A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes.

Shunsuke Nashimoto, Saori Yagi, Naoki Takeda, Miku Nonaka, Yoh Takekuma, Mitsuru Sugawara, Yuki Sato

Biochimica et biophysica acta. Biomembranes 2021 Feb 01

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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 a Xenopus laevis oocyte 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. Copyright © 2020 Elsevier B.V. All rights reserved.

Citation

Shunsuke Nashimoto, Saori Yagi, Naoki Takeda, Miku Nonaka, Yoh Takekuma, Mitsuru Sugawara, Yuki Sato. A new system to evaluate characteristics of Niemann-Pick C1 Like 1-mediated cholesterol transport using Xenopus laevis oocytes. Biochimica et biophysica acta. Biomembranes. 2021 Feb 01;1863(2):183508