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

Kota Sato, Mitsuru Nakazawa, Kimio Takeuchi, Sayuri Mizukoshi, Sei ichi Ishiguro

Research output: Contribution to journalArticle

13 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)54-62
Number of pages9
JournalExperimental Eye Research
Volume91
Issue number1
DOIs
Publication statusPublished - 2010 Jul
Externally publishedYes

Keywords

  • 11-cis-retinal
  • Degradation
  • N-glycosylation
  • RPE65
  • S-opsin

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

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