Marchesini Norma, Luberto Chiara, Hannun Yusuf A
Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston 29425, USA.
J Biol Chem. 2003 Apr 18;278(16):13775-83. doi: 10.1074/jbc.M212262200. Epub 2003 Feb 3.
Neutral sphingomyelinase (N-SMase) is one of the key enzymes involved in the generation of ceramide; however, the gene(s) encoding for the mammalian N-SMase is still not well defined. Previous studies on the cloned nSMase1 had shown that the protein acts primarily as lyso-platelet-activating factor-phospholipase C. Recently the cloning of another putative N-SMase, nSMase2, was reported. In this study, biochemical characterization of the mouse nSMase2 was carried out using the overexpressed protein in yeast cells in which the inositol phosphosphingolipid phospholipase C (Isc1p) was deleted. N-SMase activity was dependent on Mg(2+) and was activated by phosphatidylserine and inhibited by GW4869. The ability of nSMase2 to recognize endogenous sphingomyelin (SM) as substrate was investigated by overexpressing nSMase2 in MCF7 cells. Mass measurements showed a 40% decrease in the SM levels in the overexpressor cells, and labeling studies demonstrated that nSMase2 accelerated SM catabolism. Accordingly, ceramide measurement showed a 60 +/- 15% increase in nSMase2-overexpressing cells compared with the vector-transfected MCF7. The role of nSMase2 in cell growth was next investigated. Stable overexpression of nSMase2 resulted in a 30-40% decrease in the rate of growth at the late exponential phase. Moreover, tumor necrosis factor induced approximately 50% activation of nSMase2 in MCF7 cells overexpressing the enzyme, demonstrating that nSMase2 is a tumor necrosis factor-responsive enzyme. In conclusion, these results 1) show that nSMase2 is a structural gene for nSMase, 2) suggest that nSMase2 acts as a bona fide N-SMase in cells, and 3) implicate nSMase2 in the regulation of cell growth and cell signaling.
中性鞘磷脂酶（N-SMase）是参与神经酰胺生成的关键酶之一；然而，编码哺乳动物N-SMase的基因仍未明确界定。先前对克隆的nSMase1的研究表明，该蛋白主要作为溶血血小板活化因子磷脂酶C发挥作用。最近有报道克隆了另一种假定的N-SMase，即nSMase2。在本研究中，使用在缺失肌醇磷酸鞘脂磷脂酶C（Isc1p）的酵母细胞中过表达的蛋白对小鼠nSMase2进行了生化特性分析。N-SMase活性依赖于Mg(2+)，并被磷脂酰丝氨酸激活，被GW4869抑制。通过在MCF7细胞中过表达nSMase2，研究了nSMase2识别内源性鞘磷脂（SM）作为底物的能力。质量测量显示，过表达细胞中SM水平降低了40%，标记研究表明nSMase2加速了SM的分解代谢。因此，神经酰胺测量显示，与载体转染的MCF7相比，nSMase2过表达细胞中的神经酰胺增加了60±15%。接下来研究了nSMase2在细胞生长中的作用。nSMase2的稳定过表达导致对数生长期后期生长速率降低30-40%。此外，肿瘤坏死因子在过表达该酶的MCF7细胞中诱导nSMase2激活约50%，表明nSMase2是一种肿瘤坏死因子反应性酶。总之，这些结果1）表明nSMase2是N-SMase的结构基因，2）表明nSMase2在细胞中作为真正的N-SMase发挥作用，3）表明nSMase2参与细胞生长和细胞信号传导的调节。
