Wu Bill X, Chen Yumei, Chen Ying, Fan Jie, Rohrer Baerbel, Crouch Rosalie K, Ma Jian-Xing
Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Invest Ophthalmol Vis Sci. 2002 Nov;43(11):3365-72.
In the photic visual cycle, retinal G protein-coupled receptor (RGR) isomerizes all-trans retinal to 11-cis retinal in the retinal pigment epithelium (RPE) after illumination. It is unclear, however, how all-trans retinal, the substrate for RGR, is generated in the RPE, because no all-trans retinol dehydrogenase (atRDH) has been identified in the RPE. This study was conducted to identify the atRDH that generates all-trans retinal in the RPE.
The full-length cDNA encoding a novel atRDH, RDH10, was cloned by PCR based on an expressed sequence tag (EST). Cellular localization was determined at the mRNA level by Northern blot analysis, RT-PCR, and in situ hybridization and at the protein level by immunohistochemistry with an antibody specific to RDH10. The activity was measured by an RDH activity assay with recombinant RDH10 expressed in COS cells.
The full-length RDH10 was cloned from the human, cow, and mouse. These cDNAs encode a protein of 341 amino acids and have significant sequence homology with other short-chain dehydrogenases/reductases (SDRs). The human RDH10 shares 100% and 98.6% amino acid sequence identity with the bovine and mouse proteins, respectively, suggesting a highly conserved sequence during evolution. RDH10 is predominantly expressed in the microsomal fraction of the RPE. Human RDH10 expressed in COS cells oxidized all-trans retinol to all-trans retinal. RDH10 displayed substrate specificity for all-trans retinol and preferred nicotinamide adenine dinucleotide phosphate (NADP) as the cofactor.
RDH10 is a novel retinol oxidase expressed in the RPE. This enzyme can generate all-trans retinal from all-trans retinol and may play an important role in the photic visual cycle.
在光视觉循环中，视网膜G蛋白偶联受体（RGR）在光照后于视网膜色素上皮（RPE）中将全反式视黄醛异构化为11-顺式视黄醛。然而，尚不清楚作为RGR底物的全反式视黄醛是如何在RPE中生成的，因为在RPE中尚未鉴定出全反式视黄醇脱氢酶（atRDH）。本研究旨在鉴定在RPE中生成全反式视黄醛的atRDH。
基于表达序列标签（EST）通过PCR克隆编码新型atRDH即RDH10的全长cDNA。通过Northern印迹分析、RT-PCR和原位杂交在mRNA水平以及使用针对RDH10的特异性抗体通过免疫组织化学在蛋白质水平确定细胞定位。通过在COS细胞中表达的重组RDH10的RDH活性测定来测量活性。
从人、牛和小鼠中克隆出全长RDH10。这些cDNA编码一种341个氨基酸的蛋白质，并且与其他短链脱氢酶/还原酶（SDR）具有显著的序列同源性。人RDH10与牛和小鼠蛋白质的氨基酸序列同一性分别为100%和98.6%，表明在进化过程中序列高度保守。RDH10主要在RPE的微粒体部分表达。在COS细胞中表达的人RDH10将全反式视黄醇氧化为全反式视黄醛。RDH10对全反式视黄醇表现出底物特异性，并且优选烟酰胺腺嘌呤二核苷酸磷酸（NADP）作为辅因子。
RDH10是一种在RPE中表达的新型视黄醇氧化酶。这种酶可以从全反式视黄醇生成全反式视黄醛，并且可能在光视觉循环中起重要作用。
