Differences in RDS trafficking, assembly and function in cones versus rods: insights from studies of C150S-RDS

Chakraborty, Dibyendu; Conley, Shannon M.; Stuck, Michael W.; Naash, Muna I.

doi:10.1093/hmg/ddq410

Cited by 44 publications

(83 citation statements)

References 45 publications

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“…9, A and B, shows fractions from nonreducing sucrose gradients separated on reducing SDS-PAGE. As we have observed previously (31,32), cones in nrl Ϫ/Ϫ have a higher total percent of RDS in the large oligomer fractions 1-3 than rods (e.g. (Fig.…”

Section: Rds and Rom-1 Levels Are Decreased Significantly In Rds N/nsupporting

confidence: 76%

“…Specifically, cones have a larger percentage of total RDS present in high molecular weight homomeric complexes compared with intermediate and tetrameric heteromers (18,31,32). In addition, results from C150S transgenic mice (in which RDS cannot form intermolecular disulfide bonds) suggest that, in cones, covalent intermolecular disulfide bonds in RDS⅐ROM-1 complexes are formed earlier in the RDS⅐ROM-1 biosynthetic pathway than in rods (18,31). Rods and cones also tend to respond differently to different disease mutations.…”

Section: Discussionmentioning

confidence: 99%

“…Antibodies-The antibodies that were used in this study were as follows: RDS-CT (rabbit polyclonal, generated in-house, 1:1000) for immunofluorescence (IF) (18,31), RDS-2B7 (mouse monoclonal, generated by Precision Antibodies, 1:1000) for Western blot (WB) (31,38), ROM1-CT (rabbit polyclonal, generated in-house, 1:1000) for WB (14), ROM1-2H5 (mouse monoclonal, generated in-house,1:10) for WB (32) Immunofluorescence-For IF analysis, eyes were enucleated following euthanasia and fixed in 4% paraformaldehyde prior to dissection and cryoprotection as described previously (31). Eye cups were sectioned at 10 m and subjected to IF staining with procedures described previously (39).…”

Section: Methodsmentioning

confidence: 99%

“…Transmission Electron Microscopy and Immunogold Labeling-Samples at P30 or P180 were processed for EM and immunogold labeling as described previously (18,19,31). Plastic-embedded thin sections (600 -800 Å) were used for EM/immunogold labeling, and thick sections (0.75 m) were used for light microscopy.…”

Section: Methodsmentioning

confidence: 99%

“…The D2 loop contains seven cysteines, six of which form intramolecular disulfide bonds that stabilize the complex three-dimensional structure of the D2 loop and promote proper folding (36). The seventh cysteine at position 150 is responsible for the intermolecular disulfide bonds that mediate the formation of larger oligomers (18,31,33,(35)(36)(37).…”

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confidence: 99%

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

Stuck

Conley

Naash

2015

Journal of Biological Chemistry

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Background: Mutations in retinal degeneration slow (RDS) lead to rod and cone-dominant retinal degeneration by mechanisms that remain unknown. Results: Knockin mice carrying unglycosylated RDS have reduced RDS levels and functional defects in cones. Conclusion: RDS glycosylation is important for cone but not rod function. Significance: Differential reliance on glycosylation may help explain why some RDS disease mutations target cones and others target rods.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

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Section: Rds and Rom-1 Levels Are Decreased Significantly In Rds N/nsupporting

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Stuck

Conley

Naash

2015

Journal of Biological Chemistry

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Genotype–phenotype associations in a large PRPH2 ‐related retinopathy cohort

et al. 2020

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Molecular variant interpretation lacks disease gene‐specific cohorts for determining variant enrichment in disease versus healthy populations. To address the molecular etiology of retinal degeneration, specifically the PRPH2‐related retinopathies, we reviewed genotype and phenotype information obtained from 187 eyeGENE® participants from 161 families. Clinical details were provided by referring clinicians participating in the eyeGENE® Network. The cohort was sequenced for variants in PRPH2. Variant complementary DNA clusters and cohort frequency were compared to variants in public databases to help us to determine pathogenicity by current American College of Medical Genetics and Genomics/Association for Molecular Pathology interpretation criteria. The most frequent variant was c.828+3A>T, which affected 28 families (17.4%), and 25 of 79 (31.64%) variants were novel. The majority of missense variants clustered in the D2 intracellular loop of the peripherin‐2 protein, constituting a hotspot. Disease enrichment was noted for 23 (29.1%) of the variants. Hotspot and disease‐enrichment evidence modified variant classification for 16.5% of variants. The missense allele p.Arg172Trp was associated with a younger age of onset. To the best of our knowledge, this is the largest patient cohort review of PRPH2‐related retinopathy. Large disease gene‐specific cohorts permit gene modeling for hotspot and disease‐enrichment analysis, providing novel variant classification evidence, including for novel missense variants.

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