Abstract
Purpose: :
Cone photoreceptors have a faster light response than rods and a high demand for 11-cis retinal (11cRAL), the chromophore of visual pigments. RPE65 is the isomerohydrolase in the retinal pigment epithelium (RPE) which converts all-trans retinyl ester (atRE) to 11-cis retinol (11cROL), a key step in the visual cycle to regenerate 11cRAL. Accumulating evidence suggests that cone-dominant species express an alternative isomerase, likely in retinal Müller cells in order to meet the high demand for chromophore by cones. The purpose of this study is to clone and characterize a novel isomerase (isomerase-2, (ISM-2)) in the retina of cone-dominant species.
Methods: :
A candidate gene from the cone-dominant zebrafish was cloned into an expression plasmid and transfected into 293A cells. The expression levels were confirmed by Western blot analysis and semi-quantified by densitometry. The enzymatic activities were measured by in vitro isomerohydrolase activity assay, and the generated retinoids were analyzed by HPLC. Further, we examined substrate specificity, metal ion-dependency and subcellular fractionation of ISM-2. To identify the distribution of ISM-2 in the retina, we generated a polyclonal antibody against ISM-2, and verified its specificity by Western blot analysis and immunohistochemistry.
Results: :
We identified and characterized a novel isomerase, ISM-2, from the cone-dominant zebrafish retina. Recombinant ISM-2 efficiently converted atRE to 11cROL and 13cROL in the in vitro isomerase activity assay. ISM-2 used atRE as its intrinsic substrate and required iron as a co-factor for its enzymatic activity. Subcellular fractionation analysis showed that ISM-2 was predominantly present in the membrane fraction. Immunohistochemistry detected ISM-2 exclusively in Müller cells and not in the RPE.
Conclusions: :
These results suggest that ISM-2 is a candidate for the long-sought alternative isomerase in the intra-retinal visual cycle in cone-dominant species.
Keywords: retinoids/retinoid binding proteins • enzymes/enzyme inhibitors • retina