Glycosylation of rhodopsin is necessary for its stability and incorporation into photoreceptor outer segment discs

Abstract

Rhodopsin, a G-protein coupled receptor, most abundant protein in retinal rod photoreceptors, is glycosylated at asparagines-2 and 15 on its N-terminus. To understand the role of rhodopsin's glycosylation in vivo, we generated and characterized a transgenic mouse model that expresses a non-glycosylated form of rhodopsin. We show that lack of glycosylation triggers a dominant form of progressive retinal degeneration. Electron microscopic examination of retinas at postnatal day 17 revealed the presence of vacuolar structures that distorted rod photoreceptor outer segments and became more prominent with age. Expression of non-glycosylated rhodopsin alone showed that it is unstable and is regulated via ubiquitin-mediated proteasomal degradation at the base of outer segments. We observed similar vacuolization in outer segments of transgenic mice expressing human rhodopsin with a T17M mutation (hT17M), suggesting that the mechanism responsible for the degenerative process in mice expressing the non-glycosylated rhodopsin and the RHO(hT17M) mice is likely the cause of phenotype observed in retinitis pigmentosa patients carrying T17M mutation.

Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model

“…2B). In control studies, we found that Rho proteins migrated through the gel as a diffuse series of bands probably composed of dimers and trimers as well as heterogeneously glycosylated protein compared to rod cell opsin [11,40-42] and is consistent with the literature [11,43]. Figs.…”

Hydrogen/Deuterium Exchange Mass Spectrometry of Human Green Opsin Reveals a Conserved Pro-Pro Motif in Extracellular Loop 2 of Monostable Visual G Protein-Coupled Receptors

“…97,98 In bovine rhodopsin, N-terminal glycosylation has been studied extensively, and its importance in trafficking and signaling has been demonstrated. 99,100 It was previously proposed that green cone opsin, expressed in Sf9 insect cells, is glycosylated at its N-terminal region like rhodopsin, but confirmation of this has been lacking. 37 The MS/MS analysis of green opsin reported here verified the proposed N-terminal glycosylation at asparagine residues 32 and 34.…”

Lack of Overt Retinal Degeneration in a K42E Dhdds Knock-In Mouse Model of RP59

“…Here, we have presented the generation and initial characterization of a novel mouse model of RP59, where we have achieved global homozygous knock-in of the K42E Dhdds mutation specifically associated with RP59 [3][4][5]. Based upon the clinical presentation of RP59 in human patients [3][4][5], as well as the demonstrable importance of dolichol-dependent protein glycosylation in maintaining the normal structure and function of the vertebrate retina [11][12][13][14], we expected to observe retinal degeneration and retinal thinning in Dhdds K42E/K42E mice, particularly in mice homozygous for the K42E mutation. This expectation was also predicated on a preliminary report [23], using a similar K42E mouse knock-in model, that claimed nearly 50% loss of OS length and reduction in ONL thickness by about two-thirds at PN 3 months of age, compared to WT mouse retinas.…”

“…to the growing Dol-PP-linked oligosaccharide chains in the ER. Mutations in rhodopsin that block its glycosylation have been shown to cause retinal degeneration in vertebrate animals [11,12]. In addition, pharmacological inhibition of protein N-glycosylation with tunicamycin has been shown to disrupt retinal photoreceptor outer segment (OS) disc membrane morphogenesis in vitro [13], as well as to cause retinal degeneration with progressive shortening and loss of photoreceptor OSs in vivo [14].…”