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
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
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DOI：
10.1074/jbc.274.21.14624
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发表时间：
1999-05-21
影响因子：
4.8
通讯作者：
Paik, YK
中科院分区：
文献类型：
--
作者：
Bae, SH;Lee, JN;Paik, YK
编码471个氨基酸的大鼠7-脱氢胆固醇还原酶（DHCR）的cDNA已经被克隆和测序，并且已经推导出该酶的一级结构，DHCR是一种与哺乳动物中胆固醇生物合成和发育异常（例如Smith-Lemli-Opitz综合征）有关的酶。通过荧光原位杂交将DHCR基因定位于染色体8q2.1，大鼠DHCR与小鼠和人的DHCR具有相似的氨基酸同源性，其分子量为54.15-kDa（96%和87%，与其他相关蛋白质相比，（例如真菌固醇δ（14）-还原酶），并表现出高疏水性（>68%），具有9个跨膜结构域。5个推测的甾醇敏感结构域被预测定位在跨膜结构域4-8中，其与3-羟甲基戊二酰辅酶A还原酶、甾醇调节元件结合蛋白裂解激活蛋白和补丁蛋白中发现的结构域高度同源。DHCR cDNA编码的多肽在酵母中表达为55.45 kDa的myc标签融合蛋白，该蛋白被抗myc单克隆抗体9 E10识别，并显示具有完全的DHCR活性，对NADPH的依赖性和对DHCR抑制剂的敏感性。北方印迹分析表明DHCR mRNA在肝脏中的表达量最高，其次是肾脏和脑。在大鼠脑中，DHCR mRNA表达水平以中脑最高，其次是脊髓和髓质。5%消胆胺和0.1%洛伐他汀混合饲料可使DHCR mRNA表达水平提高3倍，肝脏DHCR酶活性提高5倍。当大鼠喂食0.1%（w/w）AY-9944（在食物中）14天时，观察到DHCR活性完全抑制和血清总胆固醇水平显著降低。然而，肝脏DHCR mRNA水平仅略有下降，表明在这些条件下，AY-9944在蛋白质水平上的作用可能比在DHCR基因转录水平上的作用更快。
The cDNA encoding the 471-amino acid rat 7-dehydrocholesterol reductase (DHCR), an enzyme that has been implicated in both cholesterol biosynthesis and 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 polypeptide, shares a close amino acid identity with mouse and human DHCRs (96 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 predicted to be localized in transmembrane domains 4-8, which are highly homologous to those found in 3-hydroxymethylglutaryl-CoA reductase, sterol regulatory element-binding protein cleavage-activating protein, and patched protein. 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 monoclonal antibody 9E10 and shown to possess full DHCR activity with respect to dependence on NADPH and sensitivity to DHCR inhibitors. Northern blot analysis 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 encoding DHCR was detected in the midbrain, followed by the spinal cord and medulla, 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 increase of the enzymic activity in the liver. When rats were fed 0.1% (w/w) AY-9944 (in chow) for 14-days, a complete inhibition of DHCR activity and a significant 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 transcription of the DHCR gene under these conditions.