Retinoids Assist the Cellular Folding of the Autosomal Dominant Retinitis Pigmentosa Opsin Mutant P23H

Abstract

Abstract: : Purpose: To determine the effect of 9 and 11–cis retinals on the intracellular folding of the autosomal dominant retinitis pigmentosa opsin mutant P23H. Methods: For all experiments, HEK 293 tetracycline inducible stable cell lines that separately expressed wild–type and P23H opsin were used. After induction, no retinals were added in the control experiments until the cells were harvested. To other cells, concurrent to the induction of opsin expression, either 9– or 11–cis retinal was added to the culture media. All cells were then harvested at 48 hours post–induction, solubilized and the respective rhodopsins were purified. The samples were analyzed by UV/visible spectroscopy for pigment formation and thermal stability. Additionally, the glycosylation patterns of purified samples were analyzed on immunoblots. Results: The P23H cells produce low levels of rhodopsin when retinal is added following induction and harvesting of the protein in stably transfected HEK 293 cells. When either 9– or 11–cis retinal was added during expression, P23H rhodopsin levels were many fold higher. Addition of the retinals during induction promoted the Golgi–specific glycosylation of P23H opsin and transport of the protein to the cell surface. The purified P23H rhodopsins containing 9– or 11–cis–retinal were thermally unstable. Conclusions: We demonstrate the mutant P23H opsin is effectively rescued by 9– or 11–cis–retinal, the native chromophore. The proline to histidine substitution in P23H destabilizes rhodopsin. We suggest that retinal binds and stabilizes the protein early in its biogenesis to promote its cellular folding and trafficking. The implications of this study for treating retinitis pigmentosa will be discussed.

Keywords: color pigments and opsins • proteins encoded by disease genes • retinal degenerations: hereditary