Elongation of Very Long Chain Fatty Acids Protein 1 (ELOVL1) and Fatty Acid Transport Protein 4 (FATP4) are Inhibitors of RPE65

Abstract

Purpose: : RPE65, the retinoid isomerase, is an abundant protein in the RPE. However, its activity is significantly lower than that of other visual cycle enzymes in the RPE. One of the possible explanations for this low activity is that RPE expresses inhibitor(s) of RPE65. The purpose of this study was to prove this hypothesis by indentifying and characterizing negative regulator(s) of RPE65.

Methods: : Expression screening of a bovine RPE cDNA library was done in 293T-LRC cells stably expressing LRAT, RPE65 and CRALBP. RPE65 activity was measured by monitoring synthesis of 11-cis retinol (11cROL) from all-trans retinol or all-trans retinyl palmitate substrate. Retinoids were analyzed by high-performance liquid chromatography. Expression of FATP family members and FATP4 in the mouse ocular tissues was determined by real-time RT-PCR, immunoblot analysis and immunohistochemistry. Dark-adaptation kinetics and rod visual function in the wild-type (WT) and FATP4 knockout (KO) mice were determined through scotopic electroretinography (ERG). Retinal morphologies of the WT and KO mice exposed or not exposed to high-intense light were observed by optical coherence tomography (OCT) and light microscope.

Results: : We isolated ELOVL1, FATP4 and 11-cis retinol dehydrogenase (RDH5) as inhibitors of RPE65. The amount of 11cROL in the cells transfected with these clones was significantly lower than that of the control cells transfected with mock vector. The amount of 11-cis retinal (11cRAL) in the cells transfected with RDH5 clone was nearly 9-fold higher than that in the cells transfected with mock vector, ELOVL1 or FATP4 clone, indicating that RDH5, but not ELOVL1 and FATP4, catalyzed oxidation of 11cROL to 11cRAL. ELOVL1 and FATP4 slightly promoted synthesis of all-trans retinyl ester but significantly inhibited synthesis of 11cROL. Kinetic assays showed that FATP4 functions as a mixed-type inhibitor of RPE65. FATP4 was predominantly expressed in the mouse RPE, and its mRNA content in the RPE was at least 5-fold higher than that of other FATPs. The KO mice showed higher isomerase activity and faster dark adaptation rate compared to WT mice. No significant morphological defect was observed in the KO retina. However, the KO mice exhibited significantly increased susceptibility to light-induced retinal degeneration and contained higher content of cytotoxic retinaldehydes.

Conclusions: : ELOVL1 and FATP4 negatively regulate the visual cycle by inhibiting synthesis of 11cROL. FATP4, the dominant FATP in the RPE, functions as a mixed-type inhibitor of RPE65 and is required for preventing retinal degeneration and accumulation of cytotoxic retinaldehyde induced by long-time exposure to high intense light.

Keywords: retinoids/retinoid binding proteins • retinal pigment epithelium • enzymes/enzyme inhibitors