The role of rhodopsin glycosylation in protein folding, trafficking, and light-sensitive retinal degeneration

Abstract

Several mutations in the N terminus of the G-protein-coupled receptor rhodopsin disrupt NXS/T consensus sequences for N-linked glycosylation (located at N2 and N15) and cause sector retinitis pigmentosa in which the inferior retina preferentially degenerates. Here we examined the role of rhodopsin glycosylation in biosynthesis, trafficking, and retinal degeneration (RD) using transgenic Xenopus laevis expressing glycosylation-defective human rhodopsin mutants. Although mutations T4K and T4N caused RD, N2S and T4V did not, demonstrating that glycosylation at N2 was not required for photoreceptor viability. In contrast, similar mutations eliminating glycosylation at N15 (N15S and T17M) caused rod death. Expression of T17M was more toxic than T4K to transgenic photoreceptors, further suggesting that glycosylation at N15 plays a more important physiological role than glycosylation at N2. Together, these results indicate that the structure of the rhodopsin N terminus must be maintained by an appropriate amino acid sequence surrounding N2 and may require a carbohydrate moiety at N15. The mutant rhodopsins were rendered less toxic in their dark inactive states, because RD was abolished or significantly reduced when transgenic tadpoles expressing T4K, T17M, and N2S/N15S were protected from light exposure. Regardless of their effect on rod viability, all of the mutants primarily localized to the outer segment and Golgi and showed little or no endoplasmic reticulum accumulation. Thus, glycosylation was not crucial for rhodopsin biosynthesis or trafficking. Interestingly, expression of similar bovine rhodopsin mutants did not cause rod cell death, possibly attributable to greater stability of bovine rhodopsin.

Figure 1.

Expression of human T4K and hT17M rhodopsin causes RD in transgenic X. laevis rod photoreceptors. A, Solubilized retinal extracts from primary transgenic tadpoles (n = 29 per group) expressing human WT, T4K, or T17M rhodopsin were spotted on membranes and probed with mAb B630N (recognizes total rhodopsin) or mAb 1D4 (recognizes only transgenic rhodopsin). Varying levels of transgenic rhodopsin signals were obtained within and between the different groups. B, Fluorescent signals from dot blots were quantified and used to derive plots of transgenic rhodopsin expression levels versus total rhodopsin levels. Expression of hWT rhodopsin did not affect total rhodopsin levels, whereas increasing expression of both hT4K and hT17M rhodopsin correlated with a loss of total rhodopsin, which is indicative of RD. C, Confocal micrographs of cryosections from transgenic retinas stained with wheat germ agglutinin. Top row shows the entire eye, and bottom row shows higher magnification of the central retina. Retinas expressing hWT rhodopsin (left) appeared healthy with long closely packed ROS. In contrast, retinas expressing hT4K (middle) and hT17M (right) rhodopsin exhibited retinal degeneration as seen by loss or shortening of ROS. rpe, Retinal pigment epithelium; ros, rod outer segments; ris, rod inner segments; onl, outer nuclear layer. Scale bars: top row, 100 μm; bottom row 20 μm.

Figure 2.

Expression of bovine T4R and T17M rhodopsins does not cause retinal degeneration in transgenic X. laevis rod photoreceptors. A, Plot of transgenic rhodopsin versus total rhodopsin derived from dot blots of solubilized retinal extracts from primary transgenic tadpoles (n = 29 per group) expressing bovine WT, T4R, and T17M rhodopsin. No reduction in total rhodopsin was observed in any of the groups regardless of expression level or transgene. B, Confocal micrographs of cryosections of transgenic retinas expressing bWT (top), bT4R (middle), and bT17M (bottom) stained with wheat germ agglutinin. Retinal degeneration was not observed regardless of which transgene was expressed; bT4R and bT17M expressing retinas were similar to bWT expressing retinas with respect to ROS length and density. Scale bar, 20 μm.

Figure 3.

Dark rearing rescues retinal degeneration caused by expression of hT4K and hT17M rhodopsin. Transgenic tadpoles expressing either hT4K or hT17M rhodopsin were generated and reared in either constant dark or cyclic light for 14 d. Solubilized retinal extracts were dot blotted and subjected to Western blot analysis (n = 40 per group). A, C, Fluorescent signals were quantified and used to derive plots of total and transgenic rhodopsin. In contrast to when tadpoles were reared in cyclic light, no loss of total rhodopsin was observed when tadpoles were reared in the dark. B, D, Confocal micrographs of cryosections of transgenic retinas stained with wheat germ agglutinin. Retinas of animals reared in cyclic light exhibited varying degrees of retinal degeneration (bottom). However, retinas of animals reared in constant dark retained long closely packed ROS similar to those of wild-type retinas (top). Scale bar, 20 μm.

Figure 4.

hT4K and hT17M rhodopsin exhibit normal biosynthesis and trafficking. A, Transgenic rhodopsin levels were determined from dot blots of retinal extracts from primary transgenic animals expressing either hT4K or hT17M rhodopsin (n = 29 per group) and reared in constant dark for 14 d. Mean expression levels of hWT, hT4K, and hT17M were not statistically significant. B, Confocal micrographs of retinal cryosections labeled with mAb 2B2 (green), which recognizes transgenic rhodopsin only, and counterstained with wheat germ agglutinin (red) and Hoechst nuclear stain (blue). The overlap of green and red signals is represented by yellow. Human WT rhodopsin localized primarily to the ROS and Golgi membranes (arrowheads). The same pattern of distribution was exhibited by hT4K and hT17M rhodopsins regardless of whether tadpoles were raised in cyclic light or constant dark. In contrast, hP23H rhodopsin was retained in the ER of the rod inner segments, a distribution highly suggestive of protein misfolding. Thus, both hT4K and hT17M rhodopsin appear to fold and traffic properly. Fluorescent signals derived from antibody labeling were adjusted linearly, whereas signals derived from wheat germ agglutinin and Hoechst staining were adjusted nonlinearly to better represent the architecture of the retina. rpe, Retinal pigment epithelium; ros, rod outer segments; ris, rod inner segments; onl, outer nuclear layer. Scale bar, 5 μm.

Figure 5.

Comparison of hT4K and hT17M with respect to toxicity and glycosylation state. Transgenic animals expressing either hT4K or hT17M (n = 40 per group) were raised in cyclic light. Solubilized retinal extracts were analyzed by dot blot. A, Plot of hT4K and hT17M expression levels versus total rhodopsin levels. The average total rhodopsin levels are significantly lower in animals expressing hT17M rhodopsin than in those expressing hT4K rhodopsin, suggesting that the hT17M is more toxic than hT4K. The hT17M curve is shifted to the left of the hT4K curve, indicating that a given amount of hT17M causes a larger decrease in total rhodopsin than does the same amount of hT4K. B, Western blots of transgenic retinas expressing hWT, hT4K, or hT17M rhodopsins probed with mAb 1D4 (recognizes transgenic rhodopsin only). Solubilized retinal extracts were incubated with or without PGNaseF, which removes N-linked carbohydrates from proteins. A sample of bovine ROS was also included as a control. hT4K and hT17M rhodopsins migrated at rates intermediate to fully glycosylated and deglycosylated hWT rhodopsin, but their migration rates were different from each other. When deglycosylated with PNGaseF, both hT4K and hT17M rhodopsins migrated at the same rate as hWT and bovine rhodopsin. C, Western blot of transgenic retinas expressing bWT, bT4R, or bT17M rhodopsin. Both bT4R and bT17M rhodopsins appear as two distinct bands, each of which are intermediate in size between fully glycosylated and deglycosylated bovine rhodopsin.

Figure 6.

Glycosylation of rhodopsin is not required for biosynthesis or trafficking but may be required at N15 for rod cell viability. Transgenic animals expressing hWT, hN2S, hN15S, or hN2S/N15S (n = 22 per group) were generated and raised in cyclic light. Solubilized eyes were analyzed by dot blot and immunohistochemistry. A, Plot of transgenic versus total rhodopsin levels. Expression of hWT or hN2S rhodopsin did not affect total rhodopsin levels, whereas expression of hN15S or hN2S/N15S resulted in a decrease of total rhodopsin. B, C, Confocal micrographs of cryosections from transgenic retinas expressing hN2S, hN15S, or hN2S/N15S rhodopsins. Cryosections were stained with wheat germ agglutinin (B) or labeled with mAb 2B2 (green; recognizes transgenic rhodopsin only) and counterstained with wheat germ agglutinin (red) and Hoescht nuclear dye (blue) (C). Retinas expressing hN2S appeared healthy with long, densely packed ROS. However, many retinas expressing either hN15S or hN2S/N15S exhibited retinal degeneration characterized by short or absent ROS. Immunolabeled cryosections demonstrate that all three mutants localized primarily to the ROS and Golgi membranes (arrowheads) regardless of whether or not they induced retinal degeneration. Fluorescent signals derived from antibody labeling were adjusted linearly, whereas signals derived from wheat germ agglutinin and Hoechst staining were adjusted nonlinearly to better represent the architecture of the retina. rpe, Retinal pigment epithelium; ros, rod outer segments; ris, rod inner segments; onl, outer nuclear layer. Scale bars: B, 20 μm; C, 5 μm.

Figure 7.

The amino acid sequence of the extreme N terminus rather than glycosylation of N2 is crucial for rod cell viability. Transgenic animals expressing hWT, hN2S, hT4N, or hT4V (n = 22 per group) were generated and raised in cyclic light. A, Solubilized retinal extracts were analyzed by dot blot to derive plot of transgenic versus total rhodopsin levels. Expression of hWT or hN2S rhodopsin did not affect total rhodopsin levels as shown in Figure 6. Similarly, total rhodopsin levels in retinas expressing hT4V remained constant. However, expression of hT4N resulted in a decrease of total rhodopsin. B, C, Cryosections were stained with wheat germ agglutinin (B) or labeled with mAb 2B2 (green; recognizes transgenic rhodopsin only) and counterstained with wheat germ agglutinin (red) and Hoescht nuclear dye (blue) (C). Retinas expressing hT4V appeared healthy with long, densely packed ROS. However, many retinas expressing hT4N exhibited retinal degeneration characterized by short or absent ROS. In cryosections labeled with mAb 2B2, both mutants localized primarily to the ROS and Golgi membranes (arrowheads). Fluorescent signals derived from antibody labeling were adjusted linearly, whereas signals derived from wheat germ agglutinin and Hoechst staining were adjusted nonlinearly to better represent the architecture of the retina. rpe, Retinal pigment epithelium; ros, rod outer segments; ris, rod inner segments; onl, outer nuclear layer. Scale bars: B, 20 μm; C, 5 μm.

Figure 8.

Dark rearing significantly rescues retinal degeneration induced by nonglycosylated hN2S/N15S rhodopsin. Transgenic animals expressing hN2S/N15S rhodopsin were generated and raised in either cyclic light or constant dark (n = 17 per group). A, Solubilized retinal extracts were analyzed by dot blot to derive a plot of transgenic versus total rhodopsin levels. Although a reduction in total rhodopsin was seen in both rearing conditions, the extent of rhodopsin loss was much greater in cyclic light. B, Confocal micrographs of cryosections of transgenic retinas stained with wheat germ agglutinin. Although retinas of animals from both groups exhibited retinal degeneration, on average, the severity was significantly greater in animals reared in cyclic light (bottom) than those reared in constant dark (top). Scale bar, 20 μm.