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 -carote ne 15,15'-dioxygenase activity from chicken, as well as from Drosophila, ha s been reported. Here, we describe in detail our attempt to enrich the chic ken beta,beta -carotene 15,15'-dioxygenase to such an extent,as to allow de termination of partial amino acid sequences, which were then used to design degenerate oligonucleotides. Screening of a chicken duodenal expression li brary yielded a full-length clone containing a coding sequence of 1578 bp. Functional expression in Escherichia coli and in eukaryotic cell lines conf irmed 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 be ta,beta -carotene 15,15'-dioxygenase can be considered as being an enzyme t hat is evolutionarily rather well conserved. We established the expression pattern of beta,beta -carotene 15,15'-dioxygenase in chicken and mouse tiss ues with a combination of Northern blots and in situ hybridization. The mRN A for beta,beta -carotene 15,15'-dioxygenase was localized primarily in duo denal villi, as well as in liver and in tubular structures of lung and kidn ey. These new findings demonstrate that beta,beta -carotene 15,15'-dioxygen ase is also expressed in epithelial structures, where it serves to provide the tissue-specific vitamin A supply.