Upstream of Growth and Differentiation Factor 1 (uog1), a Mammalian Homolog of the Yeast Longevity Assurance Gene 1 (LAG1), RegulatesN-Stearoyl-sphinganine (C18-(Dihydro)ceramide) Synthesis in a Fumonisin B1-independent Manner in Mammalian Cells*
Upstream of Growth and Differentiation Factor 1 (uog1), a Mammalian Homolog of the Yeast Longevity Assurance Gene 1 (LAG1), RegulatesN-Stearoyl-sphinganine (C18-(Dihydro)ceramide) Synthesis in a Fumonisin B1-independent Manner in Mammalian Cells*
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DOI：
10.1074/jbc.m205211200
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发表时间：
2002-09
期刊：
影响因子：
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通讯作者：
Krishnan Venkataraman;C. Riebeling;J. Bodennec;H. Riezman;J. Allegood;M. Sullards;A. Merrill;A. Futerman
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文献类型：
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作者：
Krishnan Venkataraman;C. Riebeling;J. Bodennec;H. Riezman;J. Allegood;M. Sullards;A. Merrill;A. Futerman
长寿保证基因 (LAG1) 及其同源基因 (LAC1) 是酵母中酰基辅酶 A 依赖性合成含有极长酰基链（例如 C26）脂肪酸的神经酰胺所必需的，LAG1 的同源基因 ASC1 赋予植物对神经酰胺合成抑制剂伏马菌素 B1 的抗性。为了进一步了解神经酰胺合成的调节机制，我们现在表征了 LAG1 的哺乳动物同源物，即生长和分化因子 1 (uog1) 的上游。 uog1 的 cDNA 克隆从表达序列标记的克隆中获得，并亚克隆到哺乳动物表达载体中。用 uog1 瞬时转染人胚肾 293T 细胞，然后用 [4,5-3H] 二氢鞘氨醇或 1-3-[3H] 丝氨酸进行代谢标记，证明 uog1 赋予细胞继续产生神经酰胺的能力，从而赋予伏马菌素 B1 抗性。令人惊讶的是，这种神经酰胺被引导到中性糖鞘脂中，而不是神经节苷脂中。电喷雾串联质谱证实了鞘脂的升高，并揭示了uog1转染细胞的神经酰胺和中性糖鞘脂主要含有硬脂酸（C18），FB1进一步增加了这种富集，并且鞘磷脂中硬脂酸的量也增加了。 UOG1 定位于内质网，表明脂肪酸选择性和伏马菌素 B1 抗性并非由于与之前发现的神经酰胺合酶活性不同的亚细胞定位。此外，体外试验显示，当使用硬脂酰辅酶A而不是棕榈酰辅酶A作为底物时，转染细胞的神经酰胺合酶活性升高。我们提出UOG1在调节C18-神经酰胺（N-硬脂酰-二氢鞘氨醇）合成中的作用，我们注意到，这不仅是哺乳动物细胞中具有如此高的脂肪酸特异性的神经酰胺形成的第一例，而且UOG1产生的N-硬脂酰-二氢鞘氨醇对中性糖鞘脂的合成影响最显着。
The longevity assurance gene (LAG1) and its homolog (LAC1) are required for acyl-CoA-dependent synthesis of ceramides containing very long acyl chain (e.g. C26) fatty acids in yeast, and a homolog of LAG1, ASC1, confers resistance in plants to fumonisin B1, an inhibitor of ceramide synthesis. To understand further the mechanism of regulation of ceramide synthesis, we now characterize a mammalian homolog of LAG1,upstream of growth and differentiation factor-1 (uog1). cDNA clones of uog1 were obtained from expression sequence-tagged clones and sub-cloned into a mammalian expression vector. Transient transfection of human embryonic kidney 293T cells with uog1 followed by metabolic labeling with [4,5-3H]sphinganine orl-3-[3H]serine demonstrated thatuog1 conferred fumonisin B1 resistance with respect to the ability of the cells to continue to produce ceramide. Surprisingly, this ceramide was channeled into neutral glycosphingolipids but not into gangliosides. Electrospray tandem mass spectrometry confirmed the elevation in sphingolipids and revealed that the ceramides and neutral glycosphingolipids ofuog1-transfected cells contain primarily stearic acid (C18), that this enrichment was further increased by FB1, and that the amount of stearic acid in sphingomyelin was also increased. UOG1 was localized to the endoplasmic reticulum, demonstrating that the fatty acid selectivity and the fumonisin B1 resistance are not due to a subcellular localization different from that found previously for ceramide synthase activity. Furthermore, in vitro assays ofuog1-transfected cells demonstrated elevated ceramide synthase activity when stearoyl-CoA but not palmitoyl-CoA was used as substrate. We propose a role for UOG1 in regulating C18-ceramide (N-stearoyl-sphinganine) synthesis, and we note that not only is this the first case of ceramide formation in mammalian cells with such a high degree of fatty acid specificity, but also that theN-stearoyl-sphinganine produced by UOG1 most significantly impacts neutral glycosphingolipid synthesis.