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

糖缀合从细胞与细胞和细胞与基质相互作用的前沿。其中，神经节苷类（含鞘糖脂的唾液酸）在细胞生长分化、胚胎发生、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），该基因编码了关键的糖基转移酶——人神经节苷脂GM3合成酶（sialyltransferase-1: ST3GalV），然后编码了负责GM3生物合成的小鼠ST3GalV。随后，通过5′- race分析，在小鼠中检测到3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，并明确了小鼠组织特异性表达：L型转录本在肝脏中特异性表达，b1型转录本普遍在各器官中检测到，b2型边缘表达。通过筛选从染色体DNA制备的BAC和【lambda bar】文库，分别确定了人、鼠ST3GalV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。在天然和人工合成唾液糖化合物（即新糖脂）的开发和医学应用中，化学合成分子改变了神经节苷脂GM3的疏水（脂肪酸）部分，以提高其生物活性，在20多种人工合成唾液糖脂化合物中，α-唾液胆固醇和α-唾液二甘油酯对人白血病细胞均表现出显著的分化和凋亡活性。人类黑色素瘤的反义寡核苷酸治疗开始使用GD3合成酶cDNA，因为据报道GD3显性黑色素瘤预后较差。我们可以制备可溶性的ST3GalV作为MBP-/ gst融合蛋白，以用于碳水化合物链自动合成器的开发。我们还设计了新的环形线圈逆流色谱法，以分离极性较低的碱不稳定糖脂和灵敏度较高的蛋白质。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