Retinal pigment epithelium 65 kDa protein (RPE65): An update

Vertebrate vision critically depends on an 11-cis-retinoid renewal system known as the visual cycle. At the heart of this metabolic pathway is an enzyme known as retinal pigment epithelium 65 kDa protein (RPE65), which catalyzes an unusual, possibly biochemically unique, reaction consisting of a coupled all-trans-retinyl ester hydrolysis and alkene geometric isomerization to produce 11-cis-retinol. Early work on this isomerohydrolase demonstrated its membership to the carotenoid cleavage dioxygenase superfamily and its essentiality for 11-cis-retinal production in the vertebrate retina. Three independent studies published in 2005 established RPE65 as the actual isomerohydrolase instead of a retinoid-binding protein as previously believed. Since the last devoted review of RPE65 enzymology appeared in this journal, major advances have been made in a number of areas including our understanding of the mechanistic details of RPE65 isomerohydrolase activity, its phylogenetic origins, the relationship of its membrane binding affinity to its catalytic activity, its role in visual chromophore production for rods and cones, its modulation by macromolecules and small molecules, and the involvement of RPE65 mutations in the development of retinal diseases. In this article, I will review these areas of progress with the goal of integrating results from the varied experimental approaches to provide a comprehensive picture of RPE65 biochemistry. Key outstanding questions that may prove to be fruitful future research pursuits will also be highlighted.     

Dynamic processes of visual transduction

Rhodopsin is one of those rare macromolecules whose inherent chromophore, 11-cis retinaldehyde, allows one to naturally observe triggered macromolecular changes on the timescale of picoseconds to minutes. Investigations of these molecular processes have been carried out with laser monochromatic light under conditions where the photon flux used for photolysis was carefully measured. The formation of bleaching intermediates has been examined as a function of fluence. Under conditions where the formation of intermediates is unaffected by photon reversal the following observations hold: Upon the absorption of a photon, the initial photochemical event results in production of metastable bathorhodopsin within 6 psec. Artificial rhodopsin regenerated with 9-cis retinal forms a distinct bathorhodopsin which must reflect distortions at the active site differing from those generated with 11-cis retinal. Bathorhodopsin thermally decays through lumirhodopsin and meta I-rhodopsin, to meta II-rhodopsin through a series of coupled equilibria. The final meta I-meta II equilibrium is stable for seconds. The process provides a unique model for utilization of energy to drive (trigger) a biological cascade of events.     

Retinol dehydrogenases (RDHs) in the visual cycle

The isomerization of 11-cis retinal to all-trans retinal in photoreceptors is the first step in vision. For photoreceptors to function in constant light, the all-trans retinal must be converted back to 11-cis retinal via the enzymatic steps of the visual cycle. Within this cycle, all-trans retinal is reduced to all-trans retinol in photoreceptors and transported to the retinal pigment epithelium (RPE). In the RPE, all-trans retinol is converted to 11-cis retinol, and in the final enzymatic step, 11-cis retinol is oxidized to 11-cis retinal. The first and last steps of the classical visual cycle are reduction and oxidation reactions, respectively, that utilize retinol dehydrogenase (RDH) enzymes. The visual cycle RDHs have been extensively studied, but because multiple RDHs are capable of catalyzing each step, the exact RDHs responsible for each reaction remain unknown. Within rods, RDH8 is largely responsible for the reduction of all-trans retinal with possible assistance from RDH12. retSDR1 is thought to reduce all-trans retinal in cones. In the RPE, the oxidation of 11-cis retinol is carried out by RDH5 with possible help from RDH11 and RDH10. Here, we review the characteristics of each RDH in vitro and the findings from knockout models that suggest the roles for each in the visual cycle.     

The colors of the visual pigment chromophores

11-cis retinal is the chromophore of visual pigments, whose absorption maxima (λ_max) lie between approximately 440 and 565 nm. To explain these color differences, Kropf and Hubbard (1958) postulated that retinal forms a protonated Schiff's base with an amino group on opsin and that the colors of the visual pigments depend on secondary interactions between the opsin and the π-electron system of the chromophore. This hypothesis has since been strengthened by: (1) Identification of the ?-amino group of a lysine residue as the chromophoric site on opsin; (2) theory and experiments showing that λ_max of protonated Schiff bases of retinal can lie at 490 nm and beyond; (3) changes in λ_max of rhodopsin resulting from conformational changes of opsin; (4) observations on squid retinochrome, a retinylidene chromoprotein whose λ_max can be changed reversibly by reagents which interact with the protein.     

Advances in understanding the molecular basis of the first steps in color vision

Serving as one of our primary environmental inputs, vision is the most sophisticated sensory system in humans. Here, we present recent findings derived from energetics, genetics and physiology that provide a more advanced understanding of color perception in mammals. Energetics of cis–trans isomerization of 11-cis-retinal accounts for color perception in the narrow region of the electromagnetic spectrum and how human eyes can absorb light in the near infrared (IR) range. Structural homology models of visual pigments reveal complex interactions of the protein moieties with the light sensitive chromophore 11-cis-retinal and that certain color blinding mutations impair secondary structural elements of these G protein-coupled receptors (GPCRs). Finally, we identify unsolved critical aspects of color tuning that require future investigation.     

The distribution and proportions of vitamin A compounds during the visual cycle in the rat

Radioisotopes and t.l.c. were used to indirectly determine the distribution and proportions of Vitamin A compounds in the rat eye. In the dark adapted eye there are Vitamin A compounds in the pigment epithelium; these compounds are likely within the rod outer segment discs being digested there. During light adaptation. 11-cis retinal decreases, alltrans retinal increases and is reduced to retinol, and retinol is esterified to fatty acids in the retina and in the pigment epithelium. Essentially the reverse occurs during dark adaptation. It is suggested that the metabolism of Vitamin A compounds in the eye functions to return chromophore to the newly synthesized visual pigment at the base of the rod outer segment.     

Membrane-binding and enzymatic properties of RPE65

Regeneration of visual pigments is essential for sustained visual function. Although the requirement for non-photochemical regeneration of the visual chromophore, 11-cis-retinal, was recognized early on, it was only recently that the trans to cis retinoid isomerase activity required for this process was assigned to a specific protein, a microsomal membrane enzyme called RPE65. In this review, we outline progress that has been made in the functional characterization of RPE65. We then discuss general concepts related to protein–membrane interactions and the mechanism of the retinoid isomerization reaction and describe some of the important biochemical and structural features of RPE65 with respect to its membrane-binding and enzymatic properties.     

Reconstitution of squid and cattle rhodopsin by the use of metaretinochrome in their respective membranes

Retinochrome is, even in membranes, converted to metaretinochrome by exposure to orange light. Upon incubation of metaretinochrome in membranes with cattle opsin in rod outer segment membranes, cattle rhodopsin is reconstituted in the dark. When opsin is present in molar excess to metaretinochrome, about 80% of the prosthetic retinal of retinochrome present initially is utilized for the reconstitution of cattle rhodopsin. One reason why all of the prosthetic retinal is not used for the rhodopsin reconstitution is that metaretinochrome transforms slowly to retinochrome during incubation in the dark and another is that metaretinochrome is in a photoequilibrium mixture with a trace of retinochrome after exposure to orange light.Squid rhodopsin is reconstituted when a mixture of metaretinochrome and squid opsin in their respective membranes is incubated in the dark. The reconstituted rhodopsin is converted to acid or alkaline metarhodopsin by exposure to orange light at neutral or alkaline pH, respectively.Three possible mechanisms for the transference of 11-cis retinal from metaretinochrome in a membrane to opsin in a different membrane were considered: (1) the migration of 11-cis retinal through an aqueous medium between the separate membranes, (2) the migration of 11-cis retinal from metaretinochrome to opsin in a fused membrane and (3) the transfer of retinal from membrane to membrane in close contact. In conclusion, the first two mechanisms were inapplicable and the third appeared to explain the present experimental findings.The possibility is discussed that the photoproduct of retinochrome may contribute to the rhodopsin synthesis as an effective donor of 11-cis retinal to opsin in the squid retina.     

Isolation and characterization of a water-soluble photopigment from honeybee compound eye

By preparative electrophoresis on polyacrylamide gel, a water-soluble light-sensitive pigment was isolated from honeybee heads incubated with tritiated vitamin A. Thin layer chromatography (TLC) on silica gel demonstrated the presence of retinal isomers as a chromophore of the bee pigment. It seems that all- trans-retinal is bound to the protein and an isomerization to 11- cis-retinal occurs in the light. Preliminary spectrophotometric measurements show that illumination of the extracted pigment is followed by a decrease in absorbance with a maximum at about 440 nm. A Schiff's base linkage is suggested for the binding between retinal and protein. Comparisons with visual pigments from other insects and with squid retinochrome are discussed, together with the possibility that the bee pigment could be involved in the turnover of the visual pigment of the honeybee eye.     

Leber congenital amaurosis due to RPE65 mutations and its treatment with gene therapy

Leber congenital amaurosis (LCA) is a rare hereditary retinal degeneration caused by mutations in more than a dozen genes. RPE65, one of these mutated genes, is highly expressed in the retinal pigment epithelium where it encodes the retinoid isomerase enzyme essential for the production of chromophore which forms the visual pigment in rod and cone photoreceptors of the retina. Congenital loss of chromophore production due to RPE65-deficiency together with progressive photoreceptor degeneration cause severe and progressive loss of vision. RPE65-associated LCA recently gained recognition outside of specialty ophthalmic circles due to early success achieved by three clinical trials of gene therapy using recombinant adeno-associated virus (AAV) vectors. The trials were built on multitude of basic, pre-clinical and clinical research defining the pathophysiology of the disease in human subjects and animal models, and demonstrating the proof-of-concept of gene (augmentation) therapy. Substantial gains in visual function of clinical trial participants provided evidence for physiologically relevant biological activity resulting from a newly introduced gene. This article reviews the current knowledge on retinal degeneration and visual dysfunction in animal models and human patients with RPE65 disease, and examines the consequences of gene therapy in terms of improvement of vision reported.     

On the rhodopsin cycle

The particulate oxido-reductase of cattle rod outer segment membranes, most likely involved in the rhodopsin cycle, appears to be quite stereospecific. It is shown that 11-cis-retinol is only oxidized by this enzyme after previous isomerization, since incubation with 11-cis-retinol and NADP⁺ only yields all-trans-retinal and a minor amount of the 13-cis isomer. A simplified rhodopsin cycle taking into account these observations, is proposed in which isomerization to the 11-cis configuration, essential for visual pigment regeneration, involves only all-trans-retinal in the rod outer segment.     

Biochemical analysis of a rhodopsin photoactivatable GFP fusion as a model of G-protein coupled receptor transport

Rhodopsin is trafficked to the rod outer segment of vertebrate rod cells with high fidelity. When rhodopsin transport is disrupted retinal photoreceptors apoptose, resulting in the blinding disease autosomal dominant retinitis pigmentosa. Herein, we introduce rhodopsin-photoactivatable GFP-1D4 (rhodopsin-paGFP-1D4) for the purposes of monitoring rhodopsin transport in living cells. Rhodopsin-paGFP-1D4 contains photoactivatable GFP (paGFP) fused to rhodopsin’s C-terminus and the last eight amino acids of rhodopsin (1D4) appended to the C-terminus of paGFP. The fusion protein binds the chromophore 11-cis retinal and photoisomerizes upon light activation similarly to rhodopsin. It activates the G-protein transducin with similar kinetics as does rhodopsin. Rhodopsin-paGFP-1D4 localizes to the same compartments, the primary cilium in cultured IMCD cells and the outer segment of rod cells, as rhodopsin in vitro and in vivo. This enables its use as a model of rhodopsin transport and details the importance of a free rhodopsin C-terminus in rod cell localization and health.     

The isomeric configuration of the bacteriorhodopsin chromophore

Oesterhelt and Stoeckenius in 1971 found a pigment in Halobacterium halobium which they called bacteriorhodopsin because it resembles rhodopsin in many aspects. The isomeric configurations of its chromophore were studied by thin layer chromatography of the retinal and of the retinal oxime derivatives. The dark form of bacteriorhodopsin (R₅₆₀) contains 13- cis retinal, in contrast to the 11- cis retinal of rhodopsin. Both the R₅₇₀ and the M₄₁₂ forms of bacteriorhodopsin were found to contain all- trans retinal. Implications of the different isomeric forms found in bacteriorhodopsin are discussed.     

The effect of retinal isomers on the VER and ERG of vitamin A deprived rats

Male weanling Long-Evans and Sprague-Dawley rats were administered a vitamin A deficient diet and the electroretinogram (ERG) and/or visual evoked response (VER) were recorded weekly. After 6–10 weeks, when the animal showed a greatly decreased VER and ERG, the animal was injected intraperitoneally with a retinal isomer. Following dark adaptation for 18 hr, the ERG and VER were recorded. The rat was then sacrificed and the retina dissected. The retina was used for either solubilizing the visual pigment in 2% Ammonyx LO or extracting the retinals in methylene chloride. The visual pigments were characterized by the absorbance spectra and the retinal isomers were identified by high pressure liquid Chromatography (HPLC). The physiologically occurring isomers of retinal (11- cis and all- trans) are incorporated in the retina of deprived animals and the VER and ERG amplitudes for a given light intensity, diminished as a result of vitamin A deprivation, are restored to their normal values. 9- cis retinal is also incorporated into the retina, as shown by extraction of the retina with organic solvent and identification of the isomers by HPLC; a pigment is formed which has a λ_max blue-shifted from that of rhodopsin. The ERG and VER amplitudes of deprived rats injected with 9- cis retinal are restored to normal. No incorporation of 13- cis retinal into the retina is observed and the ERG and VER amplitudes are not significantly affected.     

Can isorhodopsin be produced in the living rat?

This experiment is an attempt to produce isorhodopsin in the living rat by administering, to a vitamin A-deficient animal, pure 9-cis retinaldehyde. The crystalline material dissolved in soybean oil was given by stomach tube or, dissolved in ethanol, was injected subcutaneously. In both cases the visual deficiency, as monitored by the electroretinographic threshold, was repaired in a few days and visual pigment was restored to the retina. This pigment was normal rat rhodopsin without any evidence of isorhodopsin. Another isomer, 13-cis retinal, which in vitro does not combine with opsin to form a photopigment, gave the same result. The metabolic pathways whereby these two isomers lead to the production of 11-cis retinal are unknown. Some of the possibilities are pointed out of what could be done if some way were found of producing an “isorhodopsin rat”.     

Halide control of color of the chicken cone pigment iodopsin

The chicken retina contains rhodopsin, a rod cell visual pigment and iodopsin, which is believed to be a cone pigment. The spectrum of iodopsin is controlled by the binding of a halide ion. At physiological salt concentrations iodopsin is in the long wavelength form (562 nm); if chloride is removed rigorously the spectrum shifts to 520 nm. At intermediate chloride concentrations the two forms exist in equilibrium. the chloride-depleted form can be titrated back to the original long wavelength species with half saturation at 1·7 mm-chloride. The spectral shift is observed either in detergent solubilized preparations or in the native membrane, sonicated to reduce light scattering. Bromide can replace chloride, titrating iodopsin to a very similar long wavelength form with a very similar apparent binding constant. Fluoride and iodide cannot shift iodopsin to the long wavelength form. Changes of cation seem to have no effect. In the same system there are no changes of the rhodopsin spectrum with changes of halide concentration. Both the long wavelength form and the short wavelength form of iodopsin regenerate rapidly after bleaching and subsequent addition of 11-cis retinal. Chloride binding protects the iodopsin chromophore from hydroxylamine; while both forms are attacked by hydroxylamine, the chloride-free form is attacked much more rapidly. This fact and the large spectral shift are consistent with but do not prove an anion binding site very close to the retinal chromophore.     

Vitamin A activates rhodopsin and sensitizes it to ultraviolet light

The visual pigment, rhodopsin, consists of opsin protein with 11-cis retinal chromophore, covalently bound. Light activates rhodopsin by isomerizing the chromophore to the all-trans conformation. The activated rhodopsin sets in motion a biochemical cascade that evokes an electrical response by the photoreceptor. All-trans retinal is eventually released from the opsin and reduced to vitamin A. Rod and cone photoreceptors contain vast amounts of rhodopsin, so after exposure to bright light, the concentration of vitamin A can reach relatively high levels within their outer segments. Since a retinal analog, β-ionone, is capable of activating some types of visual pigments, we tested whether vitamin A might produce a similar effect. In single-cell recordings from isolated dark-adapted salamander green-sensitive rods, exogenously applied vitamin A decreased circulating current and flash sensitivity and accelerated flash response kinetics. These changes resembled those produced by exposure of rods to steady light. Microspectrophotometric measurements showed that vitamin A accumulated in the outer segments and binding of vitamin A to rhodopsin was confirmed in in vitro assays. In addition, vitamin A improved the sensitivity of photoreceptors to ultraviolet (UV) light. Apparently, the energy of a UV photon absorbed by vitamin A transferred by a radiationless process to the 11-cis retinal chromophore of rhodopsin, which subsequently isomerized. Therefore, our results suggest that vitamin A binds to rhodopsin at an allosteric binding site distinct from the chromophore binding pocket for 11-cis retinal to activate the rhodopsin, and that it serves as a sensitizing chromophore for UV light.     

Retinyl and 3-dehydroretinyl esters in the crayfish retina

We have studied chemical nature and localization of retinyl esters stored in the retina of the crayfish, Procambarus clarkii, which has a rhodopsin-porphyropsin visual pigment system. The crayfish kept at 10 degrees C in the constant dark had 3-dehydroretinal along with retinal in the retina as the chromophore of visual pigment. Both retinyl and 3-dehydroretinyl esters were found in the retina, more than 95% of them in the 11-cis configuration. Of three kinds of fatty acid detected in the esters, the major component (about 80%) was the polyunsaturated fatty acid, docosahexaenoic acid (C22 = 6). Observations with electron and fluorescence microscopy and the results of fractionation experiments showed that the esters were stored in photoreceptor cells as oil droplets. The ratio of 3-dehydroretinal/retinal as visual pigment chromophore was always higher than that of 3-dehydroretinol/retinol in the stored esters. This result suggests a mechanism of selective utilization of 3-dehydroretinol for the chromophore of visual pigment in the crayfish retina.     

Interactions of retinol-binding protein with various chromophores and with thyroxine-binding protein. A model for visual pigments

The properties of retinol-binding protein from human serum were compared with those of rhodopsin in an attempt to learn more about the role of protein in visual pigments. In the serum, retinol-binding protein (mol wt. 21 000) tightly binds one molecule of all-trans-retinol and one molecule of the tetrameric protein prealbumin (mol. wt. of tetramer, 54 000). Apo-retinol binding protein was found to be completely dissociated from prealbumin under conditions in which native retinol-retinol-binding protein and reconstituted all-trans-retinol-retinol-binding protein were tightly bound. This difference in binding to prealbumin between apo- and holo-retinol-binding protein is due most probably to different conformations of the apo- and holo-protein. By analogy, it is suggested that the incorporation of the retinal chromophore into apo-visual pigment might act as recognition marker for the incorporation of visual pigment into the photoreceptor disc membrane.Apo-retinol binding protein was shown to form stable 1 : 1 molar complexes with retinols, retinals, retinoic acid, retinyl acetate and retinyl oxime, but not with retinyl palmitate. Only the retinol isomer and retinoic acid-retinol-binding protein complexes were bound to prealbumin at physiological ionic strength, while the other chromophores-retinol·binding protein complexes were not. The various chromophores were all bound to the same site on the retinol-binding chromophore, the binding was noncovalent and irreversible under normal physiological conditions.All the chromophores-retinol-binding protein complexes showed a large induced optical activity of the chromophore absorption band upon binding to the protein. The rotatory strength of the circular dichroism bands of the various chromophore retinol-binding protein complexes was of the same order of magnitude as that of rhodopsin. The binding of retinol and retinal to bovine serum albumin did not produce optical activity in the chromophore absorption band.