Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase-defective lymphoid cells
Expression cloning of a human cDNA restoring sphingomyelin synthesis and cell growth in sphingomyelin synthase-defective lymphoid cells
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DOI：
10.1074/jbc.m401205200
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发表时间：
2004-04-30
影响因子：
4.8
通讯作者：
Okazaki, T
中科院分区：
文献类型：
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作者：
Yamaoka, S;Miyaji, M;Okazaki, T
鞘磷脂 (SM) 合酶被认为通过调节促凋亡介质神经酰胺和促生存介质二酰基甘油来参与细胞死亡和存活。然而，由于缺乏 SM 合酶基因的分子克隆，其精确功能并不明确。我们分离了 WR19L/Fas-SM(-) 小鼠淋巴样细胞，由于缺乏 SM 合酶活性和对 SM 定向溶细胞蛋白赖赛素诱导的细胞死亡的抵抗力，该细胞在质膜上显示出 SM 缺陷。与能够合成SM的WR19L/Fas-SM(+)细胞相比，WR19L/Fas-SM(-)细胞对甲基-β-环糊精(MbetaCD)也高度敏感。通过使用WR19L/Fas-SM(-)细胞的表达克隆方法和基于MbetaCD的选择，我们成功克隆了负责SM合酶活性的人cDNA。该cDNA编码一个由413个氨基酸组成的肽，名为SMS1（推定分子量，48.6 kDa），它在N端区域附近包含一个无菌α基序结构域和四个预测的跨膜结构域。表达SMS1 cDNA的WR19L/Fas-SM(-)细胞(WR19L/Fas-SMS1)恢复了对MbetaCD的抵抗力、质膜上SM的积累以及通过将磷酸胆碱从磷脂酰胆碱转移到神经酰胺来恢复SM合成。此外，补充外源SM的WR19L/Fas-SMS1细胞以及WR19L/Fas-SM(-)细胞在无血清条件下恢复了细胞生长能力，而WR19L/Fas-SM(-)细胞的生长受到严重抑制。结果表明，SMS1 负责哺乳动物细胞中的 SM 合酶活性，并在小鼠淋巴细胞的细胞生长中发挥关键作用。
Sphingomyelin (SM) synthase has been assumed to be involved in both cell death and survival by regulating pro-apoptotic mediator ceramide and pro-survival mediator diacylglycerol. However, its precise functions are ambiguous due to the lack of molecular cloning of SM synthase gene(s). We isolated WR19L/Fas-SM(-) mouse lymphoid cells, which show a defect of SM at the plasma membrane due to the lack of SM synthase activity and resistance to cell death induced by an SM-directed cytolytic protein lysenin. WR19L/Fas-SM(-) cells were also highly susceptible to methyl-beta-cyclodextrin (MbetaCD) as compared with the WR19L/Fas-SM(+) cells, which are capable of SM synthesis. By expression cloning method using WR19L/Fas-SM(-) cells and MbetaCD-based selection, we have succeeded in cloning of a human cDNA responsible for SM synthase activity. The cDNA encodes a peptide of 413 amino acids named SMS1 ( putative molecular mass, 48.6 kDa), which contains a sterile alpha motif domain near the N-terminal region and four predicted transmembrane domains. WR19L/Fas-SM(-) cells expressing SMS1 cDNA (WR19L/Fas-SMS1) restored the resistance against MbetaCD, the accumulation of SM at the plasma membrane, and SM synthesis by transferring phosphocholine from phosphatidylcholine to ceramide. Furthermore, WR19L/Fas-SMS1 cells, as well as WR19L/ Fas-SM(-) cells supplemented with exogenous SM, restored cell growth ability in serum-free conditions, where the growth of WR19L/ Fas-SM(-) cells was severely inhibited. The results suggest that SMS1 is responsible for SM synthase activity in mammalian cells and plays a critical role in cell growth of mouse lymphoid cells.