Joint Research on Acidic Glycoconjugates on Cell Surfaces

本研究项目的目的是阐明表面糖缀合物的酸性聚糖链，如脱氨神经氨酸（KDN）、聚唾液酸和硫酸唾液酸，在动物细胞系统中的细胞识别和粘附功能。得到了以下结果：1。酸聚糖链在精卵粘附中的结构与功能：(1)在非还原端O-8位置有一个硫酸盐基团的Oligo/polysialic acid （Neu5Gc）链被发现与卵子的精子受体有关，该链抑制海胆受精。在海胆精子中发现了一种新型的四asialyl (Neu5Ac) Glc-Cer，这种糖脂对受精进程有延迟作用；(2)首次在禽类未受精卵中发现ZP3和ZP1 2个同源物。这些ZP同源物参与精子与卵子的相互作用目前正在进行中。表面聚唾液酸和含KDN糖缀合物的表达调控机制建立酶法合成新KDN糖链的方法：(1)建立了从细菌中分离KDN残基特异性水解酶KDNase的纯化工艺。该酶与先前开发的单克隆抗体的结合使我们能够识别哺乳动物组织中微量的KDN残基。同时，通过新开发的荧光（DMB）化学方法也显示了KDN在哺乳动物细胞中的存在；(2)制备了一种新的寡核苷酸特异性Neu5Ac单克隆抗体。利用该抗体，首次指出了低聚硅酸残基在各种哺乳动物组织中的普遍存在；(3)确定了KDN单糖的生物合成途径，并发现该途径所涉及的酶也与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.