Functional analysis of glycosphingolipids by molecular biological approaches

1. 神经酰胺葡萄糖基转移酶的启动子分析在一些细胞系中，神经酰胺被认为是第二信使。在应激或DNA损伤的情况下，细胞内神经酰胺水平升高并诱导细胞凋亡。另一方面，神经酰胺葡萄糖基转移酶GlcT-1使神经酰胺失活并抑制细胞凋亡。因此，我们构建了启动子与荧光素酶的嵌合体基因，并将其导入多种动物细胞系。利用这些细胞系，我们检测了多种基因毒性物质。在B16细胞中，2 μ m喜树碱的活性增加了2倍，10μM依托泊苷的活性增加了3.2倍。我们还检测了40种植物提取物，并鉴定出两种抑制启动子活性的植物提取物。GlcT-1分子定位于高尔基膜及其底物神经酰胺。该酶被认为通过信号锚定序列与膜结合。为了证明这一假设，我们构建了缺乏该序列的cDNA，并将其引入GlcT-1缺失的GM95细胞。缺少信号锚定序列的GlcT-1没有显示出活性，说明该序列的重要性。突变酶的定位目前正在研究中。抑制细胞凋亡蛋白的表达克隆我们试图用逆转录病毒介导的表达克隆方法分离抑制神经酰胺诱导的细胞凋亡的基因。在研究过程中，我们碰巧分离出抑制过氧化氢诱导细胞凋亡的基因。我们将小鼠7天cDNA文库感染到NIH/3T3细胞上，并用双氧水进行筛选。通过PCR从存活细胞的基因组DNA中提取cdna。我们已经分离出11个基因，其中两个功能未知。

1. The promoter analysis of ceramide glucosyltransferaseIn several cell lines, ceramide are believed to serves as second messenger. In the case of stress or DNA damage, intracellular ceramide level elevates and induces apoptosis. In the other hand, ceramide glucosyltransferase, GlcT-1 inactivates ceramide and inhibits apoptosis. Thus, we constructed the chimera gene of the promoter and luciferase and introduced into several animal cell lines. Using these cell lines we examined a variety of genotoxic agents. In B16 cells 2μM camptothcine increase the activity twice and 10μM etoposide increased 3,2 times. We also examined 40 kinds of plant extracts and identified two kinds of plant extract that suppress the promoter activity.2. The functional analysis of GlcT-1 molecule by genetic engineeringGlcT-1 localizes in the Golgi membrane as well as its substrate ceramide. The enzyme is believed to binds to the membrane by the signal anchor sequence. To prove this hypothesis, we constructed cDNA lacking the sequence and introduced it to GlcT-1 deficient GM95 cells. The GlcT-1 lacking the signal anchor sequence did not show the activity, indicating the importance of the sequence. Localization of the mutant enzyme is now under examination.3. Expression cloning of proteins which inhibit apoptosisWe tried to isolate genes that can inhibit ceramide induced apotosis using retroviral-mediate expression cloning. In the course of the study, we happened to isolate genes that inhibit hydrogen peroxide induced apoptosis. We infected mouse 7-day cDNA library to NIH/3T3 cells and selected with hydrogen peroxide. The cDNAs were recovered by PCR from the genomic DNA of the survived cells. We have isolated 11 genes and two were functionally unknown.