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Joanna J. Kaylor, Quan Yuan, Jeremy D. Cook, Jacob Makshanoff, Anh Miu, Tongzhou Xu, Gabriel H. Travis; Identification of the Vitamin A Isomerase in Retinal Müller Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):2218.
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© ARVO (1962-2015); The Authors (2016-present)
The 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 to 11-cis-retinol. This visual cycle for the regeneration of rhodopsin is well defined in RPE cells. However, several lines of evidence suggest that cones regenerate visual pigments by a mechanism independent of the RPE that occurs in the Müller cells of the retina. Prior work from our laboratory indicated the existence of an isomerase (distinct from Rpe65) in cone-dominant chicken and ground squirrel retinas that catalyzes the direct conversion of all-trans-retinol to 11-cis-retinol. The enzyme responsible for this activity, "isomerase-2", has not been identified. The goal of this research was to identify and study isomerase-2.
A normalized chicken retina cDNA library was created using a mammalian expression vector. Library pools were transfected into HEK 293T cells and screened for isomerase-2 activity by in vitro homogenate assays using all-trans-retinol as substrate. Retinoids were extracted from assays with hexane and high performance liquid chromotography (HPLC) was used to identify the production of 11-cis-retinol by comparison with standards. Once a single clone was identified multiple experimental techniques were used to characterize the enzyme. These include gene expression (qRT-PCR and Western), immunocytochemistry, coimmunoprecipitation, etc... Modulation of isomerase-2 activity in the presence of other known retinoid processing enzymes (ie. CRALBP) was studied by both transient and stable transfection of HEK 293T cells.
Desaturase 1 (DES1) isolated from chicken retina by expression screening has isomerase-2 activity as shown by its ability to directly isomerize all-trans-retinol to 11-cis-retinol. DES1 was expressed in the retinas of every species tested (mouse, chicken, bovine, and human). DES1 clones from other species (mouse and human) also had isomerase-2 activity. DES1 catalytic activity produces a thermodynamic equilibrium mixture of cis-retinol products (9/11/13) from all-trans-retinol substrate. Immunocytochemistry of mouse retina revealed co-labelling of DES1 with CRALBP, which is present in the Müller cells of the retina. Co-immunoprecipitation of DES1 revealed binding with CRALBP, an 11-cis-specific retinoid binding protein. DES1 enzymatic activity in the presence of CRALBP was more 11-cis-retinol specific.
Desaturase 1 (DES1) is expressed in the retina of all species studied and has the unprecedented ability to directly isomerize Vitamin A retinol.
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