Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.
Molecular cloning and functional expression of human acyl-coenzyme A:cholesterol acyltransferase cDNA in mutant Chinese hamster ovary cells.
复制标题
DOI：
10.1016/s0021-9258(19)36846-2
复制
发表时间：
1993-10
期刊：
影响因子：
--
通讯作者：
C. Chang;H. Huh;K. Cadigan;Ta-Yuan Chang
中科院分区：
文献类型：
--
作者：
C. Chang;H. Huh;K. Cadigan;Ta-Yuan Chang
胆固醇酯在巨噬细胞和平滑肌细胞内以细胞质脂滴的形式积聚是动脉粥样硬化斑块早期病变的特征。在细胞内，从胆固醇形成胆固醇酯的基本要素是酶酰基辅酶A：胆固醇酰基转移酶（ACAT）。ACAT是位于内质网中的膜蛋白。ACAT蛋白从未被纯化至均一，也没有针对ACAT的抗体被报道。编码这种酶的基因尚未分离。该实验室先前曾报告分离表达人ACAT活性的中国仓鼠卵巢细胞。从这些细胞的DNA中，我们克隆了一个1.2 kb的外显子人类基因组DNA。这导致从人巨噬细胞cDNA文库中最终克隆出4-kb cDNA克隆（K1）。在ACAT缺陷的突变中国仓鼠卵巢细胞中转染K1补充了突变缺陷，并导致人ACAT活性的表达。K1含有一个1650 bp的开放阅读框，编码一个550个氨基酸的完整膜蛋白。蛋白质同源性分析表明，预测的K1蛋白共享同源肽序列与其他酶参与催化酰基腺苷酸形成，然后酰基硫酯形成和酰基转移。这些结果表明K1编码ACAT的结构基因。这里报告的cDNA将有助于未来ACAT的分子研究。
Accumulation of cholesterol esters as cytoplasmic lipid droplets within macrophages and smooth muscle cells is a characteristic feature of early lesions of atherosclerotic plaque. Intracellularly, an essential element in forming cholesterol ester from cholesterol is the enzyme acyl-coenzyme A:cholesterol acyltransferase (ACAT). ACAT is a membrane protein located in the endoplasmic reticulum. The ACAT protein has never been purified to homogeneity, and no antibodies directed against ACAT have been reported. The gene(s) encoding this enzyme had not been isolated. This laboratory had previously reported the isolation of Chinese hamster ovary cells expressing human ACAT activity. From DNAs of these cells, we have cloned a 1.2-kb exonic human genomic DNA. This led to the eventual cloning of a 4-kb cDNA clone (K1) from a human macrophage cDNA library. Transfection of K1 in ACAT-deficient mutant Chinese hamster ovary cells complemented the mutant defect and resulted in the expression of human ACAT activity. K1 contained an open reading frame of 1650 bp encoding an integral membrane protein of 550 amino acids. Protein homology analysis showed that the predicted K1 protein shared homologous peptide sequences with other enzymes involved in the catalysis of acyl adenylate formation followed by acyl thioester formation and acyl transfer. These results indicate that K1 encodes a structural gene for ACAT. The cDNA reported here should facilitate future molecular studies on ACAT.