Development of a universal sequencing method for sulfated glycosaminoglycans

各种生长/分化因子通过与细胞表面相应的高亲和力受体结合而显示其生物学活性。对于这些因子中的一些，它们需要首先结合到细胞表面的糖胺聚糖链(GAG)上。然而，复杂的结构阻碍了对这些Gag链的结构分析，特别是测序。因此，我们开展了以下几项研究，以期建立简单、适用范围广的GAG链测序方法。(1)从鱿鱼软骨硫酸软骨素中分离得到5个新的硫化四糖序列，并对其结构进行了定义。(2)从猪肠肝素中分离得到一个新的硫化四糖、一个五糖、八个六糖、三个八糖和四个八糖丝氨酸，结构上定义为新的结构。此外，从污染…的皮肤素硫酸酯链中分离出几种硫酸化的四糖和六糖(3)从鳗鱼脊索中分离得到8种新的硫酸化四糖和5种结构明确的六糖。(4)在上述肝素低聚糖的结构分析过程中，证明了N-硫化氨基葡萄糖在弱碱性条件下非酶转化为N-硫化甘露糖胺。(5)从普氏杆菌中分离到一种新的肝素酶和从黄杆菌中分离到的软骨素酶C的底物特异性。(6)用荧光团2-氨基苯甲酰胺对GAG链还原末端的荧光标记，建立了GAG链的微量分析方法。此外，将该标记方法与酶消化法相结合，建立了一种高灵敏度的GAG链测序方法。(7)利用上述新开发的方法，在线虫中发现了非硫酸软骨素和高硫酸化硫酸肝素。较少

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