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Paul S Bernstein, Raji Shyam, Aruna Gorusupudi, Kelly Nelson, Martin Horvath; RPE65 Has a Secondary Activity as the Lutein to meso-Zeaxanthin Isomerase in the Vertebrate Eye. Invest. Ophthalmol. Vis. Sci. 2017;58(8):3579.
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meso-Zeaxanthin is a macula-specific carotenoid that has no common dietary sources. Lutein is hypothesized to be the precursor for meso-zeaxanthin, but the process by which this reaction occurs under physiological conditions is not understood. Our previous work determined that the production of meso-zeaxanthin in the chicken embryonic eye occurs in a developmentally regulated manner. We showed that the production of meso-zeaxanthin specifically begins at E17 in the RPE/choroid and that the amount of this carotenoid steadily increases in the eye as the embryo ages. In the present study, we determined the biochemical mechanism underlying meso-zeaxanthin production.
In order to determine whether lutein is the precursor to meso-zeaxanthin, primary chicken RPE cultures or homogenates of RPE/choroid from E21 embryos were used. RNA-sequencing was conducted on E16 and E21 RPE/choroid isolates to identify candidate isomerase enzymes. Transient transfection of plasmids containing candidate genes, followed by their treatment with pure lutein, were conducted in HEK293T cells. A pharmacological inhibitor, ACU5200, was used to knock down RPE65 activity in chicken embryos, and its effect on meso-zeaxanthin production was determined. Structural docking experiments were conducted to determine whether lutein can interact with the candidate enzyme.
Treatment of E21 chicken primary RPE cells with pure lutein showed the production of meso-zeaxanthin. Similar results were obtained in RPE/choroid homogenates of E21 embryos. RPE65 was identified as one of the top meso-zeaxanthin isomerase candidates in our RNA sequencing. Its transcript levels were upregulated 23-fold between E16 and E21. When HEK293T cells overexpressing chicken RPE65 were treated with lutein, they produced meso-zeaxanthin. In order to abolish RPE65 activity, a pharmacological inhibitor, ACU5200 (a more potent analog of emixustat), was injected into the embryos at E17. The injected embryos showed significant reduction in the meso-zeaxanthin levels in their RPE/choroid, whereas the levels of lutein and zeaxanthin were comparable to those of control embryos. Molecular modeling experiments demonstrated that the epsilon ring of lutein can dock into the active site of RPE65.
Our results indicate that RPE65 is both necessary and sufficient for the production of meso-zeaxanthin from lutein in the vertebrate eye.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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