Joint Research on Acidic Glycoconjugates on Cell Surfaces

本研究的目的是阐明细胞表面糖复合物的酸性聚糖链对细胞识别和粘附的调控机制。研究结果如下：1.阐明了寡聚/聚唾液酸单位在脑和神经系统中的生物合成机制：（1）在哺乳动物胚胎和成年脑中，有几种糖蛋白含有寡聚/聚唾液酸结构。该表位在神经细胞系统生物学活性中的作用研究正在进行中;（2）制备了10多个新的单克隆抗体，这些抗体可区别识别不同链长的二/寡唾液酸，作为功能/结构研究的探针。建立了两种高灵敏度的荧光染料化学法检测唾液酸. KN-聚糖或硫酸化唾液酸聚糖在受精配子相互作用中的作用：（1）制备富含糖脂的低密度洗涤剂不溶性膜组分，并表征其独特的脂质和蛋白质组成特征。（2）克隆了3个与多唾液酸合成有关的唾液酸转移酶的cDNA序列，并在鱼类卵母细胞中进行了表达;（3）在卵泡颗粒细胞中合成了一种禽蛋包膜酸性糖蛋白（ZPC）.含有polySia或KN的聚糖链在肿瘤细胞中的表达和作用：（1）KN-聚糖链被证明是肿瘤发育抗原;（2）在富含甘露糖的培养基中培养的哺乳动物细胞中观察到细胞内KDN的表达升高。KDN水平高的癌细胞可能存在甘露糖代谢的改变;（3）成功克隆了鳟鱼精巢CMP-KDN合成酶的cDNA。这种类型的酶的KDN的底物特异性被建议用于确定哺乳动物正常和恶性细胞中的KDN水平。

The objective of this research project was to elucidation of regulatory mechanism for cell recognition and adhesion by acidic glycan chains of cell surface glycoconjugates. The results follow :1. Clarification of biosynthetic mechanism of oligo/polysialic acid units in brain and nerve system : (1) Several glycoproteins were shown to contain oligo/polysialic acid structures in fetal and adult mammalian brains. Studies on role of this epitope in biological activities in nerve cell system are now underway ; (2) More than 10 new monoclonal antibodies that differentially recognized various di/oligosialic acid with different chain lengths were prepared as probes for function/structure studies. Two highly sensitive chemical methods to detect di/oligosialic acid using fluorescent dye were developed.2. Role of KDN-glycans or sulfated sialoglycans in gamete interactions at fertilization : (1) The low density detergent-insoluble membrane fractions which were rich in glycolipids were prepared and characterized for unique features of lipid and protein compositions. This membrane fractions are suggested to mediate sperm-egg interaction as adhesion units on the cell surface ; (2) Three distinct cDNAs for sialyltransferases that are involved in synthesis of polysialic and in fish oocyte were cloned ; (3) An avian egg envelope acidic glycoprotein (ZPC) was shown to be biosynthesized in granulosa cells of follicles.3. Expression and roles of polySia- or KDN-containing glycan chains in tumor cells : (1) KDN-glycan chains were shown to be a oncodevelopmental antigen ; (2) Elevated expression of intracellular KDN in mammalian cells cultured in mannose-rich medium was observed. Some cancer cells whose KDN level is high may have alternations of mannose metabolism ; (3) A cDNA for CMP-KDN synthetase from trout testis was successfully cloned. Substrate specificity with KDN of this type of enzyme is suggested to determine the level of KDN in mammalian normal and malignant cells.