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Exploitation on Biological Activities of Sialoglycosphingolipids and Their Synthetic Family Compounds (Neoglycollipids)

唾液酸鞘糖脂及其合成家族化合物（新糖脂）的生物活性开发

基本信息

批准号：
09359001
负责人：
SAITO Masaki
金额：
$ 20.03万
依托单位：
National Cancer Center Research Institute (1998-1999)Hokkaido University (1997)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

Glycoconjugates from the frontier of cell-to-cell and cell-to-substratum interactions. Among them, gangliosides (sialic acid containing glycosphingolipids) 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 ellaborately-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: ST3GalV) and then, murine ST3GalV, which is responsible for GM3 biosynthesis. Subsequently, 3 kinds of transcripts (L-,B1-and B2-type) of the gene were detected in mice by 5'-RACE analyses … More whereas a single transcript detectable in human organs, and murine tissue-specific expressions were clarified: L-type transcript was specifically expressed in liver while B1-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 【lambda bar】 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 the programs for exploitation of natural and synthetic sialoglycocompounds, i.e. neoglycolipids, in the applications to the medical fields, the hydrophobic (fatty acid) moiety of ganglioside GM3 was changed in the chemically-synthesized molecule in order to enhance its biological activity, and, among more than 20 synthetic sialoglycolipid compounds, both α-sialo-cholesterol and α-sialodiglyceride were shown to exhibit significant differentiation-including and apoptosis-including activities to human leukemia cells. Anti-sense oligonucleotide therapy for human melanomas is initiated using GD3 synthase cDNA since GD3-dominant melanoma was reported worse in the prognosis. We could produce ST3GalV soluble forms as MBP-/GST-fusion proteins in order to utilize in the development of automatic carbohydrate-chain synthesizers. We have also devised newly the toroidal coil counter-current chromatography to isolate less polar alkali-labile glycolipids and proteins with higher sensitivity. Less

来自细胞与细胞和细胞与基质相互作用前沿的糖缀合物。其中，神经节苷脂（含唾液酸的鞘糖脂）在细胞生长和分化、胚胎发生和癌发生等多种生物学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神经节苷脂GM 3合成的，我们以前曾证明GM 3对人髓性白血病细胞具有分化诱导活性。在这个项目中，使用精心设计的表达克隆方法，我们首次成功地分离和分子表征了一个新的和相关的基因（cDNA和基因组），它编码一个关键的糖基转移酶，人神经节苷脂GM 3合成酶（唾液酸转移酶-1：ST 3GalV），然后，小鼠ST 3GalV，这是负责GM 3的生物合成。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-、B1-和B2-型 ...更多信息而在人类器官中可检测到单个转录物，并阐明了小鼠组织特异性表达：L型转录物在肝脏中特异性表达，而B1型通常在各种器官中检测到，B2型少量表达。通过分别筛选从染色体DNA制备的BAC和[lambda bar]文库，确定了ST 3 GalV的人和小鼠基因组结构。有趣的是，将这种酶cDNA转染到神经节苷脂缺陷型小鼠肺癌3LL细胞中，可将富含GM 3的膜结构域特征性脱落引入培养基中。在天然和合成的唾液酸糖化合物（即新糖脂）的开发计划中，在医学领域的应用中，疏水性的唾液酸糖化合物是天然的和合成的唾液酸糖化合物的衍生物。在化学合成的分子中改变神经节苷脂GM 3的（脂肪酸）部分以增强其生物活性，并且，在超过20种合成的唾液酸糖脂化合物中，α-唾液酸胆固醇和α-唾液酸甘油二酯均显示出对人白血病细胞的显著的包括分化和包括凋亡的活性。人类黑色素瘤的反义寡核苷酸治疗开始使用GD 3合酶cDNA，因为GD 3-显性黑色素瘤的预后更差。我们可以生产ST 3GalV可溶形式作为MBP-/GST-融合蛋白，以便用于自动碳水化合物链合成仪的开发。我们还设计了新的环形线圈逆流色谱分离低极性碱不稳定的糖脂和蛋白质具有较高的灵敏度。少