27-Oxygenation of C27-sterols and 25-hydroxylation of vitamin D3 in kidney: cloning, structure and expression of pig kidney CYP27A.
This paper describes the molecular cloning of a cytochrome P450 enzyme in pig kidney that catalyses the hydroxylations of vitamin D(3) (cholecalciferol) and C(27)-sterols. DNA sequence analysis of the cDNA revealed that the enzyme belongs to the CYP27 family. The first 36 amino acids have many hallmarks of a mitochondrial signal sequence. The mature pig kidney CYP27 protein contains 498 amino acids. The M(r) of the mature protein was calculated to be 56607. The structure of pig kidney CYP27, as deduced by DNA sequence analysis, shows 77-83% identity with CYP27A in rat, rabbit and human liver. Transfection of the renal CYP27A cDNA into simian COS cells resulted in the synthesis of an enzyme that catalysed the 25-hydroxylation of vitamin D(3) and the 27-hydroxylation of 5beta-cholestane-3alpha,7alpha,12alpha-triol, and the further oxidation of the product into the corresponding C(27)-acid 3alpha,7alpha,12alpha-trihydroxy-5beta-cholestanoic acid. As part of these studies, the enzymic activities of cultured human embryonic kidney cells were examined using vitamin D(3) and C(27)-sterols as substrates. The cells were found to express CYP27A mRNA and to convert the respective substrates into the same products as recombinantly expressed CYP27A, i.e. 25-hydroxyvitamin D(3) and 27-oxygenated C(27)-sterols. The results of the present study describing the structure and expression of CYP27A in kidney suggest that this enzyme is involved in the renal metabolism of vitamin D(3) and that the kidney plays a role in the metabolism of cholesterol and other C(27)-sterols.
Postlind
H
H
Division of Biochemistry, Department of Pharmaceutical Biosciences, University of Uppsala, Box 578, S-751 23 Uppsala, Sweden.
Hosseinpour
F
F
Norlin
M
M
Wikvall
K
K
D000595:Amino Acid Sequence; D000818:Animals; D015139:Blotting, Southern; D019556:COS Cells; D002460:Cell Line; D002762:Cholecalciferol / Q000737:chemistry / Q000378:metabolism*; D053493:Cholestanetriol 26-Monooxygenase; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D006900:Hydroxylation; D007668:Kidney / Q000166:cytology / Q000196:embryology / Q000201:enzymology* / Q000378:metabolism; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D010100:Oxygen / Q000378:metabolism; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D016415:Sequence Alignment; D013250:Steroid Hydroxylases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D013261:Sterols / Q000737:chemistry / Q000378:metabolism*; D013552:Swine / Q000235:genetics*
D012333:RNA, Messenger; D013261:Sterols; D002762:Cholecalciferol; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase; D010100:Oxygen