Expression pattern and localization of beta,beta-carotene 15,15'-dioxygenase in different tissues.
Beta,beta-carotene 15,15'-dioxygenase cleaves beta,beta-carotene into two molecules of retinal, and is the key enzyme in the metabolism of beta,beta-carotene to vitamin A. The enzyme has been known for more than 40 years, yet all attempts to purify the protein to homogeneity have failed. Recently, the successful cloning and sequencing of an enzyme with beta,beta-carotene 15,15'-dioxygenase activity from chicken, as well as from Drosophila, has been reported. Here, we describe in detail our attempt to enrich the chicken beta,beta-carotene 15,15'-dioxygenase to such an extent as to allow determination of partial amino acid sequences, which were then used to design degenerate oligonucleotides. Screening of a chicken duodenal expression library yielded a full-length clone containing a coding sequence of 1578 bp. Functional expression in Escherichia coli and in eukaryotic cell lines confirmed that we had cloned the first vertebrate dioxygenase that cleaves beta,beta-carotene at the central 15,15'-double bond. By performing a sequence homology search, the cDNA sequence of the mouse homologue was found as an expressed sequence tag (EST) in the gene bank. At the amino-acid level, the degree of homology between the chicken and mouse sequences is 81%. Thus beta,beta-carotene 15,15'-dioxygenase can be considered as being an enzyme that is evolutionarily rather well conserved. We established the expression pattern of beta,beta-carotene 15,15'-dioxygenase in chicken and mouse tissues with a combination of Northern blots and in situ hybridization. The mRNA for beta,beta-carotene 15,15'-dioxygenase was localized primarily in duodenal villi, as well as in liver and in tubular structures of lung and kidney. These new findings demonstrate that beta,beta-carotene 15,15'-dioxygenase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply.
F. Hoffmann-La Roche Ltd., Vitamins & Fine Chemicals Division, 4070 Basel, Switzerland. adrian.wyss@roche.com
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Wyss
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Wirtz
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Woggon
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Brugger
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Wyss
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Friedlein
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Riss
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Bachmann
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Hunziker
D000595:Amino Acid Sequence; D000818:Animals; D015152:Blotting, Northern; D016466:CHO Cells; D002645:Chickens; D003001:Cloning, Molecular; D006224:Cricetinae; D004847:Epithelial Cells / Q000378:metabolism; D017403:In Situ Hybridization; D007413:Intestinal Mucosa / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D012333:RNA, Messenger / Q000096:biosynthesis; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014162:Transfection; D050559:beta-Carotene 15,15'-Monooxygenase
D012333:RNA, Messenger; D010105:Oxygenases; C493359:Bco1 protein, mouse; D050559:beta-Carotene 15,15'-Monooxygenase