Abstract
Mutations in the gene encoding 11-cis-retinol dehydrogenase (RDH5; EC ) are associated with fundus albipunctatus, an autosomal recessive eye disease characterized by stationary night blindness and accumulation of white spots in the retina. In addition, some mutated alleles are associated with development of cone dystrophy, especially in elderly patients. The numbers of identified RDH5 mutations linked to fundus albipunctatus have increased considerably during recent years. In this work, we have characterized the biochemical and cell biological properties of 11 mutants of RDH5 to understand the molecular pathology of the disease. All RDH5 mutants showed decreased protein stability and subcellular mislocalization and, in most cases, loss of enzymatic activity in vitro and in vivo. Surprisingly, mutant A294P displays significant enzymatic activity. Cross-linking studies and molecular modeling showed that RDH5 is dimeric, and co-expression analyses of wild-type and mutated alleles showed that the mutated enzymes, in a trans-dominant-negative manner, influenced the in vivo enzymatic properties of functional variants of the enzyme, particularly the A294P mutant. Thus, under certain conditions, nonfunctional alleles act in a dominant-negative way on functional but relatively unstable mutated alleles. However, in heterozygous individuals carrying one wild-type allele, the disease is recessive, probably due to the stability of the wild-type enzyme.
Highlights
Mutations in several identified genes in the visual cycle result in hereditary forms of stationary night blindness, and there are still additional forms of stationary night blindness for which the underlying genetic defect is unknown
The substrate preferences and the extraocular tissue expression have led to the suggestion that RDH5 has dual and tissue-specific roles; in the eye, RDH5 generates 11cRAL, whereas in extraocular tissues, RDH5 may participate in the generation of 9-cis-retinoic acid (9cRA) [11,12,13]
We describe some biochemical and cell biological properties of 11 RDH5 mutants associated with fundus albipunctatus
Summary
Construction of RDH5 Mutants—Human RDH5 cDNA [14] was subcloned into the eukaryotic expression vector pSG5 [15], and the expression vectors for mutant enzymes were generated by single-stranded mutagenesis [16, 17]. The cells were collected 48 h after transfection, and microsomes were prepared and resuspended in phosphate-buffered saline, as described previously [10]. Enzymatic Analysis Using the in Vivo Cell Reporter System—The catalytic properties of wild-type and mutant RDHs were explored using the recently developed co-transfection assay as described previously [19], using the GAL4-retinoid X receptor reporter system in JEG-3 and 293 cells [20]. Microsomal membrane fractions from COS-1 cells (5 g total protein/30 l incubation) expressing human RDH5 were incubated with 0.8 mM SANPAH in phosphatebuffered saline for 30 min at room temperature in the dark. Recombinant microsomal mouse RDH5 protein (8 g total protein/30 l incubation) generated in baculovirus-infected Sf9 cells [12] was incubated with 1 mM bis(sulfosuccinimidyl) suberate in 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid/ phosphate-buffered saline for 30 min at room temperature. Homologous retinol dehydrogenases were extracted from the KIND data base [23] using FASTA3 [24] and aligned using ClustalW [25]
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