Purchase this article with an account.
B. M. Tam, O. L. Moritz; Light Activation of Rhodopsin Mutants T4K and T17M Causes Retinal Degeneration Due to Loss of Structural Integrity. Invest. Ophthalmol. Vis. Sci. 2009;50(13):2287.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
We have previously demonstrated that the RP-associated rhodopsin mutations T4K and T17M cause light sensitive retinal degeneration in transgenic X. laevis. In this study, we examine the mechanisms underlying light sensitivity for these two N-terminal rhodopsin mutants.
Seven human rhodopsin mutations that prevent N-linked glycosylation at either or both consensus glycosylation sites were generated, including three mutations associated with RP (T4K, N15S and T17M). Furthermore, T17M was also placed in the context of a constitutively inactive rhodopsin that cannot bind chromophore, and a thermally stablized rhodopsin. Transgenic tadpoles expressing these rhodopsin mutants 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.
All RP-associated mutations caused retinal degeneration in transgenic X. laevis. However, while all tested mutations affecting the glycosylation consensus sequence at N15 caused rod death, some of the amino acid substitutions abolishing glycosylation at N2 were not toxic. All of the mutant rhodopsins studied, including a double mutant that abolished both glycosylation sites, were primarily localized to the outer segment disks and Golgi membranes. Human T17M rhodopsin was significantly less toxic when the molecule was prevented from adopting the active conformation (either by dark rearing or in the context of a constitutively inactive rhodopsin). Furthermore, the T17M mutation caused significantly less retinal degeneration in the context of a rhodopsin with increased thermal stability.
Our results indicate that glycosylation at N2 is not required for rhodopsin biosynthesis, targeting or trafficking, and that alterations of the amino acid sequence in this region, rather than loss of glycosylation per se, are responsible for retinal degeneration. In contrast, the carbohydrate modification at N15 rather than the amino acid sequence may be critical for structural stability. For T17M rhodopsin, our results are consistent with a cell death mechanism that involves instability of the mutant rhodopsin molecule on exposure to light. In contrast to P23H rhodopsin, this instability is not due to loss of thermodynamic stabilization via chromophore binding, but rather is specifically associated with the transition to the active conformation of rhodopsin.
This PDF is available to Subscribers Only