Development of A New Cholesterol Lowering Drug to Prevent Atherosclerosis

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Squalene epoxidase (SE) (EC 1.14.99.7) catalyzes the first oxygenation step in sterol biosynthesis and is suggested to be one of the rate-limiting enzymes in this pathway. Rat SE cDNA was isolated by selecting yeast transformants expressing rat cDNA in the presence of transformants expressing rat cDNA in the presence of terbinafine, an inhibitor specific for fungal SE.Mouse and human SE cDNA was isolated by library screening using rat SE cDNA as a probe. SE polypeptide deduced from the nucleotide sequence contains 573 amino acids, and its molecular weight is 63,950 Da. The amino acid sequence reveals one potential transmembrane domain, a hydrophobic segment (Leu27 to Tyr43) in the NH2-terminal region. This region also contains a beta 1-alpha A-bata 2 motif, which is the consensus sequence for an FAD binding domain. This deduced rat SE sequence is 30.2% identical to the ERG 1 gene, which encodes SE from an allylamine-resistant Saccharomyces cerevisiae mutant. The cDNA had an open reading frame for a 572 amino acid polypeptide with a calculated molecular mass of 63.8 kDa. The predicted amino acid sequence of the mouse enzyme contained an FAD-binding motif, and was 93%, 83% identical to those of the rat and human enzymes, respectively. Blotting analyzes showed that the mRNA is 2.8 kb in size and that a single copy of the gene is present in the mouse genome. PCR revealed that human SE gene to chromosomes 8 by using the NIGMS Human/Rodent Somatic Cell Hybrid Mapping Panel 2 as templates. To refine the localization of the human SE gene showed that the human SE gene linked with the micro-satellite marker D8S508 which was reported to be localized in 8q 24.13-q telomere (Lod score 7.87). Moreover, fluorescence in situ hybridization also map the human SE gene to 8q 24.13.