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D. Man, K. T. Gallaher, N. Yanamala, N. Waseem, B. J. Jennings, E. Reese, K. Gerwert, S. S. Bhattacharya, A. Iannaccone, J. Klein-Seetharaman; Molecular Properties and Disease Phenotypes of Rhodopsin Mutants P23H and N15S Associated With Autosomal Dominant Retinitis Pigmentosa (ADRP). Invest. Ophthalmol. Vis. Sci. 2007;48(13):3720. doi: https://doi.org/.
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We recently showed (Iannaccone et al., Vision Res 2006) that molecular properties correlate well with the severity, type, and progression rates of ADRP phenotypes associated with the rhodopsin mutations R135L and R135W (third cytoplasmic loop), and P180A and G188R (second extracellular loop). Here, we extend these studies to N15S and P23H mutations.
We characterized the in vitro behavior of the two mutants in terms of SDS-PAGE mobility, glycosylation, thermal stability, A280:A500 absorbance ratios, and secondary structure content using Attenuated Total Reflectance Fourier Transform Infrared (ATR/FT-IR) spectroscopy. Phenotypes associated with these mutations were assessed with ETDRS acuity charts, fundus photography, full-field flash electroretinograms, Goldmann kinetic and light- and dark-adapted monochromatic automated perimetry, and dark adaptometry.
Both mutations presented with a Class B1 phenotype (Cideciyan et al., PNAS 1998), but disease severity of the N15S mutation was much milder, with significant preservation of rod function and persistent altitudinal changes into the seventh decade of life. The mutants displayed distinctly aberrant glycosylation, presence of degradation products, and aggregation. A280:A500 absorbance ratio was 14 for P23H indicating more severe misfolding compared to 6 for N15S. The thermal stability of the folded portion of N15S was essentially wild type (WT)-like, while that of P23H was highly unstable. ATR/FT-IR spectroscopy showed that P23H has less helix and more sheet and random coil content compared to that of N15S and WT.
Despite proximity in sequence and some similarities in phenotypic pattern, the N15S mutation causes significantly milder disease than P23H. The WT-like structure and greater stability of the N15S mutant in vitro explain this finding mechanistically.
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