Functions of Complex Glycosphingolipids in the Cell Proliferation, Differentiation, and Cell Death Controlled at the Gene Level of Their Synthesizing Enzymes, and Their Medical Applications

(1)In本项目在我们首次成功分离和分子鉴定编码关键糖基转移酶神经节苷脂GM 3合酶（唾液酸转移酶-1：ST 3GalV）的人相关基因的基础上，我们接下来通过同源克隆技术分离负责GM 3生物合成的鼠ST 3GalV基因（位于染色体6C，跨度约58 kb）。三种成绩单通过5 '-RACE分析在小鼠中检测到该基因的L-、B1-和B2-型，另一方面，在人器官中检测到单个转录物，而人GM 3合酶基因在人基因组中跨越约56 kb，定位在2号染色体短臂的着丝粒附近（2p11.2），由7个外显子和6个内含子组成，5 '侧翼区含有多个转录调控元件，富含GC区的3个Sp1结合位点是细胞特异性表达的关键正调控元件。阐明了小鼠组织特异性表达：L型TRANSPLAY 关于我们 nscript在肝脏中特异性表达。（2）将该酶cDNA转染入人结肠癌HCT 116细胞或小鼠浸润性膀胱癌细胞中诱导凋亡，导致细胞增殖、运动性、侵袭性、而ST 3GalV cDNA转染到神经节苷脂缺陷型小鼠肺癌3LL细胞中，有趣的是，显示出诱导细胞生长和GM 3 - 1的特征性脱落。丰富的膜微区（我们称之为“神经节小体”）进入培养基。这表明，神经节苷脂GM 3的强制表达的生物学功能可能是细胞类型/器官特异性的。(3)We最近发现TNF-α诱导的脂肪细胞胰岛素抵抗伴随着GM 3合成酶活性及其mRNA的升高，提示细胞GM 3合成的增加可能参与了2型糖尿病胰岛素抵抗的病理状态，并且体外单核细胞来源的树突状细胞和巨噬细胞表达高水平的GM 3合成酶，表明动脉粥样硬化内膜中过量的GM 3至少部分由过表达GM 3合酶的细胞局部合成。（4）最后，我们成功地产生了GM 3 β-淀粉酶缺陷小鼠，这些小鼠出生时是明显正常的后代，其组织和器官完全缺乏神经节苷脂GM 3，并且显著缺乏a系列和b系列高级神经节苷脂。（5）本研究还利用逆转录病毒信号序列捕获（SST）技术成功克隆了一个与OP 9小鼠骨髓基质细胞造血功能相关的Ia型膜糖蛋白mKirre/NEPH 2。少

(1)In this project, on the basis of our first success in isolating and molecularly characterizing a human relevant gene which encodes a key glycosyltransferase, ganglioside GM3 synthase (sialyltransferase-1:ST3 GalV), we next isolated by the homology cloning technique murine ST3GalV gene (located at chromosome 6C, spanning about 58 kb), which is responsible for GM3 biosynthesis. Three kinds of transcripts (L-,B1- and B2-type) of the gene were detected in mice by 5'-RACE analyses, a single transcript detectable in human organs on the other hand, whereas human GM3 synthase gene spans approximately 56 kb in the human genome, mapped near the centromere of the short arm of chromosome 2(2p11.2), consisting of seven exons and six introns, with a number of cis-acting elements for transcription in the 5'-flanking region, 3 Sp1 binding sites in the GC-rich region being critical positive regulatory element in cell-specific expression. Murine tissue-specific expressions were clarified : L-type tra … More nscript was specifically expressed in liver. (2)Transfection of this enzyme cDNA into human colon carcinoma HCT116 cells or murine invasive bladder cancer cells induced apoptosis, resulting in the remarkable suppression of cell proliferation, motility, invasiveness, and tumorigenesis in vivo whereas the ST3GalV cDNA transfection into ganglioside-deficient mouse lung carcinoma 3LL cells was interestingly shown to induce cell growth and the characteristic shedding of GM3-rich membrane microdomains (we call them ‘gangliosomes') into the medium. This indicates that biological functions of the enforced expression of ganglioside GM3 might be cell-type/organ specific. (3)We recently found that insulin resistance induced by TNF-α in adipocytes was accompanied by elevating GM3 synthase activity and its mRNA, suggesting that the increased synthesis of cellular GM3 might participate in the pathological conditions of insulin resistance in type 2 diabetes, and that monocyte-derived dendritic cells and macrophages in vitro expressed high levels of GM3 synthase, indicating that at least part of excessive GM3 in atherosclerotic intima is locally synthesized by cells overexpressing GM3 synthase. (4)Lastly we have succeeded in producing the GM3 synthase-deficient mice, which were born as apparently normal offspring, and of which tissues and organs were completely deficient in ganglioside GM3 and remarkably deficient in a- and b-series higher gangliosides. (5)Additionally in this project, we succeeded in cloning a type la membrane glycoprotein, mKirre/NEPH2,which is involved in the hematopoietic supportive capacity of OP9 mouse stromal cells, on the basis of a retrovirus-based signal sequence trap(SST) method. Less