Cloning and characterization of a novel all-trans retinol short-chain dehydrogenase/reductase from the RPE.
OBJECTIVE
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.


METHODS
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.


RESULTS
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.


CONCLUSIONS
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.
Wu
Bill X
BX
Department of Ophthalmology, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Chen
Yumei
Y
Chen
Ying
Y
Fan
Jie
J
Rohrer
Baerbel
B
Crouch
Rosalie K
RK
Ma
Jian-Xing
JX
D000429:Alcohol Oxidoreductases / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D020224:Expressed Sequence Tags; D006801:Humans; D007124:Immunoenzyme Techniques; D017403:In Situ Hybridization; D051379:Mice; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D012333:RNA, Messenger / Q000378:metabolism; D012172:Retinaldehyde / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D014162:Transfection; D014801:Vitamin A / Q000378:metabolism
D012333:RNA, Messenger; D014801:Vitamin A; D000429:Alcohol Oxidoreductases; C113992:trans-retinol dehydrogenase; D012172:Retinaldehyde