Molecular Biology Of Outer Retina-specific Proteins

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The retinal pigment epithelium (RPE) plays a pivotal role in the development and function of the outer retina. We are interested in RPE-specific mechanisms, at both the regulatory and functional levels, and we have been studying the function and regulation of RPE65, a gene whose expression is restricted to the RPE and mutations in which cause severe blindness in humans. The phenotype of the Rpe65 knockout mouse is due to disruption of the RPE-based vitamin A visual cycle, metabolizing. Consequently, in the Rpe65 knockout mouse there is overaccumulation of all-trans-retinyl esters and total absence of11-cis-retinal. The function of RPE65 thus appears to be associated with that of the retinol isomerase, the crucial enzyme in visual pigment chromophore regeneration. We have also continued studies on beta-carotene 15,15'-monooxygenase (BCMO1). BCMO1 is closely related to RPE65 and both are members of a newly emerging diverse family of carotenoid-cleavage enzymes. We postulate that BCMO1 and RPE65 share a similar mechanism of action. In the past year we have made the following progress: a) We have established a catalytic role (in conjunction with lecithin:retinol acyltransferase (LRAT)) for RPE65 in the synthesis of 11-cis retinol, and identify it as the long-sought isomerohydrolase. To clarify RPE65?s role in isomerization we reconstituted a robust minimal visual cycle in 293-F cells. Only cells transfected with RPE65 constructs produced 11-cis retinoids but co-expression with LRAT was needed for high level production. Accumulation was significant, amounting to more than 2 nmoles 11-cis retinol per culture. Transfection with constructs harboring mutations in residues of RPE65 homologous to those required for interlinked enzymatic activity and iron coordination in related enzymes abolished this isomerization activity. Iron chelation also abolished isomerization activity. Mutating cysteines implicated in palmitoylation of RPE65 had generally little effect isomerization activity. Mutations associated with Leber congenital amaurosis/early onset blindness caused partial to total loss of isomerization activity in direct relation to their clinical effect. b) Our empirical data from site-directed mutagenesis of putative metal binding residues in BCMO1 demonstrated a crucial role in enzymatic activity for histidine and acidic residues we hypothesized to be involved in metal coordination. These observations, along with those regarding RPE65 (see above), have been corroborated by the predicted structure of a related bacterial enzyme, Synechocystis apocarotenal oxygenase. In light of this structure we are investigating how BCMO1 and RPE65 have evolved to fulfill their functions utilizing the basic structure of the carotenoid oxygenase family. c) We have generated several different lines of transgenic mice bearing RPE65 minigene transgenes that incorporate mutations in RPE65 pathogenic in humans and these have been bred onto the Rpe65 knockout background and are being tested as to their phenotypes to generate models for RPE65-related retinal dystrophy that are not null (like the knockout mouse and the Briard dog). We find that the minigene-carrying mice are slightly more light-sensitive than Rpe65 knockout mice. Further work on this project will be carried out using knock-in mice generated by homologous recombination. d) The identity of putative factors binding to transcription elements in the RPE65 gene promoter is being sought. Expression clones for these factors are being tested for their effect on activation of the RPE65 promoter. We have tested one such putative factor, ZNF-492, a KRAB-zinc finger protein that has a moderate effect on RPE65 gene transcription.