Biochemical analysis of phenotypic diversity associated with mutations in codon 244 of the retinal degeneration slow gene

Abstract

Mutations in the protein product of the retinal degeneration slow (RDS) gene cause both rod-dominant retinitis pigmentosa and different forms of cone-dominant macular dystrophies. In particular, mutations in codon 244 can cause either of these types of disease. In this study, we examine the biochemical effects of N244H and N244K in an effort to understand the mechanism underlying rod- and cone-dominant defects, respectively. COS-1 cells were cotransfected with either wild-type (WT) RDS or RDS containing an N244H or N244K mutation along with its binding partner, ROM-1 (rod outer segment membrane protein 1). Cell extracts were analyzed for mutant protein stability by Western blot, and localization was examined by immunocytochemistry. Interactions between transfected proteins were assessed by reciprocal co-immunoprecipitation, and nonreducing velocity sedimentation was used to identify the pattern of RDS complex assembly. Interactions were confirmed using GST fusion constructs of WT and mutant RDS in GST pull-down assays from WT mouse retinal extract. In COS-1 cells, recombinant N244H RDS had a weakened ability to assemble into higher-order complexes but retained the ability to co-immunoprecipitate with ROM-1 as well as localize properly throughout the cells. In contrast, recombinant N244K protein did not associate with ROM-1, showed signs of protein aggregation, and colocalized with an ER marker. These experiments support the hypothesis that RDS mutations that interrupt higher-order oligomer formation but still interact with ROM-1 and fold properly in membranes may cause dominant, gain-of-function disease phenotypes while mutations that cause RDS misfolding (and thus incorrect trafficking and assembly) may be associated with a loss-of-function haploinsufficiency phenotype.

Figure 1. N244H RDS and N244K RDS are stably expressed in transfected COS-1 cells

COS-1 cells were transiently transfected with RDS (WT, C214S, R172W, N244H, and N244K) and WT ROM-1 constructs and proteins were extracted for non-reducing (A) or reducing (B) SDS-PAGE/Western blotting. (A) WT RDS is present in oligomeric, dimeric, and monomeric forms, while C214S RDS and N244K RDS are present as monomers and large aggregates. N244H RDS is present in monomeric and dimeric form, but little to no higher-order oligomer is detected. (B) Under reducing conditions, WT RDS, N244H RDS and R172W RDS are present in monomeric form, while C214S RDS and N244K RDS display significant evidence of aggregation. ROM-1 is shown to confirm co-transfection.

Figure 2. N244H RDS is distributed throughout the cell while N244K RDS is restricted to the ER

COS-1 cells were transfected with the indicated RDS vector (A) or the indicated RDS vector and ROM-1 (B). After 48hrs, cells were fixed and stained with (A) rabbit polyclonal antibody against RDS (red) and chicken polyclonal antibody against the ER marker calreticulin (green) or (B) rabbit polyclonal antibody against ROM-1 (blue), mouse monoclonal antibody 2B7 against RDS (red) and chicken polyclonal antibody against Calreticulin (green). (A) N244K and C214S RDS co-localize with the ER marker while WT, R172W, and N244H RDS are distributed throughout the internal membranes of the cell. (B) ROM-1 partially co-localizes with WT, R172W, and N244H RDS (pink) but not with C214S or N244K RDS. Co-expression of ROM-1 with RDS does not affect RDS localization. Scale bar, 10 μm. Images are single planes from spinning disk confocal stacks.

Figure 3. N244H RDS retains the ability to bind ROM-1 while N244K RDS does not

(A) Protein was extracted from COS-1 cells transiently co-transfected with RDS constructs and ROM-1 and RDS/ROM-1 complexes were reciprocally co-immunoprecipitated using polyclonal anti-RDS-CT or polyclonal anti-ROM-1-CT. Immunoprecipitants were processed for reducing SDS-PAGE/Western blotting with antibodies as shown. As expected, WT retinal extracts and WT COS-1 extracts bound ROM-1. N244H RDS transfected COS-1 extracts also bound ROM-1 but N244K RDS extracts did not regardless of whether ROM-1-CT or RDS-CT was used for the IP. ROM-1 expression in all co-transfected cells demonstrates that lack of ROM-1 expression does not account for the observed lack of association between N244K RDS and ROM-1. (B) GST fusion constructs carrying the RDS D2 loop (with or without additional mutation) were immobilized on a glutathione column and incubated with WT retinal extracts. Column eluents were processed for reducing SDS-PAGE/Western blotting as shown. All constructs retained the ability to bind retinal RDS as shown by immunoreactivity with anti-RDS-CT (which does not recognize the D2 fusion proteins) and all constructs brought down ROM-1. RDS-D2 and GST immunoreactivity are shown to confirm the presence of the fusion proteins.

Figure 4. Complex formation in N244H and N244K mutant RDS is altered

Protein extracts from WT retinas or COS-1 cells transfected with WT RDS, N244H RDS or N244K RDS underwent non-reducing velocity sedimentation on a 5-20% (as labeled) continuous sucrose gradient. Collected fractions were processed for reducing SDS-PAGE/Western blotting. The normal fractionation pattern (oligomers fractions 1-3, intermediate complexes fractions 4-6, tetramers fractions 7-10) was detected in WT retinal extracts. Peak fractions for molecular weight markers are shown with arrows; apoferritin (443kD), beta-amylase (200kD), alcohol dehydrogenase (150kD), bovine serum albumin (66kD), and carbonic anhydrase (29kD). As expected, the tetrameric fractions from WT RDS transfected COS-1 cells shifted slightly to the right. N244H RDS and N244K RDS were present mostly as tetramers although some immunoreactivity was detected in high molecular weight fractions.