Abstract
Purpose: :
Interphotoreceptor retinoid-binding protein (IRBP) secreted by the photoreceptors has been considered essential for normal function and survival of photoreceptors. Recently, a homozygous missense mutation (D1080N) in the IRBP gene has been identified in patients with retinitis pigmentosa (RP), a genetically heterogeneous group of inherited retinal degenerations. The molecular mechanisms by which the mutation causes RP are poorly understood. In this study we aimed to define the pathogenic basis of the D1080N mutation.
Methods: :
Wild-type and the RP-associated IRBPs were expressed in 661w cells, a mouse photoreceptor-derived cell line, and the 293T-LC cells by transient transfection. Secretion of IRBP was monitored by immunoblot analysis of the culture media from the cells treated with varying temperatures or with a varying concentration of chemical chaperones. Intermolecular and intramolecular disulfide formation in IRBP was determined by immunoblot analysis in the presence or absent of reducing reagents. Triton X-100 soluble and non-soluble fractions of cells were prepared by ultracentrifugation. Protein subcellular localization was analyzed by confocal immunofluorescence microscopy. Protein interactions were determined by immunoprecipitation.
Results: :
Wild-type IRBP, but not the RP-associated IRBP, was secreted into media from both 661W and 293T-LC cells, indicating that the D1080N mutation abolished secretion of IRBP. The non-secreted mutant IRBP formed high molecular aggregation in the cells. This mutated IRBP was mainly presented in non-soluble fraction and the aggregation involved abnormal intermolecular and intramolecular disulfide formation. Such non-secretory IRBP also partially inhibited the secretion of normal IRBP by interaction with IRBP. The mutated IRBP was not allowed to be transported to Golgi apparatus, instead retained in the endoplasmic reticulum (ER) and induced up-regulation of CHOP, a response to ER stress. The mutant IRBP was preferential bound to ER chaperons including binding immunoglobulin protein and protein disulfide isomerase and degraded by ubiquitin-proteasome system. Treatment of cells with 4-phenylbutyrate (PBA) or low temperature promoted secretion of mutant IRBP and normalized cell morphology.
Conclusions: :
Our results demonstrate that loss of normal function (non-secretion) and gain of cytotoxic function (ER stress) are involved in the disease mechanisms for the RP-associated IRBP. Our data also indicate that PBA is a promising candidate for treating or preventing the RP caused by the IRBP mutation.
Keywords: retinoids/retinoid binding proteins • mutations • retinitis