Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT) - PubMed (original) (raw)
Identification of the 11-cis-specific retinyl-ester synthase in retinal Müller cells as multifunctional O-acyltransferase (MFAT)
Joanna J Kaylor et al. Proc Natl Acad Sci U S A. 2014.
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.
Conflict of interest statement
The authors declare no conflict of interest.
Figures
Fig. 1.
Synthesis of 11-_cis-_ROLs, 11-_cis-_RPs, 13-_cis-_ROLs, and 13-_cis_-RPs from all-_trans_-ROL by chicken retina homogenates. Chicken-retina homogenates were used as the enzyme source in assays for retinol isomerization and retinyl-ester synthesis by using all-_trans_-ROL and palm CoA as substrates. (A) Chromatogram showing retinyl esters synthesized by chicken retina homogenates from all-_trans_-ROL. (B) Chromatogram showing retinols synthesized from chicken-retina homogenates from all-_trans_-ROL. (C) Rates of 11-_cis-_ROL and 11-_cis-_RE synthesis (Left), and rates of 13-_cis-_ROL and 13-_cis-_RP synthesis (Right) in the same reactions (n = 3). See
Fig. S1
for peak spectra. Error bars show SEM.
Fig. 2.
Kinetic analysis of MFAT supplied with different retinol isomers. Homogenates of 293T cells expressing MFAT were assayed for palm CoA-dependent retinyl-ester synthase activities using 11-_cis_-ROL (A), 9-_cis_-ROL (B), 13-_cis_-ROL (C), or all-_trans_-ROL (D) substrate at the indicated concentrations. Nonlinear fitting of these data with the SigmaPlot Enzyme Kinetics module yielded _V_max and _K_M values for synthesis of each retinyl-ester isomer from its cognate retinol. Note the higher _V_max and lower _K_M values for 11-_cis-_ROL–dependent synthesis of 11-_cis-_RP versus 13-_cis-_RP and all-_trans_-RP. Activities are expressed as picomoles per milligram of total protein per minute (n = 3). Error bars show SEM.
Fig. 3.
MFAT expression in the retina. Immunohistochemical analysis of MFAT (green) and CRALBP (red) are shown in distal ocular sections from 6-mo-old 129/Sv mice. The merged image shows overlapping expression of DES1 and CRALBP (yellow). Nuclei were counterstained with DAPI (blue). Labels identifying retinal layers are shown to the right of the Nomarski image. ELM, external limiting membrane; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; IS, photoreceptor inner-segments; ONL, outer nuclear layer; OPL, outer plexiform layer; OS, photoreceptor outer-segments; RPE, retinal pigment epithelium. Müller-cell endfeet contact the vitreous within the GCL. Note expression of both MFAT and CRALBP in the apical microvilli of Müller cells, extending beyond the ELM into the IS layer. (Scale bars: 20 µm.)
Fig. 4.
Retinyl-ester synthesis from 11-_cis-_ROL by chicken retinas and 293T cells. (A) Chromatogram of retinyl esters were synthesized by chicken-retina homogenates using 11-_cis-_ROL as substrate and no exogenous palm CoA. The numbered peaks were identified by their elution times and spectra (
Fig. S3
). (B) Chromatogram of retinyl esters synthesized by MFAT-expressing 293T-cell homogenates. Note the similar profiles of 11-_cis-_RE fatty-acyl forms in A and B. (C) Chromatogram of retinyl esters synthesized by homogenates of 293T cells transfected with nonrecombinant pcDNA. Only peaks 1 and 2 coeluted with the 11-_cis-_RE peaks in A and B. This shows the background ARAT activity in 293T cells.
Fig. 5.
Inhibition of DES1-coupled retinyl-ester synthase activities in chicken-retina and MFAT-expressing 293T-cell homogenates. (A) all-_trans_ROL–dependent synthesis of 11-_cis-_RP by chicken-retina homogenates in the presence or absence of MFAT inhibitors. IgG control, homogenates were preincubated with 10 µg of donkey anti-mouse IgG; MFAT Ab-1, homogenates were preincubated with 10 µg of MFAT Ab-1; MFAT Ab-2, homogenates were preincubated with 10 µg of MFAT Ab-2; MFAT Ab’s-1 & -2, homogenates were preincubated with 5 µg each of MFAT Ab-1 and MFAT Ab-2; 1-hexadecanol, homogenates were preincubated with 200 µM 1-hexadecanol; untreated, chicken-retina homogenates were preincubated with buffer only. (B) Synthesis of 11-_cis-_retinoids and 13-_cis-_retinoids from all-_trans_-ROL by 293T cells expressing both DES1 and MFAT. Rates of retinoid synthesis are shown in picomoles per minute per milligram of total protein after subtracting retinoids synthesized by 293T cells not expressing MFAT (n = 3). Error bars show SEM.
Fig. 6.
Noncanonical visual cycle in Müller cells. Cone opsins use 11-_cis-_RAL as visual chromophore. Absorption of a photon by a cone opsin isomerizes the 11-_cis-_RAL to all-_trans_-RAL, as in rods. After reduction by RDH8 in the cone OS, the all-_trans_-ROL is released into the IPM and taken up by a Müller cell. Here, the all-_trans_-ROL is isomerized by DES1 to 11-_cis-_ROL, 9-_cis-_ROL, and 13-_cis-_ROL. The 11-_cis-_ROL binds to CRALBP or is fatty acylated by MFAT to yield an 11-_cis-_RE. In either event, 11-_cis-_ROL is removed from the equilibrium reaction. The other retinol isomers are reisomerized by DES1. An as-yet unidentified 11-_cis-_retinyl-ester hydrolase (REH?) hydrolyzes 11-_cis-_RP to yield 11-_cis-_ROL and palmitic acid. The 11-_cis-_REH activity was shown to be activated by apo-CRALBP and inhibited by holo-CRALBP (25). Interaction with negatively charged phospholipids on the plasma membrane causes holo-CRALBP to release its 11-_cis-_ROL ligand into the IPM, where it binds IRBP and, subsequently, is taken up by a cone outer segment (26). Binding to CRALBP provides a mechanism to protect 11-_cis-_ROL from reverse isomerization by DES1. In the cone OS, 11-_cis-_ROL is oxidized by an unknown 11-_cis-_ROL dehydrogenase (RDH?) to 11-_cis-_RAL, which combines with apo-opsin to form a new opsin pigment. Simultaneous reduction of all-_trans_-RAL and oxidation of 11-_cis-_ROL in the cone OS provides a self-renewing supply of NADPH/NADP+ cofactors.
References
- Saari JC. Vitamin A metabolism in rod and cone visual cycles. Annu Rev Nutr. 2012;32:125–145. -PubMed
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