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Two Peeling Methods for the Isolation of Photoreceptor Cell Compartments in the Mouse Retina for Protein Analysis
Published on: December 7, 2021
S-opsin protein is incompletely modified during N-glycan processing in Rpe65(-/-) mice
Kota Sato1, Mitsuru Nakazawa, Kimio Takeuchi
1Department of Biochemistry and Biotechnology, Division of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Aomori 036-8561, Japan.
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 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.
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