Point mutations in bovine opsin can be classified in four groups with respect to their effect on the biosynthetic pathway of opsin

Abstract

Expression in vitro with the recombinant baculovirus expression system showed correct biosynthesis and post-translational processing of "wild-type' bovine opsin with regard to translocation, glycosylation, palmitoylation and targeting. However, several of these processes were severely affected by point mutations. From the overall results of 16 mutants reported here, four groups were distinguished. One group significantly affected neither biosynthesis nor folding of opsin (D83N, P291A, A299C-V300A-P303G). A second group produced a truncated protein (R69H, Y301F), suggesting that these positions are essential for a correct translational process. A third group affected membrane translocation as well as glycosylation, which can be interpreted as interference with the function of a transfer signal. Substitutions at positions Glu-113, Glu-122, Glu-134, Arg-135 and Lys-248 belong to this category. A fourth group induced structural changes in the protein that led to heterogeneous distribution in the plasma membrane (E113Q/D, W265F, Y268S). Taking any functional consequences of these mutations into consideration, it seems that point mutations can have mosaic effects and therefore should be examined at several levels (folding, targeting, functional parameters).

Introduction of a rod pigment aromatic cluster does not improve the structural stability of the human green cone pigment

“…The SDS-PAGE analysis also revealed higher bands, which represent oligomers of the pigments. This phenomenon was observed earlier for rhodopsin (DeCaluwé and DeGrip, 1996) and HGH (Vissers and DeGrip, 1996) and has been described for other members of the GPCR family (Javitch, 2004). Functional expression levels of the mutants are similar to that of the wild-type protein (Table 1).…”

Next-generation sequencing in health-care delivery: lessons from the functional analysis of rhodopsin

“…In contrast to our human data, bovine R69H rhodopsin was table 2 Bioinformatic analyses using three commonly used mutation programs of pathogenicity, SIFT, 37 PolyPhen2, 38 and MutPred, 39 shown previously to produce an abnormally translated protein in a baculovirus expression system. 34 However, we have been unable to confirm a role of R69H in the pathogenicity of retinal disease in Patient 4. A c.206G>A (p.R69H) polymorphic change has been reported once in dbSNP (rs118173887; http://www.ncbi.nlm.nih.gov/projects/SNP), where the frequency of the variant allele is low (0.008, with a heterozygosity frequency of 0.017).…”

“…Similarly we report a novel R69H variant that was previously reported to be functionally abnormal in a baculovirus system where bovine rhodopsin was used. 34 However, we found that human R69H traffics and functions normally in mammalian cells, suggesting that these inconsistencies may be, in part, because of the use of different assays or, more likely, variation between human and bovine proteins. Again, it is possible that photopigment dimerization, activation/deactivation kinetics, thermal stability, or transducin activation are impaired; however, our current data suggest that this variant functionally behaves in the same way as WT rhodopsin and further analysis would be required to exclude other possible biophysical defects.…”

Stable Rhodopsin/Arrestin Complex Leads to Retinal Degeneration in a Transgenic Mouse Model of Autosomal Dominant Retinitis Pigmentosa

“…Functional studies of some rhodopsin mutants in ADRP have led to their classification into distinct groups that implicate misfolding, mistrafficking, and constitutive activity as underlying bases for photoreceptor cell death (Sung et al, 1993;Kaushal and Khorana, 1994;DeCaluwe and DeGrip, 1996;Deretic et al, 1998;Mendes et al, 2005). Yet, a functionally distinct group of mutations is suggested by the Drosophila visual system, wherein light-dependent formation of stable rhodopsin/arrestin complex was implicated in photoreceptor cell death (Alloway et al, 2000;Kiselev et al, 2000;Iakhine et al, 2004).…”