STUDY ON THE INHIBITOR FOR HEPARAN SULFATE-ACTIVE DOMAINS

已知硫酸肝素(HS)可与多种蛋白质相互作用，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过程中起着关键作用。对控制HS1功能的化合物进行研究是很重要的。重组酶法制备八糖及其抑制活性首次以完全脱除1V的肝素衍生的八糖为原料，分别与硫酸乙酰肝素-2-O-磺酸转移酶(HS2ST)和肝素-6-O-磺酸转移酶(HS6ST)在体外反应生成2-O-硫代低聚糖和6-O-硫代八糖。用肝素有限裂解法制备了2-O-和6-O-磺化八糖。利用这个寡糖文库，我们系统地研究了各种肝素结合生长因子(Fgf-2，-4…)的结合结构更多，-7，-8，-10和-18，HGF，BMP-和VEGF)通过亲和层析和表面等离子共振。结果表明，根据与低聚糖亲和力的不同，上述HB-GFS大致可分为五类。尤其是，成纤维细胞生长因子-2和成纤维细胞生长因子-10与特定的寡糖结合，前者只与6-0-硫化的八糖结合，后者与2-O-硫化的八糖结合。接下来，我们研究了这些寡糖和修饰肝素对血管内皮生长因子165、成纤维细胞生长因子-1和成纤维细胞生长因子-2受体磷酸化的影响。在培养的血管内皮细胞(HUVEC)中加入肝素可增强血管内皮生长因子165的活性，但不受任何寡糖的影响)。在培养的成纤维细胞中，低浓度肝素使成纤维细胞成纤维细胞生长因子-1依赖磷酸化增加，高浓度肝素使成纤维细胞中成纤维细胞生长因子-1依赖的磷酸化减少。由于成纤维细胞生长因子-1与一些寡糖有很强的结合，我们正在研究低聚糖对成纤维细胞生长因子-1.2活性的影响。HS2ST和HS6ST化学抑制剂的筛选我们首先从受体底物类似物中综述了HS2ST和HS6ST的化学抑制剂。甲基-β-GLCA(2SO_4)对HS6ST的体外活性有抑制作用，但对甲基-α-GLCA(2SO_4)的抑制作用不明显。接下来，为了检测该化合物是否抑制培养细胞中HS6ST的活性，我们分析了该化合物对成纤维细胞生长因子-2和血管内皮生长因子165&gt；诱导的FGFR-1和VEGFR-2磷酸化的影响。大于3 mM的甲基-β-GLCA(2SO4)对VEGFR-2的磷酸化有抑制作用。然而，在甲基-β-GLCA(2SO4)存在下生成的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介导的机制有关尚不清楚。较少

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