Comparison of the molecular properties of retinitis pigmentosa P23H and N15S amino acid replacements in rhodopsin

Abstract

Mutations in the RHO gene encoding for the visual pigment protein, rhodopsin, are among the most common cause of autosomal dominant retinitis pigmentosa (ADRP). Previous studies of ADRP mutations in different domains of rhodopsin have indicated that changes that lead to more instability in rhodopsin structure are responsible for more severe disease in patients. Here, we further test this hypothesis by comparing side-by-side and therefore quantitatively two RHO mutations, N15S and P23H, both located in the N-terminal intradiscal domain. The in vitro biochemical properties of these two rhodopsin proteins, expressed in stably transfected tetracycline-inducible HEK293S cells, their UV-visible absorption, their Fourier transform infrared, circular dichroism and Metarhodopsin II fluorescence spectroscopy properties were characterized. As compared to the severely impaired P23H molecular function, N15S is only slightly defective in structure and stability. We propose that the molecular basis for these structural differences lies in the greater distance of the N15 residue as compared to P23 with respect to the predicted rhodopsin folding core. As described previously for WT rhodopsin, addition of the cytoplasmic allosteric modulator chlorin e6 stabilizes especially the P23H protein, suggesting that chlorin e6 may be generally beneficial in the rescue of those ADRP rhodopsin proteins whose stability is affected by amino acid replacement.

Fig 1

A. Secondary structure model of rhodopsin. The residues affected by the two mutations investigated in this manuscript, N15S and P23H, are shown encircled in red and red arrows. Predicted folding core residues based on FIRST analysis [48] are also highlighted (blue). These are 9, 10, 22–27, 102–116, 166–171, 175–180, 185–188, 203–207, and 211. Based on these predictions, P23 participates directly in folding, whereas N15 does not. B. The relative folding role of the two residues is best appreciated in a 3D view of the tertiary structure of rhodopsin (1L9H) highlighting the N15 (grey) and P23 (magenta) residues using space fill representation (red arrows). The 3D tertiary structure image is shown aligned with the secondary one with respect to its position relative to the discal membrane. Folding core residues (located intradiscally at the bottom of the molecule) are shown as ball-and-stick representation (blue). Note the deep position of the P23 residue inside the folding core compared to the N15 residue, which sits below the core, further intradiscally.

Fig 2

A. Overlay of absorbance spectra of rhodopsin proteins expressed in COS-1 cells and purified using 1D4-affinity chromatography. Proteins were eluted in phosphate buffered saline in order to elute both folded and misfolded portions. B. P23H rhodopsin obtained from stable cell line P23H and eluted in 2 mM sodium phosphate pH 6, which only elutes the folded fraction. C. Rhodopsin stability measured by loss of 500 nm chromophore over time at 37°C. Absorbance spectra of WT, N15S and P23H rhodopsin expressed in the presence of 9-cis retinal at 37°C were measured over time, and the decrease in absorbance at 500 nm was expressed at percent of intact structure. D. Rhodopsin Meta-II decay monitored by the use of fluorescence spectroscopy. Fluorescence spectra of rhodopsin Meta-II decay of WT, N15S and P23H rhodopsin expressed in the presence of 9-cis retinal.

Fig 3. Deglycosylation analysis of rhodopsin.

A. Example blot with rhodopsin mutation (N15S, P23H and WT transiently in COS-1 cells), and PNGase F treatment (+/-) by lane indicated. Protein concentrations were adjusted according to expression level and approximately 1μg was loaded. B. Use of the ladder to obtain molecular weights for mobility values for the blot shown in A. (I) Peak picking was performed automatically by selecting the local maxima. (II) Linear regression performed on ln(MW) and ln(Position). This was then rearranged so molecular weights could be estimated for each pixel. C. Density profiles aligned by the process in B. for WT rhodopsin. Black and red lines show profiles before and after PNGase F treatment respectively. D. Boxplots of centres of peaks fitted to profiles (e.g. C.). Box widths are proportional to the relative area occupied by that peak in the region from 20 to 50 kDa. E. Boxplots of the area fraction of density profiles separated by lane from numerical integration between 20 and 50kDa (white) and above 50kDa (red). F. Scatterplot of the area fraction of the density profiles of all lanes in a gel from numerical integration of the categories as in E.

Fig 4. The FTIR amide I band region along with the deconvoluted spectra corresponds to different secondary structure fractions of the three samples.

A-C: Rhodopsin in H₂0, D-F: Rhodopsin in D₂0: A, D: WT; B, E: N15S; and C, F: P23H. Bands assigned to β-sheets are indicated red, β-turns green, α-helices blue, and random coil grey.

Fig 5. Circular dichroism spectroscopy of WT and mutant rhodopsin in the presence and absence of chlorin e6.

Thermal denaturation studies of rhodopsin in the presence and absence of chlorin e6 (Ce6) were evaluated using circular dichroism (CD). A. CD melting spectra of WT rhodopsin (2.5 μM) with and without 100 μM Ce6. B. CD melting spectra of P23H. C. CD melting spectra of N15S. Concentrations of protein and chlorin e6 in B and C were the same as in A.