Purpose: The protein RPE65 plays a critical role in retinoid processing in the retinal pigment epithelium (RPE). Previous studies have identified the RPE65 mRNA in salamander cones, but not in rods. The purpose of the present study was to determine whether RPE65 is expressed at the protein level in mammalian cones, as well as in those of amphibians.
Methods: The specificity of the anti-RPE65 antibody was demonstrated by Western blot analysis. RPE65 cellular localization was determined using immunohistochemistry on flatmounted retinas and retinal sections.
Results: RPE65 protein was detected in cones in flatmounted retinas of the mouse, rabbit, and cow, in addition to Xenopus laevis. The morphology and location of labeled cones in the retina were confirmed by double staining of mouse retina sections with the anti-RPE65 antibody and peanut agglutinin (PNA) lectin, which is known to label both types of cones in mouse. The double staining in the flatmounted retinas demonstrated that RPE65 was expressed in both types of the cones in the mouse retina. Under the same double-labeling conditions, however, cones in homozygous RPE65-knockout mouse were labeled by PNA lectin, but not by the anti-RPE65 antibody, indicating that the protein recognized by the anti-RPE65 antibody is encoded by the RPE65 gene rather than by another homologous gene. No RPE65 was detected in rods of any of the species tested.
Conclusions: RPE65 is expressed in mammalian cones, but not in rods. These results provide further support for physiological observations that cones may have an alternative retinoid cycle.