Abstract
Purpose: :
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.
Methods: :
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.
Results: :
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.
Conclusions: :
Desaturase 1 (DES1) is expressed in the retina of all species studied and has the unprecedented ability to directly isomerize Vitamin A retinol.
Keywords: retinoids/retinoid binding proteins • retina • photoreceptors