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Joanna Kaylor, Jacob Makshanoff, Jennifer Yong, Tran Nguyen, Roxana Radu, Gabriel Travis; Identification and Study of Two New 11-cis-Retinyl Ester Synthases in the Retina. Invest. Ophthalmol. Vis. Sci. 2013;54(15):1700.
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© ARVO (1962-2015); The Authors (2016-present)
Biochemical and electrophysiological evidence show that cones regenerate visual pigments by an enzymatic pathway that occurs in the Müller cells of the retina. Prior work from our laboratory indicates the existence of a retinol isomerase activity in cone-dominant chicken and ground squirrel retinas. When all-trans-retinol and palmitoyl coenzyme A are added to microsomes prepared from these retinas predominantly 11-cis-retinyl esters are produced. We now have preliminarily identified the retinol isomerase as Desaturase 1 (DES1). This enzyme produces an equilibrium mixture of cis-retinol products from all-trans-retinol, including 11-cis-retinol. In order for DES1’s retinol isomerase activity to be utilized for pigment regeneration the 11-cis-retinol production must be driven by mass action due to other 11-cis-specific enzymes downstream. Identification of the enzyme(s) that are functionally coupled to DES1 that produce 11-cis-retinyl esters is the goal of this project.
DES1, DGAT1 and MFAT cDNA were subcloned into mammalian expression vector pcDNA3.1. dgat1-/- mice were obtained from Dr. Robert Farese at UCSF. Enzyme activity assays were conducted by transfection of clones into HEK 293T cells (Polyfect, Qiagen). In vitro homogenate assays using different retinol isomers as substrate were carried out in pH 7.2 40 mM Tris buffer. Retinoids were extracted with hexane and high performance liquid chromotography (HPLC) was used to identify and quantify retinoids formed. Numerous experimental techniques were undertaken to understand these proteins including gene expression (qRT-PCR and Western), Michaelis-Menten kinetic analysis, and co-expression studies with DES1.
DGAT1 and MFAT catalyze the synthesis of 11-cis-retinyl esters. MFAT preferentially esterifies 11-cis-retinol better than DGAT1 based on kinetic analysis. Gene expression studies confirm they are both located in the retina of the eye. dgat1-/- mice have reduced retinyl esters in the eye and assays on homogenates of eye tissues from these mice have greatly reduced 11-cis-retinyl ester synthesis. DGAT1 or MFAT coexpressed with DES1 show production of 11-cis-retinyl esters.
MFAT and DGAT1 are robust 11-cis-retinyl ester synthases that may be functionally coupled to DES1 and act as downstream regulators of 11-cis-specificity in the alternate visual cycle of the retina.
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