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Proc. Natl. Acad. Sci. USA Vol. 94, pp. 12920-12925, November 1997

Biochemistry

Cloning and expression of rat 25-hydroxyvitamin D3-1alpha -hydroxylase cDNA

(vitamin D metabolism / 1alpha -hydroxylase / 24-hydroxylase / P450)

Toshimasa Shinki*,,, Hiroko Shimada,§, Shu Wakino, Hideharu Anazawa§, Matsuhiko Hayashi, Takao Saruta, Hector F. DeLucapar , and Tatsuo Suda*

* Department of Biochemistry, School of Dentistry, Showa University, Hatanodai, Shinagawa-ku, Tokyo 142, Japan; § Tokyo Research Laboratory, Kyowa Hakko Kogyo Co. Ltd., Asahimachi, Machida, Tokyo 194, Japan;  Department of Internal Medicine, University of Keio, Shinanomachi, Shinjuku-ku, Tokyo 160, Japan; and par  Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin, 420 Henry Mall, Madison, WI 53706

Contributed by Hector F. DeLuca, September 16, 1997

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D3-1alpha -hydroxylase (P4501alpha ), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(+). When P4501alpha cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D3-1alpha -hydroxylase activity. The sequence analysis showed that P4501alpha 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 P4501alpha mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1alpha ,25-dihydroxyvitamin D3 (rats fed a low Ca diet), P4501alpha mRNA was greatly increased in the renal proximal convoluted tubules.


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