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)本项目在首次成功分离并分子表征了编码关键糖基转移酶神经节苷脂GM3合成酶（sialyltransferase-1:ST3 GalV）的人类相关基因的基础上，通过同源克隆技术分离出小鼠负责GM3生物合成的ST3GalV基因（位于6C染色体，全长约58 kb）。通过5′- race分析，在小鼠中检测到该基因的三种转录本（L-型、B1-型和b2型），而在人体器官中检测到单一转录本，而人类GM3合成酶基因在人类基因组中全长约56 kb，位于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，在5′-侧翼区域有许多用于转录的顺式作用元件。3富含gc区的Sp1结合位点是细胞特异性表达的关键正调控元件。明确了小鼠组织特异性表达：l型tra，更多的nscript在肝脏中特异性表达。(2)将该酶cDNA转染人结肠癌HCT116细胞或小鼠侵袭性膀胱癌细胞，诱导细胞凋亡，显著抑制细胞的增殖、活力、侵袭性。有趣的是，将ST3GalV cDNA转染到神经节苷缺乏的小鼠肺癌3LL细胞中，可以诱导细胞生长和富gm3膜微域（我们称之为“神经节苷体”）的特征性脱落。这表明神经节苷脂GM3强制表达的生物学功能可能是细胞类型/器官特异性的。(3)我们最近发现脂肪细胞TNF-α诱导的胰岛素抵抗伴随着GM3合成酶活性及其mRNA的升高，提示细胞GM3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，单核细胞来源的树突状细胞和巨噬细胞在体外表达高水平的GM3合成酶。表明动脉粥样硬化内膜中过量的GM3至少部分是由过度表达GM3合成酶的细胞局部合成的。(4)最后，我们成功地培育出GM3合成酶缺陷小鼠，这些小鼠出生时表现为明显正常的后代，其组织和器官完全缺乏GM3神经节苷脂，并明显缺乏a系列和b系列高级神经节苷脂。(5)此外，在本项目中，我们利用基于逆转录病毒的信号序列陷阱（SST）方法成功克隆了一种参与OP9小鼠间质细胞造血支持能力的la膜糖蛋白mKirre/NEPH2。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