Abstract
The photoreceptor-specific glycoprotein retinal degeneration slow (RDS, also called PRPH2) is necessary for the formation of rod and cone outer segments. Mutations in RDS cause rod and cone-dominant retinal disease, and it is well established that both cell types have different requirements for RDS. However, the molecular mechanisms for this difference remain unclear. Although RDS glycosylation is highly conserved, previous studies have revealed no apparent function for the glycan in rods. In light of the highly conserved nature of RDS glycosylation, we hypothesized that it is important for RDS function in cones and could underlie part of the differential requirement for RDS in the two photoreceptor subtypes. We generated a knockin mouse expressing RDS without the N-glycosylation site (N229S). Normal levels of RDS and the unglycosylated RDS binding partner rod outer segment membrane protein 1 (ROM-1) were found in N229S retinas. However, cone electroretinogram responses were decreased by 40% at 6 months of age. Because cones make up only 3-5% of photoreceptors in the wild-type background, N229S mice were crossed into the nrl(-/-) background (in which all rods are converted to cone-like cells) for biochemical analysis. In N229S/nrl(-/-) retinas, RDS and ROM-1 levels were decreased by ~60% each. These data suggest that glycosylation of RDS is required for RDS function or stability in cones, a difference that may be due to extracellular versus intradiscal localization of the RDS glycan in cones versus rods.
Highlights
Mutations in retinal degeneration slow (RDS) lead to rod and cone-dominant retinal degeneration by mechanisms that remain unknown
To avoid these level of expression issues that could complicate our work on the role of glycosylation in RDS function, we used a knockin strategy to introduce an Ala-to-Gly missense mutation resulting in the conversion of asparagine 229 to serine (N229S) in exon 2 of the native RDS gene (Fig. 1A)
RDS from the WT retina shows a marked downshift when subjected to PNGase F digestion, whereas the RDS from the rdsN/N retina does not, and the undigested form runs at a lower molecular size, confirming that N229S RDS is not N-glycosylated (Fig. 1D)
Summary
Mutations in retinal degeneration slow (RDS) lead to rod and cone-dominant retinal degeneration by mechanisms that remain unknown. The RDS/PRPH2 gene codes for a photoreceptor-specific glycoprotein called retinal degeneration slow (RDS2 or peripherin-2), which is a member of the tetraspanin family of proteins and is critical for the proper formation of both rod and cone. RDS assembles into homo- and heterotetramers with its unglycosylated homologue rod outer segment membrane protein 1 (ROM-1) that are held together primarily through D2 loop-mediated non-covalent interactions [30, 33] These tetramers are subsequently linked into larger intermediate- and higher-order oligomers that are assembled via covalent disulfide bonds [33, 34]. Because of the high conservation of the glycosylation site within a critical oligomer-forming domain for RDS and the known differential requirements for RDS in rods versus cones, we hypothesized that the N-linked glycan was important in cone photoreceptors. These data suggest that the RDS glycan is important for fine-tuning the cone OS and further our understanding of the RDS molecule and its regulation in rods versus cones
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