Cloning and functional expression of a first inducible avian cytochrome P450 of the CYP3A subfamily (CYP3A37).
CYP3As represent a family of cytochromes P450 involved in the metabolism of both endogenous and exogenous natural and synthetic compounds. Well described in mammals, none have yet been cloned and characterized in avian species. In this paper, we report the cloning and analysis of an avian CYP3A (CYP3A37). Using an RNA differential display approach, an 80-bp phenobarbital-inducible cDNA fragment was amplified from chicken embryo liver. Based on its homology with mammalian CYP3As, this fragment was used to clone a full-length cDNA consisting of 1638 bp encoding a putative protein of 509 amino acids. The sequence shares between 57.4 and 62% identity at the amino acid level with CYP3As of other species. This cDNA was designated CYP3A37 according to the current cytochrome P450 nomenclature. When expressed in COS1 cells, the CYP3A37 cDNA produced a protein of congruent with55 kDa, which was recognized by polyclonal anti-rat CYP3A1 antiserum. In a bacterial expression system, the CYP3A37 cDNA produced a protein capable of steroid 6beta-hydroxylation. At a substrate concentration of 100 microM, progesterone, testosterone, and androstenedione were found to be 6beta-hydroxylated at a rate of 15.4, 11.7, 12.2 nmol/min/nmol P450, respectively. Used as control, the human CYP3A4 gave similar hydroxylation rates. Finally, in both chicken embryo liver and chicken hepatoma cells (LMH), CYP3A37 mRNA was increased after treatment with typical CYP3A inducers, such as metyrapone, phenobarbital, dexamethasone, and pregnenolone 16alpha-carbonitrile, but not rifampicin. CYP2H1, a well-characterized inducible chicken cytochrome P450, also was induced by the same compounds, suggesting similar regulation of CYP3 and CYP2 genes in this species.
Ourlin
J C
JC
Biozentrum, University of Basel, Klingelbergstrasse 70, Basel, 4056, Switzerland.
Baader
M
M
Fraser
D
D
Halpert
J R
JR
Meyer
U A
UA
D000595:Amino Acid Sequence; D000735:Androstenedione / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases; D001483:Base Sequence; D019556:COS Cells; D002478:Cells, Cultured; D002642:Chick Embryo; D002645:Chickens; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D004790:Enzyme Induction / Q000187:drug effects; D008969:Molecular Sequence Data; D010089:Oxidoreductases, N-Demethylating / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010634:Phenobarbital / Q000494:pharmacology; D010802:Phylogeny; D011374:Progesterone / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity; D013739:Testosterone / Q000378:metabolism
D012333:RNA, Messenger; D013739:Testosterone; D000735:Androstenedione; D011374:Progesterone; D003577:Cytochrome P-450 Enzyme System; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; D010089:Oxidoreductases, N-Demethylating; D010634:Phenobarbital