Retinal Degeneration Slow (RDS) Glycosylation Plays a Role in Cone Function and in the Regulation of RDS·ROM-1 Protein Complex Formation

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.

Keywords: N229S; glycosylation; oligomerization; peripherin-2; photoreceptor; retina; tetraspanin.

FIGURE 1.

Knockin of the N229S mutation removes RDS glycosylation while preserving normal RDS message and trafficking. A, the N229S mutation was introduced to exon 2 of the Rds gene to drive expression of unglycosylated RDS in the native regulatory environment. B, at P30, total RNA was isolated from murine retinas of the indicated genotypes, analyzed by quantitative RT-PCR for RDS, and normalized to the housekeeping gene HPRT. N.S., not significant. C, Northern blotting with radiolabeled random primed RDS cDNA as a probe was performed on equivalent RNAs in B. 28S and 18S ribosomal RNAs were used as a size reference, and 28S is shown in the bottom panel. D, retinal extracts from WT and rds^N/N were subjected to enzymatic deglycosylation by PNGase F. Actin reactivity was used as a loading control. IB, immunoblot. E, retinal cross-sections at P30 from WT and rds^N/N were immunolabeled with antibodies for S-opsin (green), Na⁺K⁺ATPase (blue), and either RDS (red, left), ROM-1 (red, center), or rhodopsin (red, right). Nuclei were counterstained with DAPI (gray). IS, inner segment; OPL, outer plexiform layer. Scale bar = 20 μm.

FIGURE 2.

Lack of RDS glycosylation does not change steady-state levels of RDS protein and reveals RDS·ROM-1 heterodimerization. A, total retinal extracts were prepared from the indicated genotypes at P30 and analyzed by reducing SDS-PAGE/Western blotting. Membranes were probed with the indicated antibodies. Band densities were analyzed and normalized to actin and set relative to the average WT values (n = 4 retinas/genotype). Values are mean ± S.E. IB, immunoblot. B and C, reciprocal co-immunoprecipitation was performed on WT and rds^N/N retinal extracts with antibodies specific to either RDS (B) or ROM-1 (C), followed by reducing SDS-PAGE/Western blotting with the indicated antibodies. In, input; B, bound; Ft, flow-through. D, retinal extracts from the indicated genotypes were analyzed by non-reducing SDS-PAGE/Western blot and probed with antibodies to RDS (left panel) or ROM-1 (right panel). Arrows highlight size shift of RDS monomers and dimers and ROM-1 dimers because of loss of the glycosylation of RDS.

FIGURE 3.

WT and N229S RDS form covalent linkages with ROM-1. Non-reducing SDS-PAGE/Western blots were performed on P30 retinal extracts of the indicated genotypes (WT, rds^N/N, and rom1^−/−, 10 μg/lane; rds^−/−, 40 μg/lane). Blots were probed simultaneously with antibodies against RDS and ROM-1 and imaged using multiplexed fluorescent secondary antibodies. D, dimer; M, monomer; IB, immunoblot. The bracketed region was subjected to profile analysis using ImageJ, and background-subtracted plot profiles are presented in the right panels. A, RDS·ROM-1 channels (red/green, respectively) combined. B and C, the same data are separated to more easily visualize the RDS and ROM-1 channels separately.

FIGURE 4.

The loss of RDS glycosylation alters the assembly of RDS·ROM-1 complexes. A and B, retinal extracts from WT (blue) and rds^N/N (red) were separated on a 5–20% sucrose gradient under non-reducing conditions. Fractions were prepared by drip collection (fraction 1, 20% to fraction 12, 5%) and analyzed by reducing SDS-PAGE/Western blot probed with antibodies specific to RDS (A) and ROM-1 (B). IB, immunoblot. C and D, retinal extracts were fractionated along non-reducing 5–20% sucrose gradients as in A and B but were then analyzed by non-reducing SDS-PAGE/Western blot and probed with antibodies to RDS (C) and ROM-1 (D). In the quantification (top panels, C and D), monomers are depicted in black/green, and dimers are depicted in blue/red. A reduction in the higher-order oligomers (arrow) and an increase in intermediate dimers (arrowheads) is observed. Each quantification graph plots RDS or ROM-1 band intensities in each gradient fraction as a percent of the total RDS or ROM-1. Presented are means (n = 4/genotype) ± S.E.

FIGURE 5.

Moderate retinal degeneration is observed by P180 in mice expressing unglycosylated RDS. A and B, rod photoreceptor function was analyzed using the mixed full-field scotopic ERG at P30 (A) and P180 (B). Under these conditions, the initial negative displacement A-wave is generated primarily by the large number of rod photoreceptors. Representative traces are shown in the left panels, with means ± S.E. shown in the right panels. For ERG, n = 14–16 mice/genotype, statistical analysis was done by one-way ANOVA. **, p < 0.01. C and D, plastic-embedded retinal sections were imaged in the central retina at P30 (C) and P180 (D). IS, inner segment; INL, inner nuclear layer; IPL, inner plexiform layer; ONH, optic nerve head. E, the thickness of the OS layer was measured in 5–10 eyes/genotype at P180. Plotted are means ± S.E. F and G, images were taken at increasing distances from the optic nerve head, and outer nuclear layer thickness (F) and outer nuclear layer nucleus counts (G) were measured in 3–5 eyes/genotype. Plotted are means ± S.E. Statistical analysis was done by two-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001. H and I, TEM images at P180 from WT and rds^N/N. OSs from multiple different rds^N/N eyes are shown in the first, second, and third panels in I. Scale bars = C and D, 50 μm; H and I, 10 μm (left) and 500 nm (right).

FIGURE 6.

Cone function is decreased at later time points in mice that lack RDS glycosylation without an associated loss of cone photoreceptor cells. A and B, cone photoreceptor function was analyzed using full-field photopic ERG at P30 (A) and P180 (B). **, p < 0.05 by one-way ANOVA with Bonferroni post hoc comparison. Shown are mean values from n = 4–16 mice/genotype. C, frozen retinal sections along the superior-inferior axis (S-I) were prepared from P180 WT and rds^N/N mice, and cones were labeled with a mixture of antibodies specific to M-opsin and S-opsin (red), with nuclei counterstained with DAPI. INL, inner nuclear layer; IPL, inner plexiform layer. D, starting at the optic nerve, total cones were counted in 200-μm² areas at the indicated distances from the optic nerve. Shown are means (n = 3/genotype) ± S.E. E and F, retinal sections at P30 or P180 were immunogold-labeled with antibodies against S-opsin to identify cone OSs. Arrows indicate cone OSs in low-magnification EM images. The right panels show higher-magnification images of cone OSs. Most cone OSs in rds^N/N exhibit normal morphology, but abnormal cone OSs are also seen (far right panel, F). Scale bars = C, 50 μm; E and F, 2 μm.

FIGURE 7.

Cone function and steady-state levels of RDS and ROM-1 are decreased in the cone-dominant nrl^−/− retina in the absence of RDS glycosylation. A, photopic ERG of nrl^−/−, rds^N/+/nrl^−/−, and rds^N/N/nrl^−/− mice was used to assess cone function at P30. Representative traces are shown in the left panel. Maximum photopic A-wave amplitudes (center panel) represent the signal generated by cone photoreceptors, whereas photopic B amplitudes (right panel) measure inner retinal responses to photoreceptor signaling. Shown are means (n = 4–16 mice/genotype) ± S.E. **, p < 0.01 by one-way ANOVA with Bonferroni post-hoc comparison. B–D, total retinal extracts were prepared from the indicated genotypes and analyzed by reducing SDS-PAGE/Western blot. Immunoblots (IB)were probed with antibodies specific for RDS (B), ROM-1 (C), S-opsin (D), and actin (B–D). Band densities were analyzed, normalized to actin, and set relative to the average nrl^−/− values. Shown are means ± S.E. from 5–18 retinas/genotype. **, p < 0.01 by one-way ANOVA with Bonferroni post-hoc comparison (rds^N/N/nrl^−/− versus nrl^−/−).

FIGURE 8.

Deglycosylation of RDS does not affect the ability of RDS to bind ROM-1 in cones. Reciprocal co-immunoprecipitation (IP) was performed on P30 nrl^−/− and rds^N/N/nrl^−/− retinal extracts using antibodies against RDS (A) or ROM-1 (B). Reducing SDS-PAGE/Western blots were probed with the indicated antibodies. In, input; B, bound; Ft, flow-through; IB, immunoblot.

FIGURE 9.

The loss of rds glycosylation induces subtle alterations in RDS·ROM-1 complex formation cones. Retinal extracts from the nrl^−/− (red) and rds^N/N/nrl^−/− (blue) were separated on non-reducing 5–20% sucrose gradients, and gradient fractions were subsequently separated by reducing SDS-PAGE/Western blot (A and B) or non-reducing SDS-PAGE/Western blot (C and D). Blots were probed with RDS (A and C) or ROM-1 (B and D). In the quantifications in A and B, nrl^−/− values are plotted in blue, and rds^N/N/nrl^−/− values are plotted in red. In the quantifications in C and D, nrl^−/− and rds^N/N/nrl^−/− monomers are plotted in black/green, respectively, whereas dimers are plotted in blue/red, respectively. Shown are means (n = 4) ± S.E. IB, immunoblot.