Department of Biochemistry and Biotechnology, Division of Cell Technology, Faculty of Agriculture and Life Science, Hirosaki University, 3 Bunkyo-cho, Aomori 036-8561, Japan.
Exp Eye Res. 2010 Jul;91(1):54-62. doi: 10.1016/j.exer.2010.03.020. Epub 2010 Apr 14.
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.
视网膜色素上皮细胞 65kDa 特异性蛋白（RPE65）是眼睛视觉循环中的关键酶。Rpe65(-/-) 小鼠缺乏 11-顺式视黄醛，表现出早期的锥体变性和锥体视蛋白的错位。本研究旨在探讨 Rpe65(-/-) 小鼠中锥体视蛋白在蛋白质水平上是否存在异常修饰。使用 3、5 和 7 周龄的 Rpe65(-/-) 小鼠的视网膜-色素上皮-脉络膜。使用冷冻切片和视网膜平面培养物进行视蛋白免疫组织化学染色。我们通过 RT-PCR 评估锥体和杆状视蛋白基因的 mRNA 水平，并通过 Western blot 评估蛋白水平。为了检查 Rpe65(-/-) 小鼠中 N-糖基化的修饰模式，用肽-N-糖苷酶（PNGase）F 消化锥体视蛋白。在野生型小鼠中，S-视蛋白主要以约 40-kDa 的条带形式检测到，而在 Rpe65(-/-) 小鼠中检测到约 42-kDa 的 S-视蛋白蛋白。经 PNGase F 处理后，野生型和 Rpe65(-/-) 小鼠的 SDS-PAGE 上 S-视蛋白蛋白的迁移率相似。此外，在 Rpe65(-/-) 小鼠中还明显检测到约 25-kDa 的 S-视蛋白多肽。相反，在 Rpe65(-/-) 小鼠中未通过免疫组织化学或 Western blot 检测到 M-视蛋白蛋白，但在 3 和 5 周时，Rpe65(-/-) 小鼠中 M-视蛋白 mRNA 的表达与野生型小鼠没有显著差异。Rpe65(-/-) 小鼠中 M-视蛋白蛋白的迁移率没有变化。我们的数据表明，在 Rpe65(-/-) 小鼠中，S-视蛋白蛋白在 N-糖基化加工过程中不完全修饰，而在缺乏不完全 N-糖基化加工的情况下，M-视蛋白蛋白严重减少。
