Expression Mechanism and Its Medical Application of Ganglioside GM3 Synthase Gene Which Is Relevantly Related With Growth and Differentiation of Hematopoietic Cells

唾液糖鞘脂是一种神经节苷类物质，在细胞生长和分化、胚胎发生和癌变等多种生物现象中具有重要作用。在脊椎动物中，几乎所有的神经节苷脂都是由一个共同的前体合成的，神经节苷脂GM3，我们之前已经证明它对人类髓性白血病细胞具有诱导分化的活性。本项目采用精心设计的表达克隆方法，首次成功分离并分子表征了一个新的相关基因（cDNA和基因组），该基因编码了一个关键的糖基转移酶——人神经节苷脂GM3合成酶（sialyltransferase-1: STSGalV），然后编码了负责GM3生物合成的小鼠STSGalV。人类GM3合成酶基因全长约56 kb，由7个外显子和6个内含子组成。在转录起始位点区域，GC含量高（81%），位于280个核苷酸以上，引物延伸确定的推定翻译起始位点，并且人GM3合成酶基因启动子不包含典型的TATA和CCAAT盒。许多用于转录的顺式作用元件在5'侧区被注意到。利用荧光素酶基因进行的瞬时报告基因分析显示，SK N-MC细胞中的启动子活性远高于hct16细胞。位于gc富集区的三个Spl结合位点似乎是人GM3合成酶基因细胞特异性表达的关键正调控元件。在人类基因组中，糖基转移酶基因定位在2号染色体短臂的着丝粒附近。随后，通过5′- race分析，在小鼠中检测到该基因的3种转录本（L-型、Bl-型和b2型），而在人体器官中检测到单一转录本，并明确了该基因在小鼠组织中的特异性表达：l型转录本在肝脏中特异性表达，而b型转录本普遍存在于各器官中，b2型边缘表达。通过筛选从染色体DNA制备的BAC和λ文库，分别确定了ST3GalV在人和小鼠中的基因组结构。有趣的是，将这种酶cDNA转染到神经节苷缺乏的小鼠肺癌3LL细胞中，可以在培养基中引入富含gm3的膜结构域的特征性脱落。为了获得足够多的酶蛋白来分析其三维结构并用于碳水化合物链自动合成仪的开发，我们最近成功地在大肠杆菌中生产了可溶性的ST3GalV作为mbpvgst融合蛋白。少

Sialoglycosphingolipids, gangliosides, are known to bear important functions in various biological phenomena such as cell growth and differentiation, embryogenesis and carcinogenesis. In vertebrates, almost all the gangliosides are synthesized from a common pre-cursor, ganglioside GM3 , which was previously shown by us to exhibit differentiation-inducing activity against human myelogenous leukemia cells. In this project, using the elaborately-devised expression cloning method, we succeeded for the first time in isolating and molecularly characterizing a new and relevant gene (cDNA and genome) which encodes a key glycosyltransferase, human ganglioside GM3 synthase (sialyltransferase-1 : STSGalV) and then, murine STSGalV, which is responsible for GM3 biosynthesis. Human GM3 synthase gene spans approximately 56 kb in the human genome and was found to consist of seven exons and six introns. The GC content in the region of the transcription start site, which was resided to 280 nucleotide up … More stream of the putative translational initiation site determined by primer extension, is high (81 %), and human GM3 synthase gene promoter contains no canonical TATA and CCAAT boxes. A number of cis-acting elements for transcription are noted in the 5'-flanking region. A transient reporter assay using the luciferase gene showedJhat promoter activity was much higher in SK N-MC cells than in HCT1 16 cells. Three Spl binding sites located in the GC-rich region seemed to be critical positive regulatory element in cell-specific expression of human GM3 synthase gene. The glycosyltransferase gene was mapped near the centromere of the short arm of chromosome 2 in the human genome. Subsequently, 3 kinds of transcripts (L-, Bl- and B2-type) of the gene were detected in mice by 5'-RACE analyses whereas a single transcript detect able in human organs, and murine tissue-specific expressions were clarified : Ltype transcript was specifically expressed in liver while Bl-type was generally detected in various organs with B2-type marginally expressed. The human and murine genomic structures of ST3GalV have been determined by screening BAC and λ library, respectively, prepared from the chromosomal DNA. Transfection of this enzyme cDNA into ganglioside-deficient mouse lung carcinoma 3LL cells was interestingly shown to introduce the characteristic shedding of GM3-rich membrane domain into the medium. In order to obtain much amount of the enzyme protein enough to analyze three dimensional structure and to utilize in the development of automatic carbohydrate-chain synthesizers, we could recently succeed in producing ST3GalV soluble forms as MBPVGST-fusion proteins in E.coli. Less