Cloning and expression of sterol Delta 14-reductase from bovine liver.
Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes. Sterol Delta 14-reductase (Delta 14-SR) is a microsomal enzyme involved in the conversion of lanosterol to cholesterol in mammals. Amino-acid sequence analysis of a 38-kDa protein purified from bovine liver in our laboratory revealed > 90% similarity with a human sterol reductase, SR-1, encoded by the TM7SF2 gene, and with the C-terminal domain of human lamin B receptor. A cDNA encoding the 38-kDa protein, similar to human TM7SF2, was identified by analysis of a bovine expressed sequence tag (EST) database. The cDNA was synthesized by RT-PCR, cloned, and sequenced. The cDNA encodes a 418 amino-acid polypeptide with nine predicted transmembrane domains. The deduced amino-acid sequence exhibits high similarity with Delta 14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta 14-SR. Northern blot analysis of bovine tissues showed high expression of mRNA in liver and brain. The polypeptide encoded by the cloned cDNA was expressed in COS-7 cells. Immunofluorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited Delta 14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta 14-SR and provide evidence that the human TM7SF2 gene encodes Delta 14-SR.
Roberti
Rita
R
Department of Internal Medicine, University of Perugia, Italy. roberti@unipg.it
Bennati
Anna Maria
AM
Galli
Giovanni
G
Caruso
Donatella
D
Maras
Bruno
B
Aisa
Cristina
C
Beccari
Tommaso
T
Della Fazia
Maria Agnese
MA
Servillo
Giuseppe
G
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002417:Cattle; D003001:Cloning, Molecular; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006801:Humans; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection
D012333:RNA, Messenger; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase