Synthesis and utilization of neo-glycoconjugates containing deaminoneuraminic acid (KDN) residues

本研究的目的是建立含有新唾液酸(脱氨基尿酸)残基的新糖偶联物的酶促合成方法，以期将其作为新的生物活性和/或有机材料加以利用。获得了以下结果：1.寻找KDN特异的糖基转移酶和糖苷酶--(1)虹鱼卵巢和精子中的两种KDN-转移酶对合成neo-KDN-糖结合物有用；-(2)从土壤细菌中发现并纯化了对KDN残基高度特异的糖苷酶，并用于首次在哺乳动物细胞和组织中鉴定KDN-糖结合物；2.建立了酶法合成neo-KDN-糖结合物的方法。以甘露糖和丙酮酸为原料，在N-酰基神经氨酸裂解酶的作用下，合成单糖KDN；以CTP和KDN为原料，在CMP-KDN合成酶的作用下，合成CMP-KDN；利用这种方法，能够制备足够数量的糖蛋白、糖脂和低聚糖，以测试其生物学和化学性质。后两个过程中所涉及的酶是我们从虹鲑鱼中发现并制备的。根据2中描述的方法制备的neo-KDN-转铁蛋白与已知对NeuAc残基具有特异性的植物凝集素(SSA)的配基活性进行了检测，表明SSA也识别KDN残基。为了在体内合成KDN糖结合物，寻找了决定KDN糖结合物合成的关键酶(S)，并提出了KDN9-磷酸合成酶的参与。

The objective of this research project was to establish methods for enzymatic synthesis of neo-glycoconjugates containing new sialic acid (deaminoneuraminic acid, KDN) residues with the aim of utilization of such neo-KDN-glycoconjugates as new bioactive and/or organic materials. The following results were obtained :1. Search for KDN-specific glycosyltransferases and glycosidases-(1) Two KDN-transferases from rainbow trout ovary and sperm were found to be useful for neo-KDN-glycoconjugate synthesis ; -(2) Glycosidase highly specific for KDN residue was found and purified from a soil bacterium, and used for first identification of KDN-glycoconjugates in mammalian cells and tissues ;2. Establishment of methods for enzymatic synthesis of neo-KDN-glycoconjugates was achieved. Three synthetic processes using bacterial and animal enzymes were involved : synthesis of monosaccharide KDN from mannose and pyruvate with N-acylneuraminate lyase, synthesis of CMP-KDN from CTP and KDN with CMP-KDN synthetase, and incorporation of KDN residues into various glycoconjugates from CMP-KDN catalyzed by KDN-transferases. Using this method, glycoproteins, glycolipids, and oligosaccharides were able to be prepared in sufficient amounts for testing the biological and chemical properties. The enzymes involved in the latter two processes were discovered and prepared from rainbow trout by us.3. Neo-KDN-transferrin, prepared according to the methods described in 2, was examined for ligand activity to a plant lectin (SSA), which is known to be specific to NeuAc residue, revealing that SSA recognized KDN residue as well.4. For the purpose of in vivo synthesis of KDN-glycoconjugates, key enzyme(s) which determines KDN-glycoconjugate synthesis was searched for, and involvement of KDN 9-phosphate synthetase was suggested.