MZ Product Accumulation

RPE65 requires membrane association for access to substrate and enzyme activity. To explore its mechanism, we and others employed transient transfection of cultured cells that normally do not express RPE65. To test for L to MZ isomerization activity, cultured HEK293T cells were transfected with pCDNA3.1 plasmid DNA encoding RPE65 from three different species: chicken, human, and mouse. Special care was taken to ensure an identical CMV promoter and Kozak sequence for each expression vector. The empty vector or the same vector with a gene encoding GFP in place of RPE65 provided negative controls for comparison. Cell culture media was replaced with media supplemented with 4 µM L 24 hours post-transfection. Cells were harvested 72 hours post-transfection, and carotenoids were extracted using a two-phase organic solvent separation, followed by drying under nitrogen and reconstitution in a 95% hexane / 5% isopropyl alcohol mobile phase. HPLC normal phase chromatography with a chiral column quantified MZ yield in relation to L. 

The dataset compares MZ yield, expressed as MZ/L, derived by the RPE65 enzyme across three species, Mouse, Chicken, and Human. Also compared are the yield for amino-acid substitution variants of the RPE65 enzyme. All RPE65-expressing cultures produced significantly noticeable MZ product relative to the GFP-expressing cultures (p < 3.0×10⁻5 for all comparisons), confirming that L to MZ isomerization is driven by RPE65 as previously reported [18]. Chicken RPE65 (Gg WT) showed the highest normalized MZ levels, human RPE65 (Hs WT) generated somewhat lower MZ levels, and these orthologs were more robust relative to the two variants of mouse RPE65 (Mm L450 and Mm M450).  The Mm L450 variant produced approximately twice as much MZ as the Mm M450 isoform (p = 1.1×10⁻4), an observation that mirrors differences in retinoid isomerohydrolase activity for inbred lines of mice expressing the L450 and M450 variants of RPE65.