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

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

Diacylglycerol O-acyltransferase type-1 synthesizes retinyl esters in the retina and retinal pigment epithelium.

Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1(-/-) mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1(-/-) mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE's) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1(-/-) mice. Further, total retinyl-ester synthase activity was reduced in both dgat1(-/-) retina and RPE

Diacylglycerol O-acyltransferase type-1 synthesizes retinyl esters in the retina and retinal pigment epithelium.

Retinyl esters represent an insoluble storage form of vitamin A and are substrates for the retinoid isomerase (Rpe65) in cells of the retinal pigment epithelium (RPE). The major retinyl-ester synthase in RPE cells is lecithin:retinol acyl-transferase (LRAT). A second palmitoyl coenzyme A-dependent retinyl-ester synthase activity has been observed in RPE homogenates but the protein responsible has not been identified. Here we show that diacylglycerol O-acyltransferase-1 (DGAT1) is expressed in multiple cells of the retina including RPE and Müller glial cells. DGAT1 catalyzes the synthesis of retinyl esters from multiple retinol isomers with similar catalytic efficiencies. Loss of DGAT1 in dgat1(-/-) mice has no effect on retinal anatomy or the ultrastructure of photoreceptor outer-segments (OS) and RPE cells. Levels of visual chromophore in dgat1(-/-) mice were also normal. However, the normal build-up of all-trans-retinyl esters (all-trans-RE's) in the RPE during the first hour after a deep photobleach of visual pigments in the retina was not seen in dgat1(-/-) mice. Further, total retinyl-ester synthase activity was reduced in both dgat1(-/-) retina and RPE

Identification of DES1 as a vitamin A isomerase in Müller glial cells of the retina.

Absorption of a light particle by an opsin-pigment causes photoisomerization of its retinaldehyde chromophore. Restoration of light sensitivity to the resulting apo-opsin requires chemical re-isomerization of the photobleached chromophore. This is carried out by a multistep enzyme pathway called the visual cycle. Accumulating evidence suggests the existence of an alternative visual cycle for regenerating opsins in daylight. Here we identified dihydroceramide desaturase-1 (DES1) as a retinol isomerase and an excellent candidate for isomerase-2 in this alternative pathway. DES1 is expressed in retinal Müller cells, where it coimmunoprecipitates with cellular retinaldehyde binding protein (CRALBP). Adenoviral gene therapy with DES1 partially rescued the biochemical and physiological phenotypes in Rpe65(-/-) mice lacking isomerohydrolase (isomerase-1). Knockdown of DES1 expression by RNA interference concordantly reduced isomerase-2 activity in cultured Müller cells. Purified DES1 had very high isomerase-2 activity in the presence of appropriate cofactors, suggesting that DES1 by itself is sufficient for isomerase activity

Retinyl-ester processing in <i>dgat1 ^-/-</i> and <i>lrat^-/-</i> mouse eyes.

<p>(A) Levels of the DGAT1 mRNA by qRT-PCR in retina samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (B) Levels of the DGAT1 mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (C) Levels of the LRAT mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. Error bars for (A), (B), and (C) represent standard deviation of the mean for four (n = 4) samples of mRNA tested (p = <0.001, 1-way ANOVA for (A), (B), and (C)). (D) Retinyl-ester synthase activities of wild-type (C57BL/6 or 129S2/Sv), <i>dgat1 ^-/-</i> and <i>lrat^-/-</i> mouse retina homogenates using all-<i>trans-</i>ROL or 11-<i>cis-</i>ROL as substrate. Activities are shown as pmoles all-<i>trans-</i>RP or 11-<i>cis-</i>RP per minute per mg protein (2-way ANOVA: (D), p = 0.04, all-<i>trans</i>-ROL; p = 0.41, 11-<i>cis</i>-ROL). (E) Retinyl-ester synthase activities of wild-type (C57BL/6 or 129S2/Sv), <i>dgat1</i>^<i>-/-</i> and <i>lrat^-/-</i> mouse RPE homogenates using the same substrates as in (D) (2-way ANOVA: (D), p = <0.001, all-<i>trans</i>-ROL; p = <0.001, 11-<i>cis</i>-ROL). Activities are shown as pmoles all-<i>trans-</i>RP or 11-<i>cis-</i>RP per minute per mg protein. Error bars for (D) and (E) represent the standard error of the mean for three (n = 3) individual ARAT assays on the mouse type and tissue specified</p

Retinal morphology in <i>dgat1 ^-/-</i> eyes.

<p>(A) Light microscopy (LM) of a retina section from a two-month-old <i>dgat1</i>^<i>-/-</i> mouse. (B) Light microscopy (LM) of a retina section from a two-month-old wild-type (129S2/Sv) mouse. Retinal layers are indicated to the right of the figure panel. (C) Electron microscopy (EM) of RPE cells, including apical microvilli, and photoreceptor outer segments (OS). RPE and photoreceptor-OS layers are indicated. RPE cells were normal. The spaces observed on the basal and lateral aspects of the cell are due to an artifact produced during perfusion of fixative through the choriocapillaris.</p

DGAT1 expression in retina and RPE.

<p>(A) Immunofluorescent analysis of DGAT1 (green) and CRALBP (red) in distal ocular sections from six-month-old BALB/c mice. Nuclei were counter-stained with DAPI (blue). The merged image shows overlapping expression of DGAT1 and CRALBP (yellow). Labels identifying retinal layers are shown to the right of the image. RPE, retinal pigment epithelium; IPM, interphotoreceptor matrix; OLM, outer limiting membrane; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer. Müller-cell endfeet contact the vitreous within the GCL. Note expression of both DGAT1 and CRALBP in the apical microvilli of Müller cells, extending beyond the OLM into the IPM. (B) Light microscopy of twenty day old primary cultured bovine Müller-cells (scale bar = 100μm, 10X). (C) Expression of MFAT, DGAT1, CRALBP, GFAP, and DES1 mRNA’s by qRT-PCR on cDNA from primary-cultured bovine Müller-cell RNA. Levels were normalized to the actin mRNA. CRALBP, GFAP, MFAT and DES1 are positive controls for Müller-cell expression. Error bars show standard deviation of the mean for four (n = 4) independent experiments (p = <0.001, 1-way ANOVA). (D) Immunoblot of <i>dgat1</i>^<i>-/-</i> and wild-type (129S2/Sv) mouse eyecup, as well as, HEK-293T cell homogenates transfected with mouse DGAT1 or non-recombinant pcDNA using antisera against mouse DGAT1.</p

Retinyl-ester processing in <i>dgat1 ^-/-</i> and <i>lrat^-/-</i> mouse eyes.

<p>(A) Levels of the DGAT1 mRNA by qRT-PCR in retina samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (B) Levels of the DGAT1 mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. (C) Levels of the LRAT mRNA by qRT-PCR in RPE samples from mice of the indicated genotypes. Levels were normalized to the GAPDH mRNA. Error bars for (A), (B), and (C) represent standard deviation of the mean for four (n = 4) samples of mRNA tested (p = <0.001, 1-way ANOVA for (A), (B), and (C)). (D) Retinyl-ester synthase activities of wild-type (C57BL/6 or 129S2/Sv), <i>dgat1 ^-/-</i> and <i>lrat^-/-</i> mouse retina homogenates using all-<i>trans-</i>ROL or 11-<i>cis-</i>ROL as substrate. Activities are shown as pmoles all-<i>trans-</i>RP or 11-<i>cis-</i>RP per minute per mg protein (2-way ANOVA: (D), p = 0.04, all-<i>trans</i>-ROL; p = 0.41, 11-<i>cis</i>-ROL). (E) Retinyl-ester synthase activities of wild-type (C57BL/6 or 129S2/Sv), <i>dgat1</i>^<i>-/-</i> and <i>lrat^-/-</i> mouse RPE homogenates using the same substrates as in (D) (2-way ANOVA: (D), p = <0.001, all-<i>trans</i>-ROL; p = <0.001, 11-<i>cis</i>-ROL). Activities are shown as pmoles all-<i>trans-</i>RP or 11-<i>cis-</i>RP per minute per mg protein. Error bars for (D) and (E) represent the standard error of the mean for three (n = 3) individual ARAT assays on the mouse type and tissue specified</p