Kihara Akio, Igarashi Yasuyuki
Department of Biomembrane and Biofunctional Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita 12-jo, Nishi 6-choume, Kita-ku, Sapporo 060-0812, Japan.
J Biol Chem. 2004 Nov 19;279(47):49243-50. doi: 10.1074/jbc.M405915200. Epub 2004 Aug 24.
Sphingolipids are essential membrane components of eukaryotic cells. Their synthesis is initiated with the condensation of l-serine with palmitoyl-CoA, producing 3-ketodihydrosphingosine (KDS), followed by a reduction to dihydrosphingosine by KDS reductase. Until now, only yeast TSC10 has been identified as a KDS reductase gene. Here, we provide evidence that the human FVT-1 (hFVT-1) and mouse FVT-1 (mFVT-1) are functional mammalian KDS reductases. The forced expression of hFVT-1 or mFVT-1 in TSC10-null yeast cells suppressed growth defects, and hFVT-1 overproduced in cultured cells exhibited KDS reductase activity in vitro. Moreover, purified recombinant hFVT-1 protein exhibited NADPH-dependent KDS reductase activity. The identification of the FVT-1 genes enabled us to characterize the mammalian KDS reductase at the molecular level. Northern blot analyses demonstrated that both hFVT-1 and mFVT-1 mRNAs are ubiquitously expressed, suggesting that FVT-1 is a major KDS reductase. We also found the presence of hFVT-1 variants, which were differentially expressed among tissues. Immunofluorescence microscopic analysis revealed that hFVT-1 is localized at the endoplasmic reticulum. Moreover, a proteinase K digestion assay revealed that the large hydrophilic domain of hFVT-1, which contains putative active site residues, faces the cytosol. These results suggest that KDS is converted to dihydrosphingosine in the cytosolic side of the endoplasmic reticulum membrane. Moreover, the topology studies provide insight into the spatial organization of the sphingolipid biosynthetic pathway.
鞘脂是真核细胞必不可少的膜成分。其合成始于L-丝氨酸与棕榈酰辅酶A的缩合，生成3-酮二氢鞘氨醇（KDS），随后由KDS还原酶将其还原为二氢鞘氨醇。到目前为止，只有酵母TSC10被鉴定为KDS还原酶基因。在此，我们提供证据表明，人类FVT-1（hFVT-1）和小鼠FVT-1（mFVT-1）是功能性的哺乳动物KDS还原酶。在TSC10基因缺失的酵母细胞中强制表达hFVT-1或mFVT-1可抑制生长缺陷，在培养细胞中过量表达的hFVT-1在体外表现出KDS还原酶活性。此外，纯化的重组hFVT-1蛋白表现出依赖NADPH的KDS还原酶活性。FVT-1基因的鉴定使我们能够在分子水平上对哺乳动物KDS还原酶进行表征。Northern印迹分析表明，hFVT-1和mFVT-1的mRNA均广泛表达，这表明FVT-1是主要的KDS还原酶。我们还发现了hFVT-1变体的存在，它们在不同组织中差异表达。免疫荧光显微镜分析显示，hFVT-1定位于内质网。此外，蛋白酶K消化试验表明，hFVT-1的大亲水结构域（包含假定的活性位点残基）面向细胞质。这些结果表明，KDS在内质网膜的细胞质一侧转化为二氢鞘氨醇。此外，拓扑学研究为鞘脂生物合成途径的空间组织提供了见解。
