (ISe). Note that some cells were viewed from the top, resulting in round spots, whereas other cells were viewed from the side. Magnification, x1000.

 
To further confirm the identity of labeled cones, labeled cells were viewed in the retinal sections from BALB/c mice. The anti-RPE65 antibody–labeled cells showed a typical cone morphology and were localized in the photoreceptor layer (Fig. 3) . The RPE65 signal was detected predominantly in the outer segments (Fig. 3A) . The RPE65-positive cells were also labeled with PNA lectin (Fig. 3C) . These results further confirm that the cells labeled by the anti-RPE65 antibody are indeed cones.



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  		Figure 3. Double staining of mouse retina sections with the anti-RPE65 antibody and PNA lectin. Mouse retina sections were double-labeled with the anti-RPE65 antibody followed by a Cy-3–conjugated secondary antibody (red) and the FITC-conjugated PNA lectin (green). The nuclei were counterstained with DAPI to show the location of the labeled cells in the retina (blue). (A) Filter showing staining with the anti-RPE65 antibody. (B) Staining with PNA lectin and DAPI in the same field. (C) A merged image of the anti-RPE65 antibody and PNA lectin staining. (D) An enlarged image of the boxed field in (C), showing double-stained cones. Arrows: the boundary between the outer nuclear layer (ONL) and inner nuclear layer (INL). Magnification, x1000.

 
Expression of RPE65 in All Types of Cones
Cells stained by the anti-RPE65 antibody were uniformly distributed over the retina of a 4-month-old BALB/c mouse (Fig. 4A) . To determine whether all cone types are labeled by the anti-RPE65 antibody, we performed double labeling with the anti-RPE65 antibody and PNA lectin in the mouse retina. Double staining with the anti-RPE65 antibody followed by Cy-3–conjugated secondary antibody and FITC-conjugated PNA lectin demonstrated that all the PNA lectin–positive cells were also labeled by the anti-RPE65 antibody in the flatmounted BALB/c mouse retina (Figs. 4B 4C 4D) . This result indicates that both types of cones in the mouse retina express RPE65, in that PNA lectin specifically binds to ß-gal(1->3)galNAc and has been shown to label both types of cone photoreceptors in the mouse.18 19 In the superimposed image of the double staining (Fig. 3D) , the RPE65-staining (red) was surrounded by the PNA lectin signal (green), which is known to be associated with the membrane of cones. This result supports the intracellular localization of RPE65 in cones and confirms that the RPE65-positive cells in the photoreceptor layer are indeed cones. The three mouse strains, 129Sv, BALB/c, and C57Bl/6, which are known to express varying amounts of RPE65 in the RPE,20 were examined. No differences were observed among the three strains in density of RPE65 positive cones in the flatmounted retina (data not shown).



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  		Figure 4. Double staining of cones with the anti-RPE65 antibody and PNA lectin in the flatmounted mouse retinas. Flatmounted retinas from the BALB/c and RPE65-/- mice were stained with the anti-RPE65 antibody followed by the Cy-3–conjugated secondary antibody (red) and FITC-conjugated PNA lectin (green). (A) Density of RPE65-positive cones. (B) Filter showing RPE65 staining only. (C) Lectin staining in the same field as in (B). (D) A merged image of the double staining. (E, F) Absence of the RPE65 staining in cones of RPE65-/- mice at ages of 1 (E) and 4 (F) months, although PNA lectin-positive cones exist in the retina. Magnification, x1000.

 
The Labeled Protein Encoded by the RPE65 Gene
Retinas from 1- and 4-month-old RPE65-/- mice were also double labeled with the anti-RPE65 antibody and PNA lectin under the same conditions as described for the BALB/c retinas. No cells were labeled by the anti-RPE65 antibody in RPE65-/- mice of both ages. However, PNA lectin-positive cones (green) were observed in these retinas (Figs. 4E 4F) . These cones showed both inner and outer segments stained by PNA lectin, suggesting that at least some cones survive in the knockout mouse at ages 1 and 4 months (Figs. 4E 4F) . The absence of RPE65 labeling in the RPE65-/- mouse indicates that the protein labeled by the anti-RPE65 antibody in cones of wild-type animals is encoded by the RPE65 gene, rather than by another unknown homologue of the RPE65 gene.


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
RPE65 plays an essential role in the processing of retinoids in the visual cycle. This study demonstrates the presence of the RPE65 protein in mammalian and Xenopus cone photoreceptors as well as in salamander cones, as reported earlier.3 The protein was not found in the rods of any of these species, further supporting the suggestion that rods and cones have distinct pathways for retinoid processing.

In previous studies, we have detected the RPE65 mRNA in salamander cones, by single cell RT-PCR.3 However, there are two questions that were not answered by these studies. First, it is not clear whether the RPE65 protein is expressed in cones, even though its mRNA is present. Second, it is unknown whether the expression of RPE65 in cones is a specific feature of amphibians—more specifically, of the salamander—or can be found in other species. The present study addressed these questions by means of immunohistochemistry with a specific antibody to RPE65. Because it is difficult to dissect the retina from RPE contamination, we could not use Western blot analysis to demonstrate reliably the presence of RPE65 protein in the retina. Immunohistochemistry detected the RPE65 protein in cones of all mammalian species analyzed in addition to amphibians. To confirm that the labeling was not due to cross-reactivity of the antibody with another protein, the RPE65-/- mouse was used as a negative control. The identity of stained cones was determined, not only by the morphology of the stained cells and their positions in the outer retina, but also by double labeling with PNA lectin, a specific marker for cone photoreceptors.18 19 21 22 In contrast, no staining was observed in rods of any of these species. This confirms that the difference between rods and cones in RPE65 expression is not limited to amphibians.

During the preparation of this manuscript, a paper by Seeliger et al.23 was published online (http://genetics.nature.com), in which they showed that cones in the retina sections from B6/129S mice are not stained by an anti-RPE65 antibody. However, our results in retina sections showed a specific staining of the cone outer segments, but not of the rods, by an affinity-purified anti-RPE65 antibody. The labeled cones were confirmed by double staining with PNA lectin. This RPE65 labeling provides a better cone morphology and definitive location of the labeled cells in the photoreceptor layer in the retina, when compared with the labeling in retina flatmounts. There are several factors that may explain the discrepancy between our results and that of Seeliger et al.23 First, our anti-RPE65 antibody was affinity-purified by using an RPE65 peptide epitope. This purified IgG generates more specific and stronger staining than does whole serum. Seeliger et al.23 did not specify which antibody was used for staining in their study. Because RPE65 levels in cones were substantially lower than that in the RPE, it may require a purified antibody to detect the protein in cones. Second, the labeling procedure could make a difference. They embedded the retina in agarose, whereas we used frozen sections. Third, we used BALB/c mice for RPE65 staining, because BALB/c mice have significantly higher RPE65 expression levels than do either 129S or B6 mice.20 Seeliger et al.23 used mice with the B6/129S background, which is expected to produce a lower RPE65 expression than BALB/c mice. The lower expression of RPE65 could make the detection of RPE65 in cones more difficult.

Most vertebrate retinas contain more than one type of cone, with most rodents, such as mouse and rabbit having two types of cones: medium (M) and short (S) wavelength sensitive.24 25 It has been demonstrated that in the mouse retina, the superior area is dominated by the M cones, whereas the inferior area contains almost all the S cones.26 In the present study, there was no variation in the density of the RPE65-positive cones between the superior and inferior areas of the mouse retina (Fig. 3A) . To confirm that both types of cones express RPE65, we double-labeled mouse retina with PNA lectin and the anti-RPE65 antibody, because PNA lectin has been shown to label both types of cones in mouse retinas. The results demonstrate that all lectin-positive cones in the mouse retina were also labeled by the anti-RPE65 antibody. Taken together, these observations show that RPE65 was expressed in both the M and S cones in the mouse retina.

Although the RPE65 gene does not have high homology with any known sequences, there is still a possibility that the labeled protein in cones is encoded by an unknown gene homologous to RPE65, rather than by the RPE65 gene itself. The present study examined this possibility in the retina from the RPE65-knockout mouse. Double labeling of the knockout retina demonstrated no RPE65 labeling in the retina, whereas the lectin-positive cones were present in the knockout mice at ages of 1 and 4 months, confirming that the labeled protein in cones of wild-type animals is indeed encoded by the RPE65 gene.

The RPE is the major source for 11-cis retinal in the visual cycle, at least in species with a rod-dominant retina (for review see Crouch et al.27 ). Although rods depend on the RPE for the supply of 11-cis retinal, it has been suggested that cones, at least those in amphibians, may have an alternative retinoid transporting and processing pathway independent of the RPE.28 29 Early studies have demonstrated a spontaneous recovery of cone sensitivity (but not of the rod sensitivity) after bleaching in the isolated retinas of turtle,30 frog,31 32 and rat.33 In isolated salamander photoreceptors, Jones et al.16 have demonstrated that the addition of 11-cis retinol can restore cone sensitivity but not rod sensitivity, suggesting the presence of 11-cis retinol dehydrogenase or its homologue in cones.34 Different from rods, salamander cones have the capacity to transport 11-cis retinal from the inner segment to the outer segment.17 These observations support the possibility that there are differences in retinoid processing and transporting systems between rods and cones of some species.

RPE65 is essential for the formation of 11-cis retinal. Previous studies have shown that the RPE of the RPE65-/- mouse fails to isomerize all-trans retinoid to 11-cis isomers.8 Our recent experiments showed that the addition of recombinant RPE65 to the tissue homogenate of RPE65-/- mouse eyecup restored the retinol isomerase activity in the knockout mouse eyecup, although RPE65 alone did not show any in vitro isomerase activity (Moiseyev G, unpublished data, 2000). This result suggests that RPE65 is essential, but not sufficient for isomerization of all-trans retinoids into 11-cis isomers and that RPE65 may require binding with its partners to show the isomerase activity. Recent results in the RPE65-deficient dog in which visual function was restored with a gene therapy approach14 further confirm the essential role of this protein. The exact function of the protein in cones as well as the RPE remains to be determined. The identification of the RPE65 protein in mammalian cones provides additional evidence that mammalian cones may contain a metabolic system that can isomerize retinoids.


   Acknowledgements
 
The authors thank T. Michael Redmond (Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, MD) for providing the RPE65-/- mice for this study.


   Footnotes
 
Supported by National Science Foundation Grant MCB9600772, National Eye Institute Grants EY12231, EY12600, and EY04939, a grant from Foundation Fighting Blindness, and an unrestricted grant from Research to Prevent Blindness, Inc. to the Department of Ophthalmology, Medical University of South Carolina.

Submitted for publication November 6, 2001; revised December 27, 2001; accepted January 10, 2002.

Commercial relationships policy: N.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Jian-xing Ma, Department of Ophthalmology, Medical University of South Carolina, 167 Ashley Avenue, Charleston, SC 29425; majx{at}musc.edu.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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