title: Photoactivation-induced instability of rhodopsin mutants T4K and T17M in rod outer segments underlies retinal degeneration in X. laevis transgenic models of retinitis pigmentosa.
abstract: Retinitis pigmentosa (RP) is an inherited neurodegenerative disease involving progressive vision loss, and is often linked to mutations in the rhodopsin gene. Mutations that abolish N-terminal glycosylation of rhodopsin (T4K and T17M) cause sector RP in which the inferior retina preferentially degenerates, possibly due to greater light exposure of this region. Transgenic animal models expressing rhodopsin glycosylation mutants also exhibit light exacerbated retinal degeneration (RD). In this study, we used transgenic Xenopus laevis to investigate the pathogenic mechanism connecting light exposure and RD in photoreceptors expressing T4K or T17M rhodopsin. We demonstrate that increasing the thermal stability of these rhodopsins via a novel disulfide bond resulted in significantly less RD. Furthermore, T4K or T17M rhodopsins that were constitutively inactive (due to lack of the chromophore-binding site or dietary deprivation of the chromophore precursor vitamin A) induced less toxicity. In contrast, variants in the active conformation accumulated in the ER and caused RD even in the absence of light. In vitro, T4K and T17M rhodopsins showed reduced ability to regenerate pigment after light exposure. Finally, although multiple amino acid substitutions of T4 abolished glycosylation at N2 but were not toxic, similar substitutions of T17 were not tolerated, suggesting that the carbohydrate moiety at N15 is critical for cell viability. Our results identify a novel pathogenic mechanism in which the glycosylation-deficient rhodopsins are destabilized by light activation. These results have important implications for proposed RP therapies, such as vitamin A supplementation, which may be ineffective or even detrimental for certain RP genotypes.
authors.affiliation: University of British Columbia Department of Ophthalmology and Visual Sciences, Vancouver, British Columbia V5Z 3N9, Canada.
authors.forename: Beatrice M
authors.initials: BM
authors.lastname: Tam
authors.affiliation: University of Massachusetts Medical School Department of Ophthalmology, Boston, Massachusetts 02118, and.
authors.forename: Syed M
authors.initials: SM
authors.lastname: Noorwez
authors.affiliation: University of Massachusetts Medical School Department of Ophthalmology, Boston, Massachusetts 02118, and.
authors.forename: Shalesh
authors.initials: S
authors.lastname: Kaushal
authors.affiliation: Medical University of South Carolina, Department of Ophthalmology, Columbia, South Carolina 29209.
authors.forename: Masahiro
authors.identifier: http://orcid.org/0000-0001-7377-1664
authors.initials: M
authors.lastname: Kono
authors.affiliation: University of British Columbia Department of Ophthalmology and Visual Sciences, Vancouver, British Columbia V5Z 3N9, Canada, olmoritz@mail.ubc.ca.
authors.forename: Orson L
authors.initials: OL
authors.lastname: Moritz
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D030801:Animals, Genetically Modified; D019556:COS Cells; D002522:Chlorocebus aethiops; D004195:Disease Models, Animal; D006801:Humans; D008027:Light; D018613:Microscopy, Confocal; D009154:Mutation / Q000235:genetics*; D012162:Retinal Degeneration / Q000178:diet therapy / Q000209:etiology*; D012174:Retinitis Pigmentosa / Q000150:complications / Q000235:genetics / Q000473:pathology; D012243:Rhodopsin / Q000235:genetics*; D012374:Rod Cell Outer Segment / Q000473:pathology*; D018709:Statistics, Nonparametric; D014162:Transfection; D014801:Vitamin A / Q000008:administration & dosage / Q000378:metabolism; D014909:Wheat Germ Agglutinins / Q000378:metabolism; D014982:Xenopus laevis
chemical_list: D014909:Wheat Germ Agglutinins; D014801:Vitamin A; D012243:Rhodopsin
keywords: cell death; glycosylation; instability; misfolding; retinitis pigmentosa; rhodopsin