Cholesterol biosynthesis from lanosterol - Molecular cloning, tissue distribution, expression, chromosomal localization, and regulation of rat 7-dehydrocholesterol reductase, a Smith-Lemli-Opitz syndrome-related protein

The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DH CR), an enzyme that has been implicated in both cholesterol biosynthesis an d developmental abnormalities (e.g. Smith-Lemli-Opitz syndrome) in mammals, has been cloned and sequenced, and the primary structure of the enzyme has been deduced. The DHCR gene was mapped to chromosome 8q2.1 by fluorescence in situ hybridization, Rat DHCR, calculated molecular mass of 54.15-kDa po lypeptide, shares a close amino acid identity with mouse and human DHCRs (9 6 and 87%, respectively) as compared with its other related proteins (e.g. fungal sterol Delta(14)-reductase) and exhibits high hydrophobicity (>68%) with 9 transmembrane domains. Five putative sterol-sensing domains were pre dicted to be localized in transmembrane domains 4-8, which are highly homol ogous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regul atory element-binding protein cleavage-activating protein, and patched prot ein. The polypeptide encoded by DHCR cDNA was expressed in yeast as a 55.45 -kDa myc-tagged fusion protein, which was recognized with anti-myc monoclon al antibody 9E10 and shown to possess full DHCR activity with respect to de pendence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysi s indicates that the highest expression of DHCR mRNA was detected in liver, followed by kidney and brain. In rat brains, the highest level of mRNA enc oding DHCR was detected in the midbrain, followed by the spinal cord and me dulla, Feeding fats 5% cholestyramine plus 0.1% lovastatin in chow resulted in both approximately a 3-fold induction of DHCR mRNA and a 5-fold increas e of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-99 44 (in chow) for 14-days, a complete inhibition of DHCR activity and a sign ificant reduction in serum total cholesterol level mere observed. However, the level of hepatic DHCR mRNA fell only slightly, suggesting that AY-9944 may act more rapidly at the protein level than at the level of transcriptio n of the DHCR gene under these conditions.