Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes, Sterol Delta14-reductase (Delta14-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 Delta14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta14-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 Delta14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta14-SR and provide evidence that the human TM7SF2 gene encodes Delta14-SR.
Cloning and expression of sterol Delta 14-reductase from bovine liver / R., Roberti; A. M., Bennati; G., Galli; D., Caruso; Maras, Bruno; C., Aisa; T., Beccari; M. A., Della Fazia; G., Servillo. - In: EUROPEAN JOURNAL OF BIOCHEMISTRY. - ISSN 0014-2956. - 269:1(2002), pp. 283-290. [10.1046/j.0014-2956.2001.02646.x]
Cloning and expression of sterol Delta 14-reductase from bovine liver
MARAS, Bruno;
2002
Abstract
Biosynthesis of cholesterol represents one of the fundamental cellular metabolic processes, Sterol Delta14-reductase (Delta14-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 Delta14-SR from yeasts, fungi, and plants (55-59%), suggesting that the bovine cDNA encodes Delta14-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 Delta14-SR activity about sevenfold higher than control cells. These results demonstrate that the cloned bovine cDNA encodes Delta14-SR and provide evidence that the human TM7SF2 gene encodes Delta14-SR.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
