S-opsin protein is incompletely modified during N-glycan processing in Rpe65−/− mice

Abstract

Retinal pigment epithelium-specific protein 65 kDa (RPE65) is a key enzyme for the visual cycle in the eye. Rpe65 −/− mice lack 11- cis-retinal, and show early cone degeneration and mislocalization of cone opsins. The present study investigated whether abnormal modification of cone opsins at the protein level is present in Rpe65 −/− mice. Retina–RPE–choroids of Rpe65 −/− mice at 3, 5 and 7 weeks old were used. Immunohistochemistry of opsins was performed using cryosections and retinal flatmounts. We evaluated levels of mRNA for cone and rod opsin genes by RT-PCR and levels of proteins by western blotting. To examine modification patterns of N-glycan in Rpe65 −/− mice, cone opsins were digested with peptide-N-glycosidase (PNGase) F. S-opsin protein was detected at ∼40-kDa as a major band in wild-type mice, whereas ∼42-kDa S-opsin protein was detected in Rpe65 −/− mice. After PNGase F treatment, mobility of S-opsin protein in wild-type and Rpe65 −/− mice on SDS-PAGE was similar. In addition, ∼25-kDa S-opsin polypeptide was notably detected in Rpe65 −/− mice. Conversely, M-opsin proteins were not observed by immunohistochemistry or western blotting in Rpe65 −/− mice, but expression of M-opsin mRNA in Rpe65 −/− mice did not differ significantly from that in wild-type mice at 3 and 5 weeks. Mobility of M-opsin protein in Rpe65 −/− mice was unchanged. Our data suggest that S-opsin protein is incompletely modified during N-glycan processing in Rpe65 −/− mice, whereas M-opsin protein is severely reduced by posttranslational degradation in the absence of incomplete N-glycan processing in Rpe65 −/− mice.