Abstract
Purpose :
Progressive rod-cone degeneration (PRCD) is a 54 amino acid protein associated with retinitis pigmentosa (RP) in humans and dogs. Our previous study demonstrated that PRCD undergoes S-palmitoylation, which is required for PRCD’s stability and trafficking to the photoreceptor outer segment (OS), where PRCD is exclusively present. Furthermore, PRCD has predicted structural domains, including a “transmembrane helix” and ‘polybasic region’. However, little is known regarding the contributions of these structural domains to PRCD’s function in the photoreceptor. The goal of this study is to focus on the polybasic region, where the R17C is linked with RP in humans.
Methods :
Constructs, including HA-tagged wild type (WT) and R17C mutant human PRCD were transfected into human retinal pigment epithelial (hRPE1) cells. Protein stability, membrane association, and subcellular localization was determined by western blot, subcellular fractionation, and immunocytochemistry (ICC). WT and R17C mutant PRCD constructs were also injected into the sub-retinal space of murine eyes followed by electroporation at postnatal (P) day 0. Injected eyes were collected at P21 and evaluated for PRCD localization by immunofluorescent staining. Palmitoylation status of R17C-PRCD in cells and injected retina was assayed by acyl-resin assisted capture (Acyl-RAC).
Results :
Protein expression of R17C-PRCD was approximately 30% lower than WT-PRCD in hRPE1 cells. Interestingly, palmitoylation status of R17C-PRCD is unchanged, and membrane fractionation studies demonstrate that R17C-PRCD protein remains associated with the membrane. Furthermore, when R17C-PRCD was co-expressed with the palmitoyltransferase zDHHC3 in cells, protein expression was up-regulated. ICC staining of WT-PRCD appears punctate and is present throughout the cytoplasm of RPE1 cells, while R17C-PRCD is present in the mitochondria and cytoplasm. Additionally, sub-retinal injection of R17C-PRCD demonstrates that the majority of R17C-PRCD is mislocalized to the photoreceptor inner segment, compared to WT-PRCD, which is localized to the OS.
Conclusions :
Our studies demonstrate that an R17C mutation in PRCD does not affect membrane association or palmitoylation. However, the mutation does affect PRCD protein stability and trafficking to the photoreceptor OS, which may be why this mutation is associated with RP in human patients.
This is a 2020 ARVO Annual Meeting abstract.