STUDY ON THE INHIBITOR FOR HEPARAN SULFATE-ACTIVE DOMAINS

已知硫酸乙酰肝素（HS）与许多蛋白质相互作用，如肝素结合生长因子（HB-GF）、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发挥关键作用。因此，研究调控HS功能的化合物具有重要意义。重组酶法制备八糖及其抑菌活性的研究我们首次从完全纯化的1V-肝素衍生八糖出发，在体外分别与硫酸乙酰肝素-2-O-磺基转移酶（HS 2ST）和硫酸乙酰肝素-6-O-磺基转移酶（HS 6ST）反应，制备了2-O-硫酸寡糖和6-O-硫酸八糖。通过肝素的有限裂解制备2-O-和6-O-硫酸化八糖。利用该寡糖库，我们系统地研究了各种肝素结合生长因子（FGF-2，-4）的特异性结合结构 ...更多信息 、-7、-8、-10和-18、HGF、BMP-1和VEGF）。结果表明，根据与寡糖亲和力的差异，上述HB-GF可大致分为五类。特别地，FGF-2和FGF-10结合至特定的寡糖，并且前者仅结合至6-0-硫酸化八糖，而后者结合至2- 0-硫酸化八糖。接下来，我们通过其受体的磷酸化来检测这些寡糖和修饰的肝素对VEGF_<165>、FGF-1和FGF-2的活性的影响。肝素<165>可增强培养的内皮细胞（HUVEC）中VEGF_2的活性，但不受任何寡糖的影响。在培养的成纤维细胞中，FGF-1依赖的磷酸化在低浓度肝素时增加，但在高浓度肝素时减少。由于FGF-1与某些低聚糖结合强烈，我们正在研究低聚糖对FGF-1.2活性的影响。HS 2ST和HS 6ST化学抑制剂的筛选我们首先从受体底物类似物出发，对HS 2ST和HS 6ST的化学抑制剂进行了综述。Metyl-β-GlcA（2SO_4）对HS 6ST的体外活性有抑制作用，而Metyl-α-GlcA（2SO_4）对HS 6ST的体外活性无抑制作用。接下来，为了检查该化合物是否抑制培养细胞中的HS 6ST活性，我们分析了该化合物对分别用FGF-2和VEGF_诱导的FGFR-1和VEGFR-2磷酸化的影响<165>。3 mM以上的Metyl-β-GlcA（2SO_4）对VEGFR-2的磷酸化有抑制作用。而在甲基-β-GlcA（2SO_4）存在下生成的HS的二糖组成没有显著变化。因此，该化合物的抑制作用是否是由于HS介导的机制仍有待澄清。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