Cloning and expression of rat 25-hydroxyvitamin D 3 -1α-hydroxylase cDNA

Abstract

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D₃-1α-hydroxylase (P4501α), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501α cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D₃-1α-hydroxylase activity. The sequence analysis showed that P4501α was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D₃-25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D₃-24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501α mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1α,25-dihydroxyvitamin D₃ (rats fed a low Ca diet), P4501α mRNA was greatly increased in the renal proximal convoluted tubules.