A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes.
A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes.
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DOI：
10.1016/j.bbamem.2020.183508
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发表时间：
2020-11
期刊：
影响因子：
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通讯作者：
Shunsuke Nashimoto;Saori Yagi;Naoki Takeda;Miku Nonaka;Y. Takekuma;M. Sugawara;Yuki Sato
中科院分区：
文献类型：
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作者：
Shunsuke Nashimoto;Saori Yagi;Naoki Takeda;Miku Nonaka;Y. Takekuma;M. Sugawara;Yuki Sato
尼曼-匹克C1样蛋白1（NPC 1 L1）参与胆固醇的肠道吸收。为了评价NPC 1 L1的功能，在以前的研究中已经使用了细胞系如Caco-2、Madin-Darby犬肾（MDCK）II和McA-RH 7777，但尚未阐明转运的详细分子机制。在这项研究中，胆固醇转运通过NPC 1 L1的特点进行了研究，除了一个传统的细胞系稳定表达非洲爪蟾lavisocyte表达系统。NPC 1 L1过表达的MDCK细胞的胆固醇摄取转运活性较mock细胞增加，但MDCK细胞表达内源性NPC 1 L1，具有较高的胆固醇转运活性。另一方面，注射cRNA的卵母细胞在培养5-6天后表达NPC 1 L1。与注水卵母细胞相比，注射NPC 1 L1 cRNA的卵母细胞胆固醇摄取的转运活性增加。此外，在注射cRNA的卵母细胞中，存在NPC 1 L1抑制剂依折麦布的情况下，胆固醇的摄入减少，但在对照卵母细胞中则不然，这表明卵母细胞中不表达内源性NPC 1 L1。此外，与注射NPC 1 L1 cDNA的卵母细胞相比，注射NPC 1 L1 L216 A cDNA的卵母细胞的胆固醇摄取显著降低，表明NPC 1 L1的216位亮氨酸对胆固醇转运很重要，并且卵母细胞表达系统可用于突变体分析。这些结果表明，卵母细胞表达系统是有用的，用于评估NPC 1 L1介导的胆固醇转运的特性，并可能有助于阐明胆固醇转运通过NPC 1 L1的详细分子机制。
Niemann-Pick C1 Like 1 (NPC1L1) is known to be involved in the intestinal absorption of cholesterol. For evaluating the function of NPC1L1, cell lines such as Caco-2, Madin-Darby canine kidney (MDCK) II, and McA-RH7777 have been used in previous studies, but the detailed molecular mechanism of transport has not been elucidated. In this study, the characteristics of cholesterol transport via NPC1L1 were investigated using aXenopus laevisoocyte expression system in addition to a conventional cell line with stable expression. The transport activity of cholesterol uptake was increased in NPC1L1-overexpressed MDCK cells compared with that in mock cells, but MDCK cells expressed endogenous NPC1L1 and had high cholesterol transport activity. On the other hand, cRNA-injected oocytes expressed NPC1L1 after culturing for 5–6 days. The transport activity of cholesterol uptake was increased in NPC1L1 cRNA-injected oocytes compared with that in water-injected oocytes. In addition, the uptake of cholesterol was decreased in the presence of ezetimibe, an NPC1L1 inhibitor, in cRNA-injected oocytes but not in control oocytes, indicating that endogenous NPC1L1 is not expressed in oocytes. Furthermore, cholesterol uptake was substantially decreased in NPC1L1 L216A cRNA-injected oocytes compared with that in NPC1L1 cRNA-injected oocytes, indicating that leucine at position 216 of NPC1L1 is important for cholesterol transport and that an oocyte expression system is useful for mutant analysis. These results indicate that the oocyte expression system is useful for evaluating the characteristics of NPC1L1-mediated cholesterol transport and may contribute to the elucidation of the detailed molecular mechanism of cholesterol transport via NPC1L1.