F81Y point mutation dramatically reduces S-opsin protein expression
The gene-targeting strategy used to create the F81Y S-opsin knockin (Opn1swF81Y/F81Y) mouse inserted a Neomycin resistance cassette between the third and fourth exons (). Successful targeting was confirmed with Southern blotting of ES cells and sequencing of genomic DNA (). Surprisingly, initial Western blotting suggested a more than 2-fold reduction in the level of expression of the Opn1swF81Y allele relative to the wild-type allele (). In contrast, there was no reduction in the expression of M-opsin, and cone-specific G-protein (Gtαc) and phosphodiesterase (PDE6c), were only modestly reduced by 28% and 26% (n = 2 littermate pairs), respectively.
F81Y point mutation in S-opsin leads to reduced expression with negligible mRNA reduction
To determine if the decrease in expression of the Opn1swF81Y
allele arose from altered transcription efficiency, we performed real-time quantitative reverse transcriptase PCR (qRT-PCR) analysis of mRNA from retinas of WT and Opn1swF81Y/F81Y
littermate pairs, using rhodopsin and β-actin as reference genes (). Six replications of the experiment with mRNA from the separately analyzed eyes of two littermate pairs yielded an average upward shift in the logarithmic threshold cycle (CT
) of +0.24 ± 0.09 (mean ± s.e.m., p
= 0.02) log2
units for Opn1swF81Y
vs. WT S-opsin mRNA, corresponding to a 15 ± 5% decrease in Opn1swF81Y
mRNA. Application of the method of Pfaffl et al. (2001)
, which takes into consideration the efficiencies of different PCR primer pairs, yielded the identical estimate of Opn1swF81Y
mRNA reduction, because the efficiencies of amplification of the target and reference genes were within 4% of ideal (Methods). In the same samples, the M-opsin mRNA in Opn1swF81Y/F81Y
mice was negligibly different from WT (6% ± 6%), and there was no detectable change in rhodopsin mRNA.
To determine precisely the reduction in expression of the protein product of the Opn1swF81Y gene we performed quantitative comparisons of S-opsin immunoblots of Opn1swF81Y/F81Y and WT littermate pairs. In each experiment 4 levels of retinal lysate from an Opn1swF81Y/F81Y and WT pair were blotted with anti-S-opsin antibodies, and the expression ratio estimated as the ratio of the slopes of the blot strength vs. protein load data for the two genotypes (METHODS; for an example, see ). Thirty-four such experiments from 12 littermate pairs revealed the average quantity of S-opsin in Opn1swF81Y/F81Y retinas to be reduced 2.6 ± 0.3 fold that of WT controls (mean ± s.e.m.; p < 0.001, for t-test comparison against an expression ratio of unity). In summary, there was a far greater reduction in the Opn1swF81Y protein than in its mRNA, suggesting post-transcriptional downregulation of the mutant gene product.
Exogenously increased 11-cis retinal restores S-opsin expression in Opn1swF81Y/F81Y to the WT level
Cones of Opn1swF81Y/F81Y mice have shifted λmax and reduced light sensitivity
Mouse rods that underexpress rhodopsin have altered outer segment structure and signaling (Calvert et al., 2001
; Liang et al., 2004
), raising the question of whether the large reduction in the expression of F81Y S-opsin alters the anatomical structure and physiological signaling of Opn1swF81Y/F81Y
cones. To assess the functionality of F81Y cone outer segments, we recorded flash responses from single cones using the suction electrode method (Nikonov et al., 2006
). Light responses of cones of the ventral retina of Opn1swF81Y/F81Y
mice were very similar to those of WT mice (, ), indicating that the outer segments of Opn1swF81Y/F81Y
cones are fully functional. As expected (Fasick et al., 2002
), the spectral sensitivity of the Opn1swF81Y/F81Y
cones was red-shifted 60 nm, having a λmax
of 420 nm as compared to the WT value of 360 nm. However, the absolute sensitivity of Opn1swF81Y/F81Y
cones at λmax
was ~ 3-fold lower than that of WT cones (p
; , ), consistent with the 2.6-fold reduction in F81Y S-opsin protein expression.
The maximal sensitivity of Opn1swF81Y/F81Y cones is shifted to 420 nm but reduced 3-fold
Properties of ventral cones of Opn1swF81Y/F81Y mice and WT controls
Opn1swF81Y/F81Y mice have a normal number of cones with normal length outer segments
The reduction in S-opsin expression and parallel loss of sensitivity of cones of the Opn1swF81Y/F81Y ventral retina reveals that F81Y S-opsin does not traffic to the outer segment in normal quantities, and raises the question of whether the number, size or other features of the cone outer segments are normal in mice expressing the mutant S-opsin. To address this question we analyzed retinal flat mounts and sections immunostained with PNA and S-opsin antibodies. The density of S-opsin expressing cones in the Opn1swF81Y/F81Y retina was normal (). In sections of eyes of littermates selected for adherence of the pigment epithelium, we measured the lengths of Opn1swF81Y/F81Y and WT ventral retinal cone outer segments, and found them to be indistinguishable (data not shown): 12.1 ± 0.02 µm (mean ± s.e.m., 114 cones, 2 mice) vs. 12.4 ± 0.02 (103 cones, 3 mice), respectively. We conclude that despite the reduction in S-opsin expression, the morphology of ventral cones of Opn1swF81Y/F81Y mice appears normal in light microscopy.
The density of cones expressing S-opsin is normal in Opn1swF81Y/F81Y mice
The global reduction in S-opsin expression in Opn1swF81Y/F81Y mice measured by Western blotting and reflected in the reduced light sensitivity of cones should also be manifest as a reduction of S-opsin immunofluorescence. To test this prediction, we quantified S-opsin immunolabeled sections of ventral retina of littermate pairs with a photon-counting, two-photon imaging system (). For one littermate pair, the average absolute S-opsin immunofluorescence of Opn1swF81Y/F81Y cone outer segments (COS) was reduced on average 1.6-fold (; p < 0.00002 for test of no difference); for a second littermate pair the average reduction was 2.2 fold (p < 0.00003).
Cones of Opn1swF81Y/F81Y mice have normal length outer segments, with proportionate reduction of S-opsin immunofluorescence throughout the cell
A substantial fraction of S-opsin resides outside the outer segment in both WT and Opn1swF81Y/F81Y cones
The apparently normal outer segment morphology of the Opn1swF81Y/F81Y
cones suggests that opsin and membrane delivery to the outer segment may be to some extent uncoupled in cones. This contrasts with the situation in rods, whose outer segment length, diameter and disc structure depend on rhodopsin expression level (Liang et al., 2004
). This possible difference between rods and cones led us to investigate the subcellular localization of opsin in different compartments of the cones, and in particular in the cell bodies and inner segments, where translation, ER and Golgi sorting, and membrane delivery to the basal discs of the outer segment take place. S-opsin immunofluorescence was readily observed in the cone inner segment, cell body, myoid region, along the axon and in the synaptic pedicles of both WT and Opn1swF81Y/F81Y
ventral cones (). We quantified the axial distribution of S-opsin immunofluorescence in oriented digitally excised “slabs” of retina: to a first approximation, there was a proportional reduction of S-opsin in each subcellular region of Opn1swF81Y/F81Y
cones (), and in both genotypes in excess of 25% of the S-opsin immunogenicity was not in the outer segment.
F81Y S-opsin expression is reduced by ER-associated degradation
The decrease in F81Y S-opsin expression, combined with the absence of a comparable decrease in its mRNA (), suggests that Opn1swF81Y
mRNA is less efficiently translated than the mRNA of WT Opn1sw
, or that newly synthesized F81Y S-opsin protein is rapidly degraded after translation. One well characterized pathway that governs protein removal early in the secretory pathway is ER-associated degradation (ERAD, Vembar and Brodsky, 2008
). ER degradation enhancing alpha-mannosidase-like 1 (EDEM1) is a lectin chaperone that accelerates ERAD by selectively targeting misfolded proteins to the site of retrotranslocation (Hosokawa et al., 2001), and has been shown to play a role in targeting immaturely glycosylated P23H rod opsin for ERAD (Kosmaoglou et al., 2009
). Another important component of the ERAD retrotranslocation machinery is the transitional AAA-ATPase VCP/P97, which provides the energy required to extract poly-ubiquitinated substrates through the ER membrane (Ye et al., 2001), and has been shown to play a role in the degradation of heterologously expressed P23H rod opsin (Griciuc et al., 2010
To test the hypothesis that the ERAD pathway is involved in the reduction of F81Y S-opsin expression, we performed immunoprecipitation assays of WT and Opn1swF81Y/F81Y retinas with an S-opsin antibody to determine whether EDEM1 and VCP are differentially complexed with S-opsin (). VCP and EDEM1 were readily detected in the immunoprecipitates from both WT and Opn1swF81Y/F81Y retinas, but were absent from those of S-opsin knockout mice, establishing the S-opsin specificity of the antibody. Comparing the blot densities of VCP and EDEM1 relative to that of S-opsin in the immunoprecipitates, we found VCP to be enhanced 3.7 ± 0.7 fold (n = 5; p < 0.01), while EDEM1 was enhanced 2.6 ± 0.7 fold (n = 4; p = 0.05). These results implicate ERAD in the reduced expression of F81Y S-opsin, and as a consequence suggest that newly translated F81Y S-opsin has a stronger tendency to misfold than WT S-opsin.
Figure 5 Molecular factors governing ER-associated degradation have elevated association with F81Y S-opsin. Results of an experiment in which S-opsin was immunoprecipitated, and the precipitate probed for two markers of ER quality control, the ER-mannosidase EDEM1, (more ...)
Exogenous 11-cis retinal, the cone opsin GPCR ligand, increases F81Y S-opsin expression to the WT level
Because the F81Y point mutation clearly alters the binding pocket for 11-cis
retinal ligand (thus affecting spectral sensitivity), we considered the hypothesis that interaction of the nascently translated S-opsin with 11-cis
retinal binding might affect the folding of F81Y S-opsin. We tested this hypothesis by systemically increasing 11-cis
retinal with intraperitoneal injections in Opn1swF81Y/F81Y
and WT mice. Littermate pairs were injected with 20 µg (70 nmol) of 11-cis
retinal on alternate days for 10 days, a time sufficient for complete renewal of their outer segments (Young, 1967
; Hollyfield, 1979
; Jonnal et al., 2010
); sham-injected, age-matched littermate pairs exposed to the identical light rearing conditions served as controls. Quantitative analysis of Western blots for S-opsin () from 10 sets of littermate pairs revealed the ratio of expression of S-opsin in Opn1swF81Y/F81Y
vs. WT for 11-cis
retinal-injected mice to be 0.96 ± 0.06 (mean ± s.e.m.), while for the sham-injected mice the corresponding ratio was 0.52, corresponding to a 1.9 ± 0.05 –fold reduction in F81Y S-opsin expression, as expected from previous results. The difference between the S-opsin expression ratios of sham- and 11-cis
retinal injected littermate pairs was highly significant (p
< 0.001, t
-test for unit ratio). Thus, exogenously elevated 11-cis
retinal caused the expression of F81Y S-opsin to increase to the WT level, effectively rescuing the Opn1swF81Y/F81Y
phenotype of reduced S-opsin expression.
Exogenous 11-cis retinal increases the expression of S-opsin in wild-type mice
During the course of the 11-cis retinal injection experiments just reported, we also performed immunoblot comparisons of the quantity of S-opsin extracted from WT mice injected with 11-cis retinal vs. WT mice that were sham-injected. These comparisons suggested that the chromophore injections might be increasing the level of WT S-opsin, and so we pursued this effect in a series of experiments with age-matched WT mice. In 16 immunoblot comparisons (as in ) of retinal lysates of 9 pairs of WT mice, the ratio of S-opsin in 11-cis retinal-injected vs. sham-injected was 1.22 ± 0.09 (mean ± s.e.m.; p = 0.023 for 1-tailed t-test). Thus, exogenous 11-cis retinal increased WT S-opsin expression, and so we conclude that during biosynthesis WT S-opsin, as well as the mutant F81Y-S-opsin, is sensitive to the ambient level of chromophore.
We also compared the quantities of rhodopsin and M-opsin of mice injected with 11-cis retinal vs. mice that were sham-injected. The average quantity of rhodopsin extracted was 430 ± 25 pmol/eye and 429 ± 18 pmol/eye (mean ± sem, n = 24 pairs) for sham-injected and 11-cis retinal injected mice, respectively. The average slope ratio of M-opsin signals in Western blots (as in ) for sham- vs. 11-cis retinal injected mice was 1.00 ± 0.06 (mean ± sem, n = 5 pairs). In both cases, the statistical power was adequate to have reliably detected a 15% change at p = 0.05. Thus, increased opsin production with exogenous 11-cis retinal injections in WT eyes appears uniquely associated with S-opsin.
11-cis retinoid production by the neural retina may contribute to cone opsin biosynthesis
To understand more deeply the organization of the retinal mechanisms that govern cone opsin biosynthesis and their relationship to native sources of 11-cis
chromophore, we examined the cell body and inner segment regions of cones with high resolution confocal immunohistochemistry, and with electron microscopy (), taking advantage of the ability of S-opsin antibodies to localize S-opsin in these subcellular regions (). The cell body region contains substantial S-opsin and a high density of rough ER (), and every cone cell body had a “cap” region, where rough ER and non-outer segment S-opsin were localized (). Remarkably, Mueller cell processes tightly apposed the ER-containing region of the cones, as revealed by immunohistochemical staining for the cellular 11-cis
retinoid binding protein, CRALBP () (Bunt-Milam and Saari, 1983
; Nawrot et al., 2004
), and well known morphological features of Mueller cells, including villous processes that project into the inner segment layer (). Mueller cells in some species have been established to be capable of synthesizing 11-cis
retinol (Das et al., 1992
), and CRALBP serves as a high-affinity cellular carrier for both 11-cis
retinol and 11-cis
retinal (Futterman et al., 1977
; Saari et al., 1982
; Saari et al., 2001
). In addition, interphotoreceptor retinoid binding protein (IRBP) is clearly present near the cell body and inner segment region of cones (). Because IRPB is secreted by photoreceptors into the interstitial space, some IRBP is no doubt also present in the cone cytoplasm (). Overall, then, these results show that the ER-containing regions of the cone, where its opsin is translated and proofread, are tightly apposed to a source of 11-cis
retinol, an immediate precursor of 11-cis
retinal, and that 11-cis
retinoid-specific carriers are present in the Mueller cell cytoplasm and in the nearby interstitial spaces.
Cellular and subcellular organization of retinal mechanisms implicated in cone opsin biosynthesis