Development of a universal sequencing method for sulfated glycosaminoglycans

硫酸化糖胺聚糖通用测序方法的开发

• 批准号: 09558082
• 负责人: SUGAHARA Kazuyuki
• 金额: $ 6.72万
• 依托单位: Kobe Pharmaceutical University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09558082/
• 关键词: chondroitin sulfate; heparin; heparin sulfate; dermatan sulfate; sulfated oligosaccharides; glycosaminoglycan; heparinase; chondroitinase; 硫酸化; 線虫; コンドロイチナーゼC; マンノサミン; C2-エピメリゼーション; NMR; デルマタン流酸; 2-アミノベンズアミド; 螢光標識

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

各种生长/分化因子通过与细胞表面上的相应高亲和力受体结合而表现出其生物学活性。对于这些因子中的一些，它们需要首先与细胞表面上的糖胺聚糖（GAG）链结合。然而，这些GAG链的结构分析特别是测序一直受到复杂结构的阻碍。因此，我们进行了以下研究，以开发简单且广泛适用的硫酸化GAG链测序方法。(1)从鱿鱼软骨硫酸软骨素中分离出五个新的硫酸化四糖序列，并对其结构进行了确定。(2)从猪肠肝素中分离出一种硫酸化四糖、一种五糖、八种六糖、三种八糖和四种八糖丝氨酸，并确定其结构为新结构。此外，已从皮肤素硫酸酯链中分离出几种硫酸化的四糖和六糖， ...更多信息 使上述猪肠肝素制剂稳定。(3)八个新的硫酸化四糖和五个六糖已被分离和结构确定从盲鳗脊索。(4)在肝素寡糖的上述结构分析过程中，已经证明在温和碱性条件下，N-硫酸化葡糖胺非酶转化为N-硫酸化甘露糖胺。(5)普雷沃氏菌肝素酶的底物特异性。和分离自黄杆菌属h.的软骨素酶C，两者都是GAG链结构研究的工具。(6)用荧光团2-氨基苯甲酰胺标记GAG链的还原末端，建立了GAG链的微量分析方法。将该标记方法与酶切方法相结合，建立了一种高灵敏度的GAG链测序方法。(7)使用上述新开发的方法，非硫酸化软骨素和高度硫酸化硫酸乙酰肝素已在C.优雅的少

项目成果

Yamada, Shuhei: "Structural determanation of sulfated tetra- and hexasaccharides containing a rate disaddharide sequence, -3GalNAc(4, 6-disulfated) β1-4IdoAα1-, isolated from porcin eintestinal dermatan sulfate"Eur. J. Biochem.. 258(2). 775-783 (1998)

Yamada, Shuhei：“从猪肠硫酸皮肤素中分离出的含有二加氢序列的硫酸化四糖和六糖的结构测定，-3GalNAc(4, 6-二硫酸化) β1-4IdoAα1-”Eur. J. Biochem.. 258(2) ）775-783（1998）。

Ueoka, Chikako: "Structural Determination of Novel Tetra- and Hexasaccharide Sequences Isolated from Chondoitin Sulfate H(Oversulfated Dermatan Sulfate) of Hagfish Notochord"Glyconjugate J.. 16(6). 291-305 (1999)

Ueoka，Chikako：“从盲鳗脊索硫酸软骨素 H（过硫酸皮肤素硫酸盐）中分离的新型四糖和六糖序列的结构测定”糖缀合物 J.. 16(6)。

津田 博美: "Substrate specificity studies of flavobacterium chondroitinase C and heparitinases towards the glycosaminoglycan-protein linkage region:Use of a sensitive analytical method developed by chromophore labeling of linkage glycoserines using dimethylam

Hiromi Tsuda：“黄杆菌软骨素酶 C 和肝素酶对糖胺聚糖-蛋白质连接区域的底物特异性研究：使用通过使用二甲胺对连接糖丝氨酸进行发色团标记而开发的灵敏分析方法

植岡 千香子: "Structural Determination of Novel Tetra- and Hexasaccharide Sequences Isolated from CHondroitin Sulfate H(Oversulfated Dermatan Sulfate) of Hagfish Notochord"Glycoconjugate J.. 16(6). 291-305 (1999)

Chikako Ueoka：“从盲鳗脊索硫酸软骨素 H（过硫酸皮肤素硫酸盐）中分离的新型四糖和六糖序列的结构测定”糖缀合物 J.. 16(6) (1999)。

Yamada, Shuhei: "Structure of Oligosaccharides Isolated from Heparan Sulfate/Heparin and Subatrate Specificities of the Degranding Enzymes of Bacterial Origin"Trend. Glycosci. Glycotechnol.. 10(52). 95-123 (1998)

山田修平：“从硫酸乙酰肝素/肝素中分离出的寡糖的结构和细菌来源的降解酶的底物特异性”趋势。