Thermal Stability of Pharmacologically–Rescued Mutant Rhodopsins Correlates With Their Autosomal Dominant Retinitis Pigmentosa Phenotype

Abstract

Abstract: : Purpose: To test whether the thermal stability of autosomal dominant retinitis pigmentosa (ADRP) mutations in rhodopsin (Rho) is correlated with their clinical severity. Methods: Mutant and WT opsins were expressed separately in HEK293 cells in the presence or absence of the rhodopsin chromophore 11–cis retinal. Levels of the expressed proteins were determined by immunoblotting of crude cell lysates. The proteins were immunoaffinity purified and analyzed by UV–visible spectroscopy and immunoblotting. The thermal stability of the purified proteins was determined by measuring the loss of pigment as a function of time at elevated temperatures. Results: When P23A, P23H and P23L opsins were expressed by cells grown in the absence of 11–cis retinal, the proteins accumulated at low levels and exhibited glycosylation defects. The yield of Rho pigment formed by adding 11–cis retinal to these cells after harvesting was also low. By contrast, when the mutant proteins were expressed in the presence of 11–cis retinal, these defects were reduced. This result is consistent with the pharmacological rescue of P23H opsin by retinoids, which we recently reported. Pro23 substitution mutants were less thermally stable than WT Rho, in the order WT > P23A > P23H ∼ P23L. F220L and A299S Rho, two mutations found in the human population not associated with ADRP, show no biosynthetic defects and are as stable as the WT protein. Clinical reports indicate that patients with P23H and P23L Rho mutations have a more severe form of ADRP than those with the P23A mutation, and patients with F220L or A299S mutations show no sign of the disease. Conclusions: We demonstrate a correlation between the thermal stability of mutant Rho and the clinical severity of ADRP, with less stable Rho proteins causing a more severe form of the disease. This study extends our previous observations of pharmacological rescue to ADRP Rho mutations other than P23H, and has important implications for the clinical diagnosis, prognosis, and treatment of Rho ADRP.

Keywords: retinitis • proteins encoded by disease genes • color pigments and opsins