S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice.
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 approximately 40-kDa as a major band in wild-type mice, whereas approximately 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, approximately 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.
Sato
Kota
K
Department of Biochemistry and Biotechnology, Division of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Aomori 036-8561, Japan.
Nakazawa
Mitsuru
M
Takeuchi
Kimio
K
Mizukoshi
Sayuri
S
Ishiguro
Sei-Ichi
S
D000818:Animals; D015153:Blotting, Western; D002352:Carrier Proteins / Q000502:physiology*; D002829:Choroid / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005136:Eye Proteins / Q000502:physiology*; D006031:Glycosylation; D007150:Immunohistochemistry; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000378:metabolism; D012160:Retina / Q000378:metabolism; D012162:Retinal Degeneration / Q000378:metabolism*; D055213:Retinal Pigment Epithelium / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012243:Rhodopsin / Q000235:genetics / Q000378:metabolism; D017299:Rod Opsins / Q000235:genetics / Q000378:metabolism*; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase
D002352:Carrier Proteins; D005136:Eye Proteins; D012333:RNA, Messenger; D012243:Rhodopsin; D017299:Rod Opsins; D019745:cis-trans-Isomerases; D000099178:Retinoid Isomerohydrolase; C095854:middle-wavelength opsin; C095853:short-wavelength opsin