Joint Research on Acidic Glycoconjugates on Cell Surfaces

本研究的目的是阐明表面糖复合物如脱氨基神经氨酸（KDN）、聚唾液酸和硫酸化唾液酸的酸性聚糖链在动物细胞系统中的细胞识别和粘附中的功能。获得了以下结果：1.精卵粘附中酸性聚糖链的结构与功能：（1）Neu 5Gc链与精子卵受体相连，Neu 5Gc链非还原端O-8位有硫酸基，具有抑制海胆受精的作用。在海胆精子中发现了新的四唾液酸（Neu 5Ac）Glc-Cer，这种糖脂对受精过程具有延缓作用;（2）首次证实ZP 3和ZP 1，2同源物存在于鸟类未受精卵中。这些ZP同源物参与精卵相互作用的研究正在进行中.含表面聚唾液酸和KDN糖复合物表达的调控机制新KDN糖链酶法合成方法的建立：（1）建立了KDN残基特异性水解酶KDNase的纯化方法。这种酶和先前开发的单克隆抗体的组合使我们能够识别哺乳动物组织中微量的KDN残基。（2）研制了一种新的寡聚Neu 5Ac单克隆抗体，首次发现寡聚唾液酸残基在哺乳动物组织中普遍存在;（3）确定了KDN单糖的生物合成途径，并发现参与该途径的酶也与N-酰基神经氨酸单糖的生物合成有关。

The objective of this research project was to elucidation of functions of acidic glycan chains of surface glycoconjugates, such as deaminoneuraminic acid (KDN), polysialic acid, and sulfated sialic acid, in cell recognition and adhesion in animal cellular systems. The following results were obtained :1. Structure and function of acidic glycan chains in sperm-egg adhesion : (1) Oligo/polysialic acid (Neu5Gc) chains capped by a sulfate group at O-8 position of non-reducing terminus, which were shown to inhibit fertilization of sea urchin, were found to be linked to egg receptor for sperm. In sea urchin sperm, novel tetrasialyl (Neu5Ac) Glc-Cer was found, and this type of glycolipid had an delaying effect on progress of fertilization ; (2) ZP3 and ZP1,2 homologues were for the first time demonstrated to be present in avian unfertilized eggs. Involvement ofthese ZP homologues in sperm-egg interaction are now underway.2. Regulatory mechanism for expression of surface polysialic acid- and KDN-containing glycoconjugates establishment of methods for enzymatic synthesis of neo-KDN-glycan chains : (1) Purification procedures of KDN residue specific hydrolytic enzyme, KDNase, from a bacterium were established. Combination of this enzyme and the previously developed monoclonal antibodies enabled us to identify a minute amount of KDN residues in mammalian tissues. At the same time, the presence of KDN in mammalian cells were also shown chemically by a newly developed fluorometic (DMB) method ; (2) A new oligo Neu5Ac specific monoclonal antibody was developed.Using this antibody, ubiquitous occurrence of oligosialic acid residues in various mammalian tissues were first indicated ; (3) Biosynthetic pathway of KDN monosaccharide was identified, and enzymes involved in the pathway were shown to be also related with biosynthesis of N-acylneuraminic acid monosaccharide.