May 2008
Volume 49, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2008
Characterization of the Light Sensitive T4K and T17M Rhodopsin Mutants in Transgenic X. Laevis Models of Retinitis Pigmentosa
Author Affiliations & Notes
  • B. M. Tam
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • O. L. Moritz
    Ophthalmology and Visual Sciences, University of British Columbia, Vancouver, British Columbia, Canada
  • Footnotes
    Commercial Relationships  B.M. Tam, None; O.L. Moritz, None.
  • Footnotes
    Support  CIHR, MSFHR, FFB-Canada
Investigative Ophthalmology & Visual Science May 2008, Vol.49, 3072. doi:https://doi.org/
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      B. M. Tam, O. L. Moritz; Characterization of the Light Sensitive T4K and T17M Rhodopsin Mutants in Transgenic X. Laevis Models of Retinitis Pigmentosa. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3072. doi: https://doi.org/.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Several mutations in the N-terminus of rhodopsin cause retinitis pigmentosa (RP) in humans. Moreover, visual loss often exhibits a sector RP phenotype that is thought to be due to variations in light exposure experienced by different retinal regions. Light sensitivity has also been observed in animal models of RP with similar mutations. In this study, we examine the in vivo characteristics of two N-terminal rhodopsin mutants, T4K and T17M.

Methods: : Transgenic tadpoles expressing bovine T4R or T17M rhodopsin or human T4K or T17M rhodopsin were generated by nuclear transplantation. Tadpoles were raised in either cyclic light or constant dark conditions. Transgenic rhodopsin levels and retinal degeneration were assessed by quantitative dot blot analysis. Frozen retinal sections were analyzed by immunocytochemistry and confocal microscopy to determine protein localization. The glycosylation states of the mutants were analyzed by glycosidase treatment and SDS-PAGE.

Results: : Both human T4K and T17M rhodopsins induced retinal degeneration when transgenic X. laevis were reared in cyclic light (12 light:12 dark) but not when the animals were raised in constant dark. Interestingly, neither bovine T4R nor T17M rhodopsin resulted in rod death regardless of lighting conditions. Although transgenic rhodopsin expression levels were relatively low suggesting protein misfolding, no accumulation of any of the mutant rhodopsins was apparent in the ER of transgenic rod photoreceptors. Mutant rhodopsins were overwhelmingly localized to the outer segment and Golgi membranes. Based on dose response curves, human T17M was more toxic than human T4K rhodopsin. As expected, mutations at T4 and T17 prohibited N-linked glycosylation at N2 and N15, respectively. The relative mobilities of T4K and T17M rhodopsin (with and without PNGaseF treatment) on SDS-PAGE indicated that the oligosaccharides linked to N2 and N15 were not identical.

Conclusions: : T4K and T17M rhodopsins cause light sensitive retinal degeneration in X. laevis similar to that observed in other animal models and human RP patients. Our results indicate that the carbohydrate moieties at positions N2 and N15 in these mutants are not structurally equivalent. Disruption of glycosylation at either site induced retinal degeneration but disruption of glycosylation at N15 was more toxic. Unlike P23H rhodopsin (another N-terminal mutant associated with light sensitive degeneration) neither T4K nor T17M rhodopsin significantly accumulated in the endoplasmic reticulum.

Keywords: photoreceptors • apoptosis/cell death • retinal degenerations: cell biology 
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