Abstract
Purpose :
Congenital Stationary Night Blindness (CSNB) is a heterogeneous inherited group of retinal disorders characterized by a non-progressive photoreceptor degeneration leading to poor vision at night (nyctalopia), decreased visual acuity, myopia and nystagmus. The inherited causes of CSNB include mutations in the Rhodopsin (RHO) gene. Specifically, the substitution of Gly in position 90 of RHO with Asp was associated to the autosomal dominant form of CSNB. We found two new RHO mutations in patients diagnosed with CSNB. Since dominant mutations in RHO cause also Retinitis Pigmentosa (RP) and are characterized by the retention of the protein in the Endoplasmic Reticulum (ER), we evaluated the structural effects of the two new CSNB mutations and assessed the chaperon effect of retinal.
Methods :
The structural effects of the new RHO mutations were investigated by equilibrium and steered molecular dynamics (MD) simulations by the CHARMM force field. In vitro analyses were performed in COS7 cells transiently transfected with cDNA of WT or mutant RHO for subcellular localization of mutant RHO and the potential chaperon-like effect of 11-cis-retinal.
Results :
The in vitro analysis in COS7 cells highlighted a different behavior of the two CSNB RHO mutants. One mutation caused some retention in the ER of the opsin protein. Administration of 11-cis-retinal reduced the ER retention to the native opsin levels. The second mutant showed a similar distribution in the cellular compartments compared to the wild type protein. Interestingly, when exposed to 11-cis-retinal a marked reduction of membrane localization and increased ER retention were detected. The computational model could predict the effects of the two mutations on ER retention in the presence of 11-cis-retinal. Moreover, MD analysis highlighted a structural communication between mutation sites and the visual arrestin binding regions.
Conclusions :
Consistent with in silico analysis, the two new mutations found in CSNB patients showed a different in vitro behavior in their ER-retention. In this respect, the first mutant could fall in the same cluster, cluster 2, of adRP RHO mutants (Behnen et al, iScience 2018). The second mutant appeared to be destabilized by 11-cis-retinal binding. In their opsin state, both mutants could be affected in visual arrestin binding.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.