Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.
Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity.
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DOI：
10.1016/s0021-9258(19)57389-6
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发表时间：
1988-01
期刊：
影响因子：
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通讯作者：
K. M. Cadigan;J G Heider;T Y Chang
中科院分区：
文献类型：
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作者：
K. M. Cadigan;J G Heider;T Y Chang
建立了从中国仓鼠卵巢细胞克隆25-RA中分离胆固醇酯缺乏细胞的方法。该细胞系在含有10%胎牛血清的培养基中生长时，曾被证明部分抵抗25-羟基胆固醇对胆固醇生成酶活性的抑制，并在细胞内积累大量的胆固醇酯（Chang, t.y ., and Limanek, j.s. (1980) J. Biol.）。化学，255,7787-7795)。与野生型中国仓鼠卵巢细胞相比，25-RA较高的胆固醇酯含量是由于胆固醇生物合成速率和低密度脂蛋白受体活性的增加，而不是由于酰基辅酶a（胆固醇酰基转移酶）异常。分离胆固醇酯缺乏突变体的方法使用两性霉素B，这是一种已知能与胆固醇结合并在膜上形成孔复合物的多烯抗生素。在无胆固醇的培养基和内源性胆固醇生物合成抑制剂中孵育后，发现25-RA细胞对两性霉素B杀伤的敏感性是含有少量胆固醇酯的突变细胞的50-500倍。12个两性霉素b抗性突变体保留了25-羟基胆固醇抗性表型。这些突变体没有表现出25-RA细胞的核周脂滴特征，脂质分析显示细胞胆固醇酯大幅减少（高达40倍）。在完整细胞或体外重构实验中，这些胆固醇酯缺乏突变体的酰基辅酶a：胆固醇酰基转移酶活性明显低于25-RA。最紧密的突变体AC29被发现具有不到亲本酰基辅酶a：胆固醇酰基转移酶活性的1%。与25-RA相比，这些突变体的固醇合成速率和低密度脂蛋白受体活性都降低了，这可能是它们的酶活性降低的结果。细胞融合实验表明，所有突变体的表型都不占优势，并且突变体都属于同一互补群。我们得出结论，这些突变体在编码酰基辅酶a：胆固醇酰基转移酶的基因或编码酶生产所需因子的基因中含有病变。
A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.