Riebeling Christian, Allegood Jeremy C, Wang Elaine, Merrill Alfred H, Futerman Anthony H
Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.
J Biol Chem. 2003 Oct 31;278(44):43452-9. doi: 10.1074/jbc.M307104200. Epub 2003 Aug 11.
Overexpression of upstream of growth and differentiation factor 1 (uog1), a mammalian homolog of the yeast longevity assurance gene (LAG1), selectively induces the synthesis of stearoyl-containing sphingolipids in mammalian cells (Venkataraman, K., Riebeling, C., Bodennec, J., Riezman, H., Allegood, J. C., Sullards, M. C., Merrill, A. H. Jr., and Futerman, A. H. (2002) J. Biol. Chem. 277, 35642-35649). Gene data base analysis subsequently revealed a new subfamily of proteins containing the Lag1p motif, previously characterized as translocating chain-associating membrane (TRAM) protein homologs (TRH). We now report that two additional members of this family regulate the synthesis of (dihydro)ceramides with specific fatty acid(s) when overexpressed in human embryonic kidney 293T cells. TRH1 or TRH4-overexpression elevated 3Hceramide synthesis from l-[3-3H]serine and the increase was not blocked by the (dihydro)ceramide synthase inhibitor, fumonisin B1 (FB1). Analysis of sphingolipids by liquid chromatography-electrospray tandem mass spectrometry revealed that TRH4 overexpression elevated mainly palmitic acid-containing sphingolipids whereas TRH1 overexpression increased mainly stearic acid and arachidic acid, which in both cases were further elevated upon incubation with FB1. A similar fatty acid specificity was obtained upon analysis of (dihydro)ceramide synthase activity in vitro using various fatty acyl-CoA substrates, although in a FB1-sensitive manner. Moreover, in homogenates from TRH4-overexpressing cells, sphinganine, rather than sphingosine was the preferred substrate, whereas no preference was seen in homogenates from TRH1-overexpressing cells. These findings lend support to our hypothesis (Venkataraman, K., and Futerman, A. H. (2002) FEBS Lett. 528, 3-4) that Lag1p family members regulate (dihydro)ceramide synthases responsible for production of sphingolipids containing different fatty acids.
生长与分化因子1上游基因（uog1）是酵母寿命保证基因（LAG1）的哺乳动物同源物，其过表达可选择性诱导哺乳动物细胞中含硬脂酰鞘脂的合成（文卡塔拉曼，K.，里贝林，C.，博德内克，J.，里兹曼，H.，阿利古德，J.C.，苏拉德兹，M.C.，梅里尔，A.H. Jr.，以及富特曼，A.H.（2002年）《生物化学杂志》277卷，35642 - 35649页）。随后的基因数据库分析揭示了一个新的蛋白质亚家族，其包含Lag1p基序，之前被鉴定为转位链相关膜（TRAM）蛋白同源物（TRH）。我们现在报告，该家族的另外两个成员在人胚肾293T细胞中过表达时，可调节含特定脂肪酸的（二氢）神经酰胺的合成。TRH1或TRH4过表达可提高l - [3 - 3H]丝氨酸合成[3H]（二氢）神经酰胺的水平，且这种增加不受（二氢）神经酰胺合酶抑制剂伏马菌素B1（FB1）的阻断。通过液相色谱 - 电喷雾串联质谱法分析鞘脂发现，TRH4过表达主要提高含棕榈酸的鞘脂水平，而TRH1过表达主要增加硬脂酸和花生酸，在这两种情况下，与FB1孵育后这些脂肪酸水平会进一步升高。使用各种脂肪酰辅酶A底物在体外分析（二氢）神经酰胺合酶活性时，也获得了类似的脂肪酸特异性，尽管是以FB1敏感的方式。此外，在TRH4过表达细胞的匀浆中，鞘氨醇而非鞘氨醇是首选底物，而在TRH1过表达细胞的匀浆中未观察到偏好。这些发现支持了我们的假设（文卡塔拉曼，K.，以及富特曼，A.H.（2002年）《欧洲生物化学学会联合会快报》528卷，3 - 4页），即Lag1p家族成员调节负责生成含不同脂肪酸鞘脂的（二氢）神经酰胺合酶。
