Identification of the 11- cis- specific retinyl-ester synthase in retinal Müller cells as multifunctional O- acyltransferase (MFAT)

Abstract

Absorption of a photon by a rhodopsin or cone-opsin pigment isomerizes its 11-cis-retinaldehyde (11-cis-RAL) chromophore to all-trans-retinaldehyde (all-trans-RAL), which dissociates after a brief period of activation. Light sensitivity is restored to the resulting apo-opsin when it recombines with another 11-cis-RAL. Conversion of all-trans-RAL to 11-cis-RAL is carried out by an enzyme pathway called the visual cycle in cells of the retinal pigment epithelium. A second visual cycle is present in Müller cells of the retina. The retinol isomerase for this noncanonical pathway is dihydroceramide desaturase (DES1), which catalyzes equilibrium isomerization of retinol. Because 11-cis-retinol (11-cis-ROL) constitutes only a small fraction of total retinols in an equilibrium mixture, a subsequent step involving selective removal of 11-cis-ROL is required to drive synthesis of 11-cis-retinoids for production of visual chromophore. Selective esterification of 11-cis-ROL is one possibility. Crude homogenates of chicken retinas rapidly convert all-trans-ROL to 11-cis-retinyl esters (11-cis-REs) with minimal formation of other retinyl-ester isomers. This enzymatic activity implies the existence of an 11-cis-specific retinyl-ester synthase in Müller cells. Here, we evaluated multifunctional O-acyltransferase (MFAT) as a candidate for this 11-cis-RE-synthase. MFAT exhibited much higher catalytic efficiency as a synthase of 11-cis-REs versus other retinyl-ester isomers. Further, we show that MFAT is expressed in Müller cells. Finally, homogenates of cells coexpressing DES1 and MFAT catalyzed the conversion of all-trans-ROL to 11-cis-RP, similar to what we observed with chicken-retina homogenates. MFAT is therefore an excellent candidate for the retinyl-ester synthase that cooperates with DES1 to drive synthesis of 11-cis-retinoids by mass action.L ight perception begins with the absorption of a photon by an opsin pigment in the membranous outer segment (OS) of a rod or cone photoreceptor cell. The light-absorbing chromophore in most vertebrate opsins is 11-cis-retinaldehyde (11-cis-RAL). Photon capture isomerizes the 11-cis-RAL to all-trans-retinaldehyde (all-trans-RAL), inducing conformational changes in the protein that lead to its active meta-II state. After a brief period of signaling through the transduction cascade, meta II decays to yield apo-opsin and free all-trans-RAL. Light sensitivity is restored to the apo-opsin when it combines with 11-cis-RAL to regenerate the pigment. Conversion of all-trans-RAL to 11-cis-RAL is carried out by a multistep enzyme pathway called the visual cycle, located in cells of the retinal pigment epithelium (RPE) (1, 2). The retinoid isomerase in this pathway is Rpe65, which converts an all-transretinyl ester (all-trans-RE), such as all-trans-retinyl palmitate (alltrans-RP), to 11-cis-retinol (11-cis-ROL) and a free fatty acid (3-5). Retinyl esters are synthesized in RPE cells by lecithin:retinol acyl transferase (LRAT), which transfers a fatty acid from phosphatidylcholine to retinol (6, ...

CRALBP supports the mammalian retinal visual cycle and cone vision

“…Indeed, it was shown recently that the retina promotes pigment regeneration and dark adaptation in cones independently of the RPE, and that pharmacological ablation of Müller cells blocks this process (5). Recent biochemical studies have identified putative retinoid isomerase (7) and ester synthase activities (8) in Müller cells consistent with a retinal visual cycle. However, the molecular mechanism involved in the trafficking and recycling of chromophore still remains largely unknown, and skepticism still remains, partly due to the lack of experimental evidence for the involvement of any of the putative molecular players in this path- -/-retinae with AAV-driven expression of control GFP (black, n = 4), Müller cell-specific CRALBP (red, n = 5), and RPE-specific CRALBP (green, n = 3).…”

“…The cone-specific visual cycle (3) has been suggested to enable cones, but not rods, to quickly recover from bright light exposure and to function over a wide range of light intensities (4)(5)(6). While an active area of research (7,8), to date, none of the putative molecular components in this pathway have been shown to actually affect mammalian cone function, casting doubt on the significance of this pathway.…”

Peropsin modulates transit of vitamin A from retina to retinal pigment epithelium

“…Tissues were cryopreserved first in 10% and then 30% sucrose solutions in PBS before embedding in OCT (TissueTek) and cut into 10-m sections. Sections were prepared as described previously (4). Primary (YenZym) and secondary (Alexa Fluor 488 donkey anti-rabbit) antibodies were used at 1:100 dilutions.…”

“…Restoration of light sensitivity to the resultant apo-opsin occurs after it recombines with another 11-cis-RAL. The conversion of all-trans-RAL back to 11-cis-RAL is carried out by multistep enzyme pathways in the retinal pigment epithelium (RPE) (1) and Müller cells of the retina (2)(3)(4). Non-enzymatic photoisomerization of retinyl lipids also contributes to 11-cis-RAL synthesis and visual pigment regeneration (5).…”

Preserved visual function in retinal dystrophy due to hypomorphicRPE65mutations

“…Electrophysiology demonstrates absent rod function but there may be residual cone function in childhood 5 10. This may reflect the alternative source of 11- cis -retinol that cones obtain from Müller cells 11. Gene therapy trials using subretinally administered recombinant adeno-associated viral vectors expressing RPE65 have so far achieved promising, although largely unsustained improvements in retinal function 12–14…”