Development of a universal sequencing method for sulfated glycosaminoglycans

各种生长/分化因子通过与细胞表面各自的高亲和力受体结合而发挥其生物活性。其中一些因子需要首先与细胞表面的糖胺聚糖（GAG）链结合。然而，由于这些GAG链的结构复杂，其结构分析特别是序列分析一直受到阻碍。因此，我们开展了以下研究，以开发简单且广泛适用的磺化GAG链测序方法。(1)从鱿鱼软骨硫酸软骨素中分离得到5个新的硫酸化四糖序列，并确定了其结构。(2)从猪肠肝素中分离到1个磺化四糖、1个五糖、8个六糖、3个八糖和4个八糖丝氨酸，并在结构上定义为新结构。此外，从皮肤硫酸酯链中分离出了几种硫酸化的四糖和六糖，这些糖污染了猪肠肝素制剂。(3)从盲鳗脊索中分离得到8个新的硫酸化四糖和5个新的硫酸化六糖，并确定了其结构。(4)以上对肝素寡糖的结构分析表明，在温和的碱性条件下，n-硫酸氨基葡萄糖可以非酶转化为n-硫酸甘露糖胺。(5)从普雷沃氏菌（Prevotella h.）分离的一种新型肝素酶和从黄杆菌（Flavobacterium h.）分离的一种软骨素酶C （chondroitinase C）的底物特异性已经得到澄清，这两种酶都是GAG链结构研究的工具。(6)利用荧光基团2-氨基苯甲酰胺在GAG链的还原端进行荧光标记，建立了GAG链的微量分析方法。此外，将这种标记方法与酶切相结合，开发了一种高灵敏度的GAG链测序方法。(7)利用上述新开发的方法，已在秀丽隐杆线虫中证实了非硫酸软骨素和高硫酸乙酰肝素。少

Various growth/differentiation factors exhibit their biological activities through binding to the respective high affinity receptors on cell surface. For some of these factors to do so, they need to first bind to glycosaminoglycan (GAG) chains on cell surface. However, the structural analysis especially the sequencing of these GAG chains has been hampered by the complicated structure. Therefore, we have carried out the following studies to develop simple and wide-applicable sequencing methods for sulfated GAG chains.(1) Five novel sulfated tetrasaccharide sequences have been isolated from squid cartilage chondroitin sulfate and structurally defined.(2) A sulfated tetrasaccharide, a pentasaccharide, eight hexasaccharides, three octasaccharides and four octasaccharide serines have been isolated from porcine intestinal heparin and structurally defined as novel structures. In addition, several sulfated tetra- and hexasaccharides have been isolated from dermatan sulfate chains which contami … More nated the porcine intestinal heparin preparation described above.(3) Eight novel sulfated tetrasaccharides and five hexasaccharides have been isolated from hagfish notochord and structurally defined.(4) It has been demonstrated during the course of the above structural analysis of heparin oligosaccharides that N-sulfated glucosamine is converted into N-sulfated mannosamine non-enzymatically under mild alkaline conditions.(5) Substrate specificities of a novel heparinase isolated from Prevotella h. and chondroitinase C isolated from Flavobacterium h., both of which are tools for structural studies of GAG chains, have been clarified.(6) A microanalytical method for GAG chains has been developed by fluorescent labeling of GAG chains at their reducing termini with a fluorophore 2-aminobenzamide. Furthermore, a sequencing method for GAG chains with high sensitivity has been developed by combining this labeling method and enzymatic digestion.(7) Using the newly developed method described above, nonsulfated chondroitin and highly sulfated heparan sulfate have been demonstrated in C. elegans. Less