Identification of RDH10, an All-trans Retinol Dehydrogenase, in Retinal Muller Cells.
Identification of RDH10, an All-trans Retinol Dehydrogenase, in Retinal Muller Cells.
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DOI：
10.1167/iovs.03-1302
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发表时间：
2004-11
影响因子：
4.4
通讯作者：
Bill X. Wu;G. Moiseyev;Ying Chen;B. Rohrer;R. Crouch;Jian-xing Ma
中科院分区：
文献类型：
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作者：
Bill X. Wu;G. Moiseyev;Ying Chen;B. Rohrer;R. Crouch;Jian-xing Ma
目的研究视网膜色素上皮（RPE）中发现的全反式视黄醇脱氢酶RDH10在视网膜Muller细胞中的表达。方法采用抗RDH10多克隆抗体，采用Western blot方法检测小鼠眼液和牛组织中RDH10蛋白水平。采用免疫组化方法确定视网膜细胞定位。采用RT-PCR和Western blot检测大鼠Muller细胞rMC-1中RDH10的表达。使用rMC-1细胞的裂解物进行全反式视黄醇脱氢酶活性测定。用高效液相色谱法分析了由氚化全反式视黄醇生成全反式视网膜的过程。结果RDH10、视网膜G蛋白偶联受体（RGR）和RPE65在BALB/c小鼠的眼窝中的表达水平均高于C57Bl/6小鼠。除了RPE外，RDH10在视网膜和肝脏中也检测到较低的水平。免疫组化显示RDH10定位于视网膜切片Muller细胞中。RDH10在rMC-1细胞中以RNA和蛋白水平被检测到。从rMC-1细胞中克隆出含有全长编码区的大鼠RDH10 cDNA。大鼠RDH10 cDNA编码一个341个氨基酸的蛋白，在氨基酸水平上与人、牛和小鼠RDH10具有99%的序列一致性。在rMC-1细胞中，微粒体部分检测到全反式视黄醇脱氢酶活性。结果表明NADP为首选辅因子，这与重组RDH10的首选辅因子一致。结论RDH10除RPE外，在视网膜Muller细胞中也有表达。RDH10产生全反式视网膜，这是Muller细胞中光异构酶RGR的底物。
PURPOSE To investigate the expression of RDH10, an all-trans retinol dehydrogenase identified in the retinal pigment epithelium (RPE), in retinal Muller cells. METHODS The RDH10 protein levels in mouse eyecups and bovine tissues were examined by Western blot analysis using a polyclonal antibody against RDH10. The cellular localization in the retina was determined by immunohistochemistry. Expression of RDH10 in rMC-1, a cell line derived from rat Muller cells, was determined by RT-PCR and Western blot analysis. All-trans retinol dehydrogenase activity assays were performed using lysates from rMC-1 cells. The generation of all-trans retinal from tritiated all-trans retinol was analyzed by HPLC. RESULTS RDH10, retinal G protein-coupled receptor (RGR), and RPE65 all had higher expression levels in the eyecups of BALB/c than in C57Bl/6 mice. In addition to the RPE, RDH10 was also detected at lower levels in the retina and liver. Immunohistochemistry showed that RDH10 was localized in Muller cells in retinal sections. RDH10 was detected in rMC-1 cells, at both the RNA and protein levels. The rat RDH10 cDNA containing the full-length coding region was cloned from rMC-1 cells. The rat RDH10 cDNA encodes a protein of 341 amino acids and shares 99% sequence identity with human, bovine, and mouse RDH10 at the amino acid level. In rMC-1 cells, all-trans retinol dehydrogenase activity was detected in the microsomal fraction. NADP was shown to be the preferred cofactor, which is identical with the cofactor preference of the recombinant RDH10. CONCLUSIONS RDH10 was expressed in retinal Muller cells, in addition to the RPE. RDH10 generates all-trans retinal, which is the substrate for the photoisomerase RGR in Muller cells.