title: Cloning and expression of sterol Delta 14-reductase from bovine liver.
abstract: 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.
authors.affiliation: Department of Internal Medicine, University of Perugia, Italy. roberti@unipg.it
authors.forename: Rita
authors.initials: R
authors.lastname: Roberti
authors.forename: Anna Maria
authors.initials: AM
authors.lastname: Bennati
authors.forename: Giovanni
authors.initials: G
authors.lastname: Galli
authors.forename: Donatella
authors.initials: D
authors.lastname: Caruso
authors.forename: Bruno
authors.initials: B
authors.lastname: Maras
authors.forename: Cristina
authors.initials: C
authors.lastname: Aisa
authors.forename: Tommaso
authors.initials: T
authors.lastname: Beccari
authors.forename: Maria Agnese
authors.initials: MA
authors.lastname: Della Fazia
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Servillo
mesh_terms: 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
chemical_list: D012333:RNA, Messenger; D010088:Oxidoreductases; C022672:delta(14)-sterol reductase