Cloning and expression of a cDNA encoding bovine retinal pigment epithelial 11-cis retinol dehydrogenase.
OBJECTIVE
Identification of a 32-kd protein in the bovine retinal pigment epithelium.


METHODS
A bovine retinal pigment epithelium cDNA library was constructed in the bacteriophage lambda ZAP Express. A monoclonal antibody, designated 21-C3/AV, was used to isolate the cDNA encoding the 21-C3/AV antigen. A positive full-length clone, designated 21-C3RDH/CD, was sequenced. Northern blot analysis was used to determine the length of the mRNA and the tissue expression pattern. The entire open reading frame of clone 21-C3RDH/CD was used to isolate a recombinant baculovirus clone and expressed in Spodoptera frugiperda insect cells. Enzymatic activity toward 11-cis retinaldehyde was investigated.


RESULTS
The complete nucleotide sequence of 21-C3RDH/CD was obtained. The deduced amino acid sequence reveals homology with short-chain alcohol dehydrogenases. Northern blot analysis detected a 1.2-kb transcript. Although the monoclonal antibody used to isolate 21-C3RDH/CD also reacts with other ocular and nonocular tissues, the authors were unable to demonstrate any reactivity with RNA samples isolated from different (non)ocular tissues. Recombinant baculovirus-infected insect cells synthesized the 21-C3/AV antigen. This protein showed 11-cis retinol dehydrogenase activity.


CONCLUSIONS
Homology to the human D-beta-hydroxybutyrate dehydrogenase precursor and other alcohol dehydrogenases shows that 21-C3RDH/CD encodes a short-chain alcohol dehydrogenase. Furthermore, tissue specificity and molecular weight of the antigen suggest that 21-C3RDH/CD encodes the bovine retinal pigment epithelial 11-cis retinol dehydrogenase. Direct proof came from experiments in which we used the baculovirus-based expression system for in vitro synthesis of the protein encoded by 21-C3RDH/CD. Protein extracts obtained from recombinant baculovirus-infected insect cells were found capable of reducing 11-cis retinaldehyde.
Driessen
C A
CA
Institute of Ophthalmology, University of Nijmegen, The Netherlands.
Janssen
B P
BP
Winkens
H J
HJ
van Vugt
A H
AH
de Leeuw
T L
TL
Janssen
J J
JJ
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000429:Alcohol Oxidoreductases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D016367:Baculoviridae; D001483:Base Sequence; D015152:Blotting, Northern; D002417:Cattle; D002478:Cells, Cultured; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics*; D015971:Gene Expression Regulation, Enzymologic; D008969:Molecular Sequence Data; D010857:Pigment Epithelium of Eye / Q000201:enzymology*; D012313:RNA / Q000302:isolation & purification; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D018411:Spodoptera
D000911:Antibodies, Monoclonal; D018076:DNA, Complementary; D012313:RNA; D000429:Alcohol Oxidoreductases; D000426:Alcohol Dehydrogenase; C022520:retinol dehydrogenase