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J. Kaylor, R. Radu, M. Jin, W. Moghrabi, G. Travis; DGAT1: A Palmitoyl Coenzyme A-dependent Retinyl-Ester Synthase in Retina and RPE. Invest. Ophthalmol. Vis. Sci. 2007;48(13):3256.
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© ARVO (1962-2015); The Authors (2016-present)
Regeneration of visual chromophore involves an enzyme pathway called the visual cycle. The retinoid isomerase in this pathway (Rpe65) converts a fatty-acyl ester of all-trans-retinol (all-trans-ROL) to 11-cis-retinol (11-cis-ROL). These all-trans-retinyl esters (all-trans-RE’s) are synthesized in the retinal pigment epithelium (RPE) by lecithin:retinol acyltransferase (LRAT). A second retinyl-ester synthase called acyl CoA:retinol acyltransferase (ARAT) has been reported in the RPE. Unlike LRAT, which uses phosphatidylcholine as a fatty-acyl donor, ARAT uses an activated fatty acid such as palmitoyl coenzyme A (palm CoA). The maximum turnover rate (Vmax) of ARAT is similar to that of LRAT. However, the Michaelis constant (KM) of ARAT for all-trans-ROL substrate is 10-fold higher than that of LRAT. These observations suggest that ARAT may complement LRAT to provide additional ester synthase capacity under conditions of high all-trans-ROL, such as following exposure to bright light. A strong candidate for ARAT is acyl CoA:diacylglyerol acyltransferase type-1 (DGAT1). In the current study we evaluated the role of DGAT1 in the processing of visual retinoids.
We measured expression of the DGAT1 gene in chicken and bovine retinas and RPE using quantitative real-time PCR. We determined levels of endogenous retinoids in animal tissues by extracting tissue homogenates into hexane and analyzing with normal-phase liquid chromatography. To assay ARAT activity, we prepared homogenates from wild-type and dgat1 -/- knockout mouse retinas and RPE. These were incubated in assay mixtures containing all-trans-ROL plus or minus palm CoA. Reactions were quenched with methanol, extracted with hexane, and analyzed for production of all-trans-RE’s by liquid chromatography. ARAT activity was determined by measuring the difference between the rates of all-trans-RE synthesis in the absence and presence of palm CoA.
The mRNA for DGAT1 was expressed in both retina and RPE. Interestingly, the dgat1 mRNA was four-fold more abundant in cone-dominant chicken versus rod-dominant bovine retinas. Endogenous all-trans-RE’s and 11-cis-RE’s were less abundant in eyecups from light-adapted dgat1 -/- versus wild-type mice. Finally, ARAT catalytic activity was 10-fold decreased in RPE from dgat1 -/- versus wild-type mice.
These observations indicate that DGAT1 accounts for at least a significant fraction of the observed ARAT activity in mouse retinas and RPE. It is less clear if DGAT1 represents the dominant palm CoA-dependent retinyl-ester synthase in the cone-dominant chicken retina.
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