THE ACTIVITY OF A NEW GLYCOSAMINOGLYCAN ON THE REGULATION OF FIBROBLAST GROWTH FACTORS.

本实验室目前正在进行的研究成果和研究项目如下：(1)糖胺聚糖(GAG)的分析方法。对于生物样品的检测，最可靠的方法之一是毛细管电泳法，我们已经建立了各种涉及毛细管电泳法检测的GAG的检测方法。核磁共振波谱可以提供糖胺多聚糖和蛋白多聚糖等糖共轭化合物的定量和结构信息。建立了测定皮肤素/肝素硫酸盐中尿酸根残留量的质子核磁共振光谱分析方法。(2)从巨螺中提取一种新的糖胺多糖制备低聚糖。从巨型蜗牛中提纯了大量的阿卡林硫酸酯，并用肝素裂解酶II对糖胺多糖进行处理，得到低聚糖。用凝胶渗透色谱(GPC)和高效液相色谱(SAX-Hplc)方法对4-10MERS的低聚糖进行了纯化，并用质子核磁共振对其进行了表征。(3)调控…更多的成纤维细胞生长因子是由糖胺聚糖合成的。成纤维细胞生长因子是重要的肝素结合、有丝分裂蛋白。成纤维细胞生长因子-2(碱性成纤维细胞生长因子)在肝素和硫酸肝素中的结合部位被描述为富含氨基葡萄糖-2-硫酸盐1-和4-艾杜糖酸-2-硫酸盐。肝素结合部位的氨基葡萄糖残基也是6-硫酸化的。对一种新的糖胺多糖--阿卡兰多糖硫酸盐进行了化学修饰，得到了一种由氨基葡萄糖-2-硫酸盐与艾杜糖酸-2-硫酸盐相连的多糖硫酸酯N-磺基-阿卡兰多糖。阿查兰硫酸盐与成纤维细胞生长因子-2的结合非常弱，而N-硫代阿卡兰硫酸酯与肝素的结合亲和力几乎相同。使用稳定转染成纤维细胞生长因子受体-1的无硫酸肝素细胞进行有丝分裂活性研究。阿查兰硫酸盐抑制肝素对成纤维细胞生长因子-2有丝分裂活性的增强，但不影响细胞存活率，而N-磺基阿查兰多糖硫酸盐表现出类似肝素的活性，但水平大大降低。这些结果表明，不需要高亲和力的糖胺聚糖与成纤维细胞生长因子-2结合的其他机制可能在其促有丝分裂活性中起重要作用。较少

The results and research projects currently underway in our laboratory are as follows : (1) Methodology for analyses of glycosaminoglycans (GAGs). One of the most reliable methods, which are useful for determination of biological samples, is CE.We have already established determination methods for various GAGs involving CE with detection. NMR spectroscopy can provide quantitative and structural information of glycoconjugates such as glycosaminoglycans and proteoglycans. The proton NMR spectroscopic method has been established for determination of uronate residues in dermatan/heparan sulfates. (2) Preparation of oligosaccharides from a new glycosaminoglycan from the giant snail. A large amount of acharan sulfate was purified from the giant snail and the glycosaminoglycan was treated with heparin lyase II to obtain oligosaccharides. The oligosaccharides ranged 4-10 mers were purified by GPC and SAX HPLC methods, and each oligosaccharide was characterized by proton NMR.(3) The regulation … More of fibroblast growth factors by glycosaminoglycans. Fibroblast growth factors are important heparin binding, mitogenic proteins. The binding site in heparin and heparan sulfate for fibroblast growth factor-2 (basic fibroblast growth factor) has been described as rich in glucosamine-2-sulfate 1->4linked to iduronic acid-2-sulfate. The glucosamine residue in the heparin binding site is also 6-sulfated. A new glycosaminoglycan, acharan sulfate, has been chemically modified to prepare a polysaccharide, N-sulfoacharan sulfate, consisting of glucosamine-2-sulfate 1->4 linked to iduronic acid-2-sulfate. Acharan sulfate binds very weakly to fibroblast growth factor-2 while N-sulfoacharan sulfate binds with nearly the same affinity as heparin. Mitogenicity studies were performed using heparan sulfate-free cells stably transfected with fibroblast growth factor receptor-1. Acharan sulfate inhibits heparin's enhancement of fibroblast growth factor-2 mitogenic activity, without affecting cell viability, while N-sulfoacharan sulfate shows heparin-like activity but at a greatly reduced level. These results suggest additional mechanisms not requiring high affinity glycosaminoglycan binding to fibroblast growth factor-2 may be important in its mitogenic activity. Less