STUDY ON THE INHIBITOR FOR HEPARAN SULFATE-ACTIVE DOMAINS

硫酸肝素（HS）已知与许多蛋白质相互作用，如肝素结合生长因子（HB-GFs），细胞外基质蛋白，蛋白酶抑制剂和各种病原体。这些相互作用已被证明在各种病理生理现象以及发育中起关键作用。研究控制HS.1功能的化合物具有重要意义。重组酶制备八糖及其抑制活性研究首先通过体外与硫酸肝素-2- o -硫转移酶（HS2ST）和硫酸肝素-6- o -硫转移酶（HS6ST）反应，将完全脱硫的肝素衍生八糖合成2- o -硫代寡糖和6- o -硫代八糖。通过肝素的有限裂解制备了2-O-和6- o -硫酸八糖。利用这个寡糖文库，我们通过亲和层析和表面等离子体共振技术系统地研究了各种肝素结合生长因子（FGF-2、-4…More、-7、-8、-10和-18、HGF、BMP-和VEGF）的特异性结合结构。结果表明，根据与低聚糖亲和力的差异，上述hb - gf可大致分为5类。其中，FGF-2和FGF-10与特异性寡糖结合，前者仅与6-0-硫酸八糖结合，后者与2- 0-硫酸八糖结合。接下来，我们研究了这些低聚糖和修饰肝素通过磷酸化VEGF_<165>、FGF-1和FGF-2受体对其活性的影响。在体外培养的内皮细胞（HUVEC）中，添加肝素可增强VEGF_<165>的活性，而低聚糖对其活性无明显影响。在培养成纤维细胞中，FGF-1依赖性磷酸化在低浓度肝素下升高，在高浓度肝素下降低。由于FGF-1与一些低聚糖结合强烈，我们正在研究低聚糖对FGF-1.2活性的影响。筛选HS2ST和HS6ST的化学抑制剂首先从受体底物类似物中筛选HS2ST和HS6ST的化学抑制剂。Metyl-β-GlcA（2SO_4）对体外HS6ST活性有抑制作用，而Metyl-α-GlcA（2SO_4）对体外HS6ST活性无抑制作用。接下来，我们分析了该化合物对FGF-2和VEGF_<165>诱导的FGFR-1和VEGFR-2磷酸化的影响，以研究该化合物在培养细胞中是否抑制HS6ST的活性。超过3 mM的meyl -β-GlcA（2SO_4）对VEGFR-2的磷酸化有抑制作用。然而，在甲基β- glca （2SO_4）存在下，HS的双糖组成没有明显变化。因此，该化合物的抑制作用是否与HS介导的机制有关，还有待进一步研究。少

Heparan sulfate (HS) are known to interact with numerous proteins, such as heparin-binding growth factors (HB-GFs), extracellular matrix proteins, protease inhibitors and various pathogens. These interactions have been shown to play pivotal role in various patho-physiological phenomenons as well as in development. It is important to survey the compounds to control the function of HS.1. Preparation of octasaccharides n by recombinant enzymes and its inhibitory activityWe first generated 2-O-sulfated oligosaccharides and 6-O-sulfated octasaccharides from completely desulfated 1V-resulfated heparin-derived octasaccharide by in vitro reaction with heparan sulfate-2-O-sulfotransfease (HS2ST) and heparansulfate-6-O-sulfotransferase (HS6ST). Both 2-O-, and 6-O-sulfated octasaccharides were prepared by limited cleavage of heparin. Using this oligosaccharide library, we investigated systematically investigated the specific binding structures for various heparin-binding growth factors (FGF-2, -4 … More , -7, -8, -10, and -18, HGF, BMP-, and VEGF) by affinity chromatography and surface plasmon resonance. The results indicated that the above listed HB-GFs could be classified roughly into five groups on the basis of the difference in affinity with the oligosaccharides. In particular, FGF-2 and FGF-10 bound to specific oligosaccharide, and the former bound to only 6-0-sulfated octasaccharide and the latter to 2-O-sulfated octasaccharide. Next, we examined the effects of these oligosaccharides and modified heparin on the activities of VEGF_<165>, FGF-1 and FGF-2 by phosphorylation of their receptors. The activity of VEGF_<165> was enhanced by addition of heparin in cultured endothelial cells (HUVEC), but was not affected by any oligosaccharides). In cultured fibroblasts, FGF-1 dependen phosphorylation was increased at low concentration of heparin but decreased at higher concentration of heparin. As FGF-1 bound strongly to some oligosaccharides, we are investigating the effects of oligosaccharides on the activity of FGF-1.2. Screening of chemical inhibitor for HS2ST and HS6STWe first surveyed the chemical inhibitors of HS2ST and HS6ST from acceptor substrate analogs. Metyl-β-GlcA(2SO_4) inhibited the activity of HS6ST in vitro, but not Metyl-α-GlcA(2SO_4). Next, to examine whether this compound inhibit the activity of HS6ST in cultured cell, we analyzed the effects of this compound on phosphorylation of FGFR-1 and VEGFR-2 induced with FGF-2 and VEGF_<165>, respectively. More than 3 mM Metyl-β-GlcA(2SO_4) showed inhibitory effect for phosphorylation of VEGFR-2. However, disaccharide compositions of HS generated in the presence of Metyl-β-GlcA(2SO_4) did not change significantly. Therefore, it remains to be clarified whether the inhibitory effect of this compound is due to the mechanism mediated by HS. Less