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

来自细胞与细胞和细胞与底物相互作用前沿的糖共轭。其中，神经节苷脂(含唾液酸鞘糖脂)在细胞生长分化、胚胎发生和肿瘤发生等多种生物现象中发挥着重要作用。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，我们首次利用设计的表达克隆方法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：ST3GalV)，然后是小鼠ST3GalV，它负责GM3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型、B1型和B2型此外，还明确了在人体器官和小鼠组织中可检测到的单一转录本：L类型的转录本在肝脏中特异表达，而B1型转录本在各种器官中普遍表达，B2型转录本的表达水平较低。人和小鼠ST3GalV的基因组结构分别通过筛选染色体DNA制备的BAC文库和[lambda bar]文库确定。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。在开发天然和合成唾液酸脂类化合物的计划中，在医学领域的应用中，为了提高其生物活性，化学合成的分子中神经节苷脂GM3的疏水(脂肪酸)部分被改变，并且在20多个合成唾液酸脂类化合物中，α-唾液酸胆固醇和α-唾液酸二甘油酯都被证明对人白血病细胞具有显著的分化-包括和凋亡-活性。人类黑色素瘤的反义寡核苷酸治疗是利用GD3合酶基因开始的，因为GD3显性黑色素瘤的预后较差。我们可以生产ST3GalV可溶性形式的MBP-/GST-融合蛋白，以用于自动碳水化合物链合成器的开发。我们还设计了新的环形线圈逆流层析来分离极性较弱的碱不稳定的糖脂和具有较高灵敏度的蛋白质。较少

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