Biosynthesis of cholesterol represents one of the funda- mental cellular metabolic processes. Sterol Delta14-reductase (D14-SR) is a microsomal enzyme involved in the con- version 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 D14-SR from yeasts, fungi, and plants (55±59%), sug- gesting that the bovine cDNA encodes D14-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. Immu- no¯uorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited D14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes D14- SR and provide evidence that the human TM7SF2 gene encodes D14-SR. Keywords: sterol biosynthesis; sterol reductase; cloning; endoplasmic reticulum.
Cloning and expression of sterol delta14-reductase from bovine liver
ROBERTI, Rita;BENNATI, Anna Maria;BECCARI, Tommaso;DELLA FAZIA, Maria Agnese;SERVILLO, Giuseppe
2002
Abstract
Biosynthesis of cholesterol represents one of the funda- mental cellular metabolic processes. Sterol Delta14-reductase (D14-SR) is a microsomal enzyme involved in the con- version 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 D14-SR from yeasts, fungi, and plants (55±59%), sug- gesting that the bovine cDNA encodes D14-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. Immu- no¯uorescence analysis of transfected cells revealed a distribution of the protein throughout the ER. COS-7 cells expressing the protein exhibited D14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes D14- SR and provide evidence that the human TM7SF2 gene encodes D14-SR. Keywords: sterol biosynthesis; sterol reductase; cloning; endoplasmic reticulum.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
