Abstract
Abstract: :
Purpose: The vertebrate retinal pigment epithelium (RPE) contributes to the visual cycle by isomerization of all–trans–retinol to 11–cis retinal for visual pigment regeneration, a metabolic process which involves both enzymes and retinoid–binding proteins. In this process, it was not fully elucidated whether RPE65, which is responsible for ∼10% of Leber congenital amaurosis, a severe visual impairment at birth, belongs to an enzymatic and/or retinoid–binding protein family. In this study, we have therefore identified protein partners of RPE65 in retina/RPE to provide new insights into its function. Methods: Yeast two hybrid analysis allows the study of protein–protein interactions in a eukaryotic in vivo environment. The complete coding sequence of human RPE65 was fused to the GAL4 DNA binding domain in the pGBKT7expression vector to transform the yeast reporter strain, AH109. RPE65–expressing cells were subsequently transformed with two cDNA two–hybrid libraries generate from human retina and porcine retinal pigment epithelium. The interaction between RPE65 and library–encoded proteins was phenotypically detectable on nutritionally deficient plates and ß–galactosidase activity. Co–transformation experiments using isolated AD/library plasmids eliminated false positives. Sequencing of the clones followed by BLAST searches allowed identification of potential partners. Some interactions are confirmed by co–immunoprecipitation experiments in vitro and in eukaryotic cell systems. Results: We have cloned 10 cDNA encoding full–length or part of good candidates for binding partners of RPE65. Some of them were identified in data banks and known as scaffolding protein, enzyme and lipid transport protein. Others have been only identified by cDNA and EST sequences so far and their characterization is underway. Binding region has also been determined by using truncated RPE65 protein as bait and in co–immunoprecipitation assays. Since RDH5 has been demonstrated to form a complex with RPE65 (A. Simon et al., J. Biol. Chem. 270: 1107–12, 1995), we used bovine RDH5 as a bait protein but we were unable to retrieve neither these RPE65 partners nor RPE65 itself. Conclusions: We have identified potential partners of RPE65 that might contribute to the activity of the protein in the endoplasmic reticulum of RPE and will help to understand the detail of macromolecular interactions that determines all–trans–retinol isomerization.
Keywords: retinal pigment epithelium • metabolism • protein structure/function