Rpe65 is the retinoid isomerase in bovine retinal pigment epithelium

Abstract

The first event in light perception is absorption of a photon by an opsin pigment, which induces isomerization of its 11-cis-retinaldehyde chromophore. Restoration of light sensitivity to the bleached opsin requires chemical regeneration of 11-cis-retinaldehyde through an enzymatic pathway called the visual cycle. The isomerase, which converts an all-trans-retinyl ester to 11-cis-retinol, has never been identified. Here, we performed an unbiased cDNA expression screen to identify this isomerase. We discovered that the isomerase is a previously characterized protein called Rpe65. We confirmed our identification of the isomerase by demonstrating catalytic activity in mammalian and insect cells that express Rpe65. Mutations in the human RPE65 gene cause a blinding disease of infancy called Leber congenital amaurosis. Rpe65 with the Leber-associated C330Y and Y368H substitutions had no isomerase activity. Identification of Rpe65 as the isomerase explains the phenotypes in rpe65-/- knockout mice and in humans with Leber congenital amaurosis.

Figure 1

Visual Cycle for Regeneration of Rhodopsin The light-sensitive protein in rods is rhodopsin, in the membranes of the outer segment. The 11-cis-retinaldehyde (11cRAL) chromophore is coupled to rhodopsin through a protonated Schiff-base linkage. Absorption of a photon (hv) induces 11-cis to all-trans isomerization of retinaldehyde to yield metarhodopsin, which activates the visual transduction cascade. The all-trans-retinaldehyde (atRAL) subsequently dissociates from apo-opsin and is reduced to all-trans-retinol (atROL) by all-trans-retinol dehydrogenase (atRDH). The atROL diffuses from the outer segment and is taken up by an RPE cell, where it is transferred to a fatty acid from phosphatidylcholine by lecithin-retinol acyl transferase (LRAT) to yield an all-trans-retinyl ester (atRE). The atRE is converted to 11-cis-retinol (11cROL) by the isomerase (shown here to be Rpe65). The 11cROL is oxidized by one of several 11cROL dehydrogenases (11cRDHs) to yield 11cRAL. 11cROL and 11cRAL are bound to CRALBP. The 11cRAL diffuses back to the outer segment where it combines with apo-opsin to form a new rhodopsin pigment molecule.

Figure 2

Immunoblot Analysis of 293T Cell Lines Stably Expressing LRAT, Rpe65, and CRALBP Proteins from the indicated 293T cell lines were separated by electrophoresis, blotted, and reacted with antisera against Rpe65, LRAT, and CRALBP. The immunoreactive band of the appropriate molecular mass is indicated by the arrowheads.

Figure 3

Expression Screen for the Isomerase in Bovine RPE (A) Primary screen of 42 pools from the bovine RPE cDNA library. Plasmid from each library pool was transfected into 293T-LRC cells and assayed for in vivo synthesis of 11cROL from atROL added to the culture medium. Levels of 11cROL in each dish of cells were normalized to the level in an equivalent dish of cells transfected with nonrecombinant pRK5. Cells transfected with pool #7 synthesized 2.8-fold more 11cROL than the pRK5 control. This pool was selected for secondary screening. (B) Secondary screen of 293T-LRC cells transfected with plasmid from pool #7. Thirty subpools containing ∼650 clones each were analyzed as in (A). Subpool #7:22 synthesized 7-fold more 11cROL than the pRK5 control and was selected for tertiary screening. (C) HPLC chromatogram of retinoids extracted from cells transfected with subpool #7:22. The peak corresponding to 11cROL is indicated by the arrow. (D) UV spectrum acquired from the 11cROL peak in (C). (E) HPLC chromatogram of retinoids extracted from cells transfected with pRK5. The 11cROL peak is indicated by the arrow. Note the 7-fold lower peak area relative to the 11cROL peak in (C). (F) Tertiary screen of subpool #7:22. Fifty-four subpools (22 shown) containing ∼36 clones each were analyzed with the in vivo isomerase assay, as in (A) and (B). Subpools #7:22:9 and #7:22:18 each synthesized ∼11-fold more 11cROL than pRK5 control-transfected cells. (G) In vitro isomerase assay of homogenates from 293T-LRC cells transfected with subpools #7:22:9 and #7:22:18 using atROL as substrate. Note that the isomerase activity of subpool #7:22:9 is only 2-fold higher than the pRK5 control while #7:22:18 is 6-fold higher. Also note the 17-fold potentiation of isomerase activity in homogenates from cells cotransfected with subpools #7:22:9 and #7:22:18. (H) Analysis of individual clones from subpool #7:22:9. Sequence analysis of plasmid from clone #7:22:9:10 showed it to be LRAT.

Figure 4

Analysis of LRAT and Rpe65 Expressed in 293T Cell Lines (A) Immunoblot analysis of homogenates from 293T-LRC cells transiently transfected with pLRAT or pRPE65 using antisera against LRAT or Rpe65. Homogenates from nontransfected 293T-LRC and 293T cells were also analyzed. Note the stronger LRAT and Rpe65 immunoreactivity in homogenates from transiently transfected 293T-LRC cells. (B) In vitro isomerase activity of homogenates from 293T-RC and 293T-LRC cells transfected with pRK5 or pRPE65. Substrates were atROL or atRP, as indicated. Histograms show 11cROL peak areas from HPLC chromatograms acquired at 318 nm. Error bars show standard deviations (n = 4). Note the absence of isomerase activity in pRPE65-transfected 293T-RC cell homogenates using atROL as substrate and the presence of activity in the same cells using atRP as substrate. Also note the presence of isomerase activity in pRPE65-transfected 293T-LRC cell homogenates using both atROL and atRP as substrate. Finally, note the reduced synthesis of 11cROL from atRP substrate in 293T-LRC, which express LRAT, versus 293T-RC cells, which do not.

Figure 5

Isomerase Activity in Sf9 Cells Expressing Rpe65 and Other Proteins (A) 11cROL production in Sf9 cells infected with recombinant baculoviruses encoding the indicated proteins from atROL added to the culture medium. (B) 11cROL production in Sf9 cells expressing the indicated proteins from atRP added to the medium. (C) In vitro isomerase assay of membranes from Sf9 cells expressing the indicated proteins with atROL substrate. Levels of 11cROL are expressed in peak-area units (mAU) at 318 nm. (D) In vitro isomerase assay of membranes from Sf9 cells expressing the indicated proteins with atRP substrate. Note the production of 11cROL from atRP by Sf9 cell membranes expressing only Rpe65. (E) Representative chromatogram acquired at 318 nm from the in vitro isomerase assay of Sf9 cell membranes expressing Rpe65 with atRP substrate (D), scaled to show the 11cROL peak (arrow). (F) UV spectrum acquired from the indicated 11cROL peak in (E). (G) Representative chromatogram from the in vitro isomerase assay of membranes from Sf9 cells expressing GFP with atRP substrate (D). (H) In vitro isomerase assay of membranes from Sf9 cells expressing Rpe65 with atRP substrate. Assay conditions were identical except for deletion or addition of 3 mM sodium cholate. Note the 9.2-fold higher isomerase activity in the presence of sodium cholate. (I) Substrate saturation curves for 11cROL production from atRP by membranes from Sf9 cells expressing Rpe65 or GFP. Initial reaction rates (V₀) were determined at the indicated concentrations of atRP. The curve was fit by the Michaelis-Menten equation for a single-substrate reaction. (J) Eadie-Hofstee transformation of the kinetic data in (I). The kinetic parameters V_max and K_M were derived from the x intercept and slope. Error bars show standard deviations (n = 4).

Figure 6

Isomerase Activity of Wild-Type and Disease-Associated Mutant Rpe65 (A) In vivo isomerase assay in 293T-LC cells transiently transfected with pRK5 (nonrecombinant), w/t Rpe65 (wild-type), C330Y (C330Y-substituted Rpe65), or Y368H (Y368H-substituted Rpe65). Error bars show standard deviations (n = 4). (B) Immunoblot of protein homogenates from cells in (A). The migration position of protein standards is shown to the left in kDa.