Cloning, tissue distribution, and functional studies of a new cytochrome P450 3A subfamily member, CYP3A45, from rainbow trout (Oncorhynchus mykiss) intestinal ceca.
In trout and mammals, the major extrahepatic expression site for CYP3A forms is in the intestine. A cDNA encoding a new CYP3A subfamily member was isolated from rainbow trout intestinal ceca by reverse transcriptase-polymerase chain reaction (RT-PCR), followed by rapid amplification of cDNA ends (RACE)-PCR. In a set of two primers for PCR, a consensus sequence in the highly conserved regions in 17 CYP3A sequences was used for one primer, and the second primer was designed based on adapter sequence ligated on the 5(') and 3(') cDNA ends. The 3(') and 5(') end nucleotide sequences of RACE-PCR products were used for the priming sites for the full-length cDNA in RT-PCR. The resulting 2615-bp cDNA contained an open reading frame of 1554 bp encoding a 518-amino acid residue protein (M(r)=59057.13, pI=6.15) with 26 amino acid differences from that of the previously cloned rainbow trout CYP3A27. The cDNA was assigned as CYP3A45 by the P450 Nomenclature Committee. The deduced amino acid sequence of rainbow trout CYP3A45 was 94% identical with trout CYP3A27, 72% with killifish CYP3A56, and 71% with both medaka CYP3A40 and killifish CYP3A30 in positional alignment comparisons. Northern blotting by a CYP3A45-specific nucleotide probe showed that the major expression site was the intestinal ceca rather than the liver in both male and female trout. Recombinant baculovirus containing a CYP3A45 cDNA (Bv-3A45) was constructed under polyhedrin promoter of the Autographa californica nuclear polyhedrosis virus and used to express CYP3A45 protein in Spodoptera frugiperda cells. The Western blot showed that the expressed CYP3A45 protein comigrated with purified LMC5 P450 and was recognized by anti-LMC5 polyclonal antibodies. The expressed CYP3A45 showed catalytic activities for the 6 beta-, 2 beta-, and 16 beta-hydroxytestosterones of 1.76, 0.193, and 0.078 nmol/min/nmol CYP3A45, respectively. In summary, a second form of CYP3A with steroid hydroxylase activity, CYP3A45, has been cloned from rainbow trout and the major site of expression was in the intestinal tissues.
Lee
Su Jun
SJ
Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR 97331-7301, USA.
Buhler
Donald R
DR
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000378:metabolism; D001483:Base Sequence; D001665:Binding Sites; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D005260:Female; D015723:Gene Library; D005822:Genetic Vectors; D007313:Insecta; D007413:Intestinal Mucosa / Q000378:metabolism; D007422:Intestines / Q000201:enzymology*; D008099:Liver / Q000201:enzymology / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D017686:Oncorhynchus mykiss; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012333:RNA, Messenger / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013739:Testosterone / Q000378:metabolism; D014018:Tissue Distribution
D018076:DNA, Complementary; D012333:RNA, Messenger; D013739:Testosterone; D004247:DNA; D003577:Cytochrome P-450 Enzyme System; C473667:cytochrome P-450 CYP3A45