Moncecchi D, Murphy E J, Prows D R, Schroeder F
Division of Pharmacology and Medicinal Chemistry, University of Cincinnati, College of Pharmacy, OH 45267-0004, USA.
Biochim Biophys Acta. 1996 Jul 26;1302(2):110-6. doi: 10.1016/0005-2760(96)00044-6.
Despite the progress made on the possible functions of sterol carrier protein (SCP-2) using assays in vitro, very little is known regarding the role of SCP-2 in intact cells. To further elucidate this role, mouse L-cell fibroblasts were transfected with cDNA encoding for mouse 15 kDa or 13.2 kDa SCP-2. The data show for the first time, that SCP-2 expression increases cholesterol uptake into transfected L-cell fibroblasts. Untransfected L-cells expressed SCP-2 at levels near or below the lower limit of detectability. SCP-2 immunoreactive protein levels were 0.030 +/- 0.004% and 0.036 +/- 0.002% of total cytosolic proteins in the 15 and 13.2 kDa stable transfectants, respectively. Both the 15 and 13.2 kDa SCP-2 expressions products were found as 13.2 kDa proteins, consistent with rapid post-translational cleavage of the putative amino terminal mitochondrial targeting sequence from the 15 kDa SCP-2. The effect of expressing either form of SCP-2 on [3H]cholesterol uptake was determined. Expression of the 15 kDa form, but not the 13.2 kDa form of SCP-2, enhanced the rate and extent of [3H]cholesterol uptake compared to control or mock-transfected L-cells. The [3H]cholesterol uptake rate in 15 kDa SCP-2 expressing cells was increased 1.3-fold, while the extent of [3H]cholesterol uptake was increased 1.4-fold after 12 h of uptake compared to control L-cells. The differences in cholesterol uptake between the cells expressing the 13.2 versus the 15 kDa protein, suggest that the 15 kDa form of SCP-2 is functionally localized within the cell, while the 13.2 kDa product is not.
尽管利用体外实验在固醇载体蛋白（SCP - 2）的可能功能方面取得了进展，但对于SCP - 2在完整细胞中的作用却知之甚少。为了进一步阐明这一作用，将编码小鼠15 kDa或13.2 kDa SCP - 2的cDNA转染到小鼠L - 细胞成纤维细胞中。数据首次表明，SCP - 2的表达增加了转染的L - 细胞成纤维细胞对胆固醇的摄取。未转染的L - 细胞表达的SCP - 2水平接近或低于可检测下限。在15 kDa和13.2 kDa稳定转染细胞中，SCP - 2免疫反应蛋白水平分别占总胞质蛋白的0.030±0.004%和0.036±0.002%。15 kDa和13.2 kDa的SCP - 2表达产物均以13.2 kDa蛋白形式存在，这与从15 kDa SCP - 2假定的氨基末端线粒体靶向序列的快速翻译后切割一致。测定了表达两种形式的SCP - 2对[3H]胆固醇摄取的影响。与对照或mock - 转染的L - 细胞相比，15 kDa形式而非13.2 kDa形式的SCP - 2的表达提高了[3H]胆固醇摄取的速率和程度。与对照L - 细胞相比，表达15 kDa SCP - 2的细胞中[3H]胆固醇摄取速率增加了1.3倍，摄取12小时后[3H]胆固醇摄取程度增加了1.4倍。表达13.2 kDa与15 kDa蛋白的细胞之间胆固醇摄取的差异表明，15 kDa形式的SCP - 2在细胞内具有功能定位，而13.2 kDa产物则没有。
