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

本实验室目前的研究成果和研究项目如下：(1)糖胺聚糖（GAGs）的分析方法。CE是测定生物样品最可靠的方法之一。我们已经建立了包括CE和检测在内的各种gag的测定方法。核磁共振光谱可以提供糖胺聚糖和蛋白聚糖等糖缀合物的定量和结构信息。建立了测定皮肤皂苷/硫酸肝素中脲酸盐残留量的质子核磁共振光谱法。(2)从巨螺中提取新的糖胺聚糖制备低聚糖。从巨螺中纯化出大量硫酸阿恰兰，用肝素裂解酶II对其进行糖胺聚糖处理，得到低聚糖。通过GPC和SAX高效液相色谱法纯化了4 ~ 10个分子量的低聚糖，并通过质子核磁共振对其进行了表征。(3)糖胺聚糖对成纤维细胞生长因子的调节作用。成纤维细胞生长因子是重要的肝素结合、有丝分裂蛋白。在肝素和硫酸肝素中，成纤维细胞生长因子-2（碱性成纤维细胞生长因子）的结合位点富含氨基葡萄糖-2-硫酸1- bbbb4，与伊杜醛酸-2-硫酸相连。肝素结合位点的氨基葡萄糖残基也被6-硫酸化。对一种新的糖胺聚糖硫酸阿恰兰进行了化学改性，制备了一种由氨基葡萄糖-2-硫酸1- bbb4与硫酸伊杜醛酸-2-硫酸连接而成的n -磺化硫酸阿恰兰多糖。硫酸Acharan与成纤维细胞生长因子-2的结合非常弱，而n -磺化硫酸Acharan与肝素的结合几乎相同。用无硫酸肝素细胞稳定转染成纤维细胞生长因子受体-1进行有丝分裂性研究。硫酸阿恰兰抑制肝素增强成纤维细胞生长因子-2的有丝分裂活性，但不影响细胞活力，而n-硫代硫酸阿恰兰表现出肝素样活性，但水平大大降低。这些结果表明，不需要高亲和力糖胺聚糖结合成纤维细胞生长因子-2的其他机制可能对其有丝分裂活性很重要。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