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

与造血细胞生长分化相关的神经节苷脂GM3合酶基因的表达机制及其医学应用

基本信息

批准号：
10470206
负责人：
SAITO Masaki
金额：
$ 8.06万
依托单位：
National Cancer Center Research Institute
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

项目摘要

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

唾液酸鞘糖脂，神经节苷脂，在细胞生长和分化、胚胎发生和癌症发生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常见的前体神经节苷脂GM3合成的，我们之前已经证明GM3对人髓系白血病细胞具有诱导分化活性。在本项目中，我们利用精心设计的表达克隆方法，首次成功地分离和鉴定了一个新的相关基因(cDNA和基因组)，该基因编码一个关键的糖基转移酶，人神经节苷脂GM3合成酶(sialylTransfer ase-1：STSGalV)，然后是负责GM3生物合成的小鼠STSGalV。人类GM3合成酶基因全长约56kb，由7个外显子和6个内含子组成。转录起始点区域的GC含量，位于280个核苷酸以上的…经引物延伸确定的推测翻译起始点有更多的序列(81%)，并且人类GM3合成酶基因启动子不包含典型的TATA和CCAAT盒。在5‘-侧翼区中发现了一些转录顺式作用元件。利用荧光素酶基因进行的瞬时报告实验表明，在SK N-MC细胞中JHAT启动子的活性明显高于在HCT16细胞中的活性。位于GC富集区的3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。糖基转移酶基因定位于人类基因组2号染色体短臂着丝粒附近。随后，通过5‘-RACE分析发现该基因在小鼠体内有3种转录本(L、Bl和B2型)，而在人体器官中只检测到一种转录本，并明确了小鼠组织特异性表达：L型转录本在肝脏中特异表达，BL型转录本在各种器官中普遍表达，B2型转录本在各器官中有微弱表达。人和小鼠ST3GalV的基因组结构分别通过筛选BAc和λ文库进行了测定。有趣的是，将该酶基因导入神经节苷脂缺陷的小鼠肺癌3LL细胞，可使富含GM3的膜结构域发生脱落。为了获得大量能够分析三维结构的酶蛋白，并将其用于全自动糖链合成仪的开发，我们最近成功地在大肠杆菌中以MBPVGST-融合蛋白的形式生产了ST3GalV可溶性蛋白。较少