A protein called RPE65 performs a key role in the trans-cis isomerization of retinol in the retinal pigment epithelium of the eye. The palmitoylation of RPE65 serves to switch off the visual cycle in darkness and to switch it on in the light.
Key words: retina; retinal pigment epithelium; vitamin A; all-trans-retinyl palmitate; all-trans-retinol; 11-cis-retinol; isomerohydrolase; lethicin; lecithin retinol acyl transferase
© 2005 International Life Sciences Institute
Since 1993,1 it has been known that the major protein of the microsomal membranes in the retinal pigment epithelium (RPE) is RPE65. Mutations in the RPE65 gene are responsible for a number of congenital eye diseases, such as Leber's congenital amaurosis,2 a severe and relatively common recessive disease resulting in blindness at birth, as well as some forms of retinitis pigmentosa.3 However, the biochemical function of RPE65 has been unknown until recently.
In the visual cycle (Figure 1), a photon triggers the transformation of rhodopsin in the retinal outer segment to metarhodopsin II, involving the isomerization of the rhodopsin-bound 11-cis-retinal (11cRAL) to all-transretinal (atRAL), an event that sends the light signal from the retina to the brain. The atRAL dissociates from rhodopsin, leaving behind apo-rhodopsin (also known as opsin). atRAL is reduced to all-trans-retinol (atROL) and transfered to the RPE, where it is esterified by dipalmitylphosphorylcholine (DPPC, also known as lecithin) to form all-trans-retinyl palmitate (atRP), the storage form of vitamin A, by the enzyme lecithin retinol acyl transferase (LRAT). AtRP serves as a substrate for the enzyme isomerohydrolase (IMH), that simultaneously hydrolyzes and isomerizes it to 11-cis-retinol (11cROL). This compound is oxidized to 11cRAL, which binds avidly to apo-rhodopsin to form rhodopsin, thereby completing the visual cycle.
The importance of the protein RPE65 in the visual cycle became apparent as a result of the work of Redmond et al.,4 who generated a RPE65-knockout mouse. RPE65-knockout mice are morphologically normal, but their retinas completely lack 11-cis-retinoids and therefore cannot form rhodopsin. The outer segments of their retinas contain only apo-rhodopsin. They accumulate atRP in their RPE.
Ma et al.5 undertook the isolation and purification of RPE65. Starting with human RPE65-cDNA, they made human RPE65-RNA, which was amplified in the baculovirus expression system to form recombinant human RPE65-DNA. This was transfected into sf9 (insect) cells in culture, from which a membrane-associated and a cytosolic form of RPE65 were isolated and purified on a nickel-nitrilotriacetic acid resin column. The membrane-associated mRPE65 (MW = 61,961 ± 150 D) and the cytosolic sRPE65 (MW = 61,161 ± 80 D) were different as a result of posttranslational modification.
Using the atRP analog all-trans-retinylchloroacetate, Jahng et al.6 showed that RPE65 binds atRP specifically and selectively at low concentrations of atRP. RPE65 is therefore the first retinyl ester-binding protein discovered, one molecule of RPE65 binding two molecules of atRP. The authors6 suggested that it served the mobilization of the atRP to assist in its intermembranous transfer, in view of the extreme hydrophobicity of the retinoid esters.
Further work by the same researchers7 used the quenching of protein fluorescence, a consequence of the binding of retinoids to protein, to study the reversible binding of atRP to RPE65. With an excitation wave-length at 275 nm and an emission wavelength at 340 nm, an increasing concentration of atRP in the presence of RPE65 resulted in an exponential decay of protein fluorescence. This made possible the calculation of the dissociation constant of the reversible binding of atRP to RPE65 (K^sub d^ = 20 pM). AtRP binding was not only strong but also specific, as atROL bound weakly (K^sub d^ = 10.8 nM), and stereospecific, as 11cRP also bound weakly (K^sub d^ = 14 nM).
Earlier work8 suggested an interaction of LRAT and RPE65. Therefore, it appeared that atROL in the RPE meets a triple combination of proteins: LRAT-RPE65-IMH. The first component, LRAT, palmitoylates atROL to form atRP; this is picked up by RPE65 and presented to IMH, which simultaneously hydrolyzes and isomerizes it to form 11cROL. In the binding of atRP, RPE65 functions stoichiometrcally, not catalytically.
Further details of the reactions of RPE65 were elucidated by Mata et al.9 They not only showed (as had Gollapalli et al.7 previously) that RPE65 bound atRP specifically, but also explored the function of RPE65 in solubilizing and mobilizing the hydrophobic atRP to enable it to react with IMH.
Liposomes (artificial vesicles) were prepared from partially purified RPE65 and a mixture of phospholipids. Using fluorescent probes, the authors9 observed interaction of RPE65 with atRP added to the liposomes. The important finding was that RPE65, present in the lipid membrane of the liposomes, interacted with and bound atRP rapidly, leading to saturation within 15 minutes. The RPE65 present in the aqueous interior of the liposomes reacted with the atRP slowly, as saturation of the binding did not occur until after 30 minutes. The membrane-associated and the aqueous-associated forms of RPE65 in liposomes clearly corresponded to the two forms of microsomes described by Ma et al.5 mentioned above, a higher-molecular weight form associated with microsomal membranes (mRPE65) and a lower-molecular weight form that is soluble in the aqueous medium (sRPE65).
Mata et al.9 concluded that "RPE65 strongly stimulates uptake of atRP into membranes and effect the transfer of atRP into the aqueous interior." Therefore, the function of RPE65 is not only to bind and stabilize atRP, but also to extract it from lipid membranes of the RPE microsomes and to deliver it to the isomerizing enzyme IMH.
Mata et al.9 then demonstrated the role of RPE65 in the isomerization reaction by incubation of solubilized RPE microsomes from wild-type mice with atRP, resulting in the transformation of atRP to 11cROL. Incubating microsomes from RPE65-knockout mice under the same conditions produced zero 11cROL; when RPE65 was then added to the reaction, isomerization occurred and as much 11cROL was formed as in the wild-type reaction. Therefore, IMH activity was absolutely dependent upon the presence of RPE65, even though RPE65 had no enzyme activity itself.
LRAT, therefore, is a bifunctional enzyme in that it can use either palmitoylated mRPE65 or DPPC as a palmitic acid donor in the esterification of retinol. The difference between mRPE65 and sRPE65 is in their state of palmitoylation, the mRPE65 being mostly palmitoylated.
In summary (Figure 2), in the dark, when 11cRAL is not needed for rhodopsin formation, 11cROL is mopped up by the palmitoylated mRPE65 and converted to 11cRP, a storage form for vitamin A and sRPE65 (reactions 7 and 8, above). Thus, accumulation of 11cROL drives the conversion of mRPE65 to sRPE65 and stops further reactions of the visual cycle through removal of 11cROL in the form of 11cRP.
In the light, rhodopsin dissociates, yielding atRAL, which is reduced to atROL. This accumulates and is bound by sRPE65 and converted to atRP by LRAT (reactions 3 and 4, above). The atRP binds to mRPE65 and is presented to IMH to be converted to 11cROL and palmitoylated mRPE65 (reactions 1 and 2, above). Thus, the formation of atROL in the light drives the conversion of sRPE65 to mRPE65 and thereby promotes the continued operation of the visual cycle.
1. Hamel CP, Tsilou E, Harris E, et al. A developmentally regulated microsomal protein specific for the pigment epithelium of the vertebrate retina. J Neurosci Res. 1993;34:414-425.
2. Marlhens F, Bareil C, Griffoin JM, et al. Mutations in RPE65 cause Leber's congenital amaurosis. Nat Genet. 1997;17:139-141.
3. Morimura H, Fishman GA, Grover SA, Fulton AB, Berson EL, Dryja TP. Mutations in the RPE65 gene in patients with autosomal recessive retinitis pigmentosa or leber congenital amourosis. Proc Natl Acad Sci USA. 1998;95:3088-3093.
4. Redmond TM, Yu S, Lee E, et al. RPE65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nat Genet. 1998;20:344-351.
5. Ma J, Zhang J, Othersen KL, et al. Expression, purification, and MALDI analysis of RPE65. Invest Ophthalmol Vis Sci. 2001;42:1429-1435.
6. Jahng WJ, David C, Nesnas N, Nakanishi K, Rando RR. A cleavable affinity biotinylating agent reveals a retinoid binding role for RPE65. Biochemistry. 2003; 42:6159-6168.
7. Gollapalli DR, Maiti P, Rando RR. RPE65 operates in the vertebrate visual cycle by stereospecifically binding all-trans-retinyl esters. Biochemistry. 2003; 42:11824-11830.
8. West KA, Van L, Shadrach K, et al. Protein database, human retinal pigment epithelium. Mol Cell Proteomics. 2003;2:37-49.
9. Mata NL, Moghrabit WN, Lee JS, et al. RPE65 is a retinyl ester binding protein that presents insoluble substrate to the isomerase in retinal pigment epithelial cells. J Biol Chem. 2004;279:635-643.
10. Xue L, Gollapalli DR, Maiti P, Jahng WJ, Rando RR. A palmitoylation switch mechanism in the regulation of the visual cycle. Cell. 2004;117:761-771.
George Wolf, DPhil
Dr. Wolf is with the Department of Nutritional Sciences and Toxicology, University of California, Berkeley.
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Copyright International Life Sciences Institute and Nutrition Foundation Mar 2005
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