Purpose: RPE65 is an abundant protein necessary for the synthesis of the chromophore 11-cis retinal by the retinal pigment epithelium (RPE). Our purpose was to identify RPE65 surface epitopes, to assess protein interactions, and to evaluate RPE65 expression in eyes from rod- and cone-dominant species using a monoclonal antibody approach.
Methods: RPE65-specific monoclonal antibodies, mAb 8B11, and mAb 1F9, were generated using bovine RPE microsomal membranes and a human RPE65 synthetic peptide as immunogen, respectively. Western analysis was performed on bovine RPE membranes, as well as yeast strains generated by transfection with RPE65 cDNAs. Competition of antibody binding by synthetic peptides was assayed using ELISAs, western analysis, and elution from immunoaffinity matrices. RPE65 structural models were generated by ab initio and comparative methods. Immunohistochemistry was performed on retina/RPE/choroid cryosections and retina flatmounts.
Results: The antigenic determinant recognized by mAb 8B11 was localized to a 10 amino acid sequence, KVNPETLETI, that competed binding with microM affinity and eluted RPE65 from an immunoaffinity matrix incubated with solubilized bovine RPE membranes or RPE65-transfected cells. Similarly, solubilized RPE65 was bound and eluted from an mAb 1F9 immunoaffinity matrix using the immunizing peptide, FHHINTYEDNGFLIV. In both cases, 11-cis retinol dehydrogenase, but not other known visual cycle proteins, appeared to co-elute with RPE65 in substoichiometric amounts. Both sequences localized to surface exposed regions of predicted RPE65 tertiary structures. RPE65 immunoreactivity was detected by mAb 8B11 and mAb 1F9 in the RPE, but not in retina, in bovine, rat, mouse, human, chicken, and Xenopus laevis, and in Nrl knockout mice whose retinas contain exclusively cone-like photoreceptor cells.
Conclusions: The identification of RPE65 surface exposed antigenic determinants represents a first step toward understanding RPE65 structure and its interaction with visual cycle proteins, and provides a means for the purification of the native protein. The finding that RPE65 immunoreactivity is present in the RPE and not retina of both rod- and cone-dominant species does not support a proposed direct role for RPE65 in cone cell function.