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STUDY ON THE BIOLOBICAL AND PHYSIOLOGICAL FUNCTION OF 6-O-SULFATION IN HEPARANSULFATE/HEPARIN

硫酸乙酰肝素/肝素中6-O-硫酸化的生物和生理功能研究

基本信息

批准号：
17570099
负责人：
HABUCHI Hiroko
金额：
$ 2.3万
依托单位：
INSTITUTE FOR MOLECULAR SCIENCE OF MEDICINE, AICHI MEDICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2006
项目状态：
已结题

项目摘要

Heparan sulfate (HS) plays critical roles in a variety of developmental, physiological, and pathogenic processes. These functions are due to interact in a structure-dependent manner with numerous growth factors that participate in cellular signaling. We examined the roles of 2-O-sulfate and 6-O-sulfate in HS using mice, chick, drosophila, and mutant cells.1.Regulation of heparin-binding growth factor (HB-GF)-induced signaling by heparan sulfate(1) We indicated that VEGF165-dependent tube formation and mitogenic activity was enhanced by 6-O-sulfate in heparan sulfate (2005, J.Biol.Chem.). (2) HS6ST-1 knockout mice exhibited the abnormal angiogenesis in placental labyrinthine zone. These defects appear to be due to the reduced expression of VEGF (2007, J.Biol.Chem.) (3) In drosophila, FGF signaling regulates tracheal system formation. 39% of Hs6st null embryos exhibited tracheal defects (2006, J.Cell.Biol.). (4) The formation of limb bud involves various HB-GFs. When HS2ST in the prospec … More tive limb of chick embryo was inhibited by siRNA, the limb buds were truncated and the expression of FGF-8 was reduced in the apical ectodermal ridge (2007, J.Biol.Chem.). (5) We examined the functions of HS 6-O-sulfation in HB-GFs signaling using fibroblast derived from HS6ST-1 ; HS6ST-2 double mutant mice (dKO-MEF). In dKO-MEFs, FGF-4-and FGF-2-dependent signaling was reduced to approximately 30% and 60% of WT-MEFs, respectively. FGF-1-dependent signaling was moderately reduced compared to that of WT-MEFs but only at lower FGF-1 concentration. The results suggest that 6-O-sulfate in HS probably regulates the signaling of some of HB-GFs, including FGFs, by inducing the differential interaction between ligands and their receptors (in submission).2.Biosynthesis of 6-O-sulfation in heparinMast cells have many proteases, which are bound to heparin in granules, and released by the interaction of the antigen with cell surface IgE. Sulfaion of Heparin is required for the interaction with some of these proteins in granules. Therefore, it is important to elucidate biosynthesis pathway of 6-O-sulfation of heparin. Structural analysis of heparin derived from ears of knockout mice showed that HS6ST1 null mice generated normal sulfated heparin. On the other hand, heparin derived from HS6ST-2 null mice was reduced to 50% of 6-O-sulfation in normal heparin. We are investigating the structure changes of heparin produced by the double knockout mice using cultured mast cell from embryonic skin. Less

硫酸乙酰肝素（HS）在多种发育、生理和致病过程中起着关键作用。这些功能是由于以结构依赖性方式与参与细胞信号传导的许多生长因子相互作用。我们使用小鼠、鸡、果蝇和突变细胞检测了2-O-硫酸盐和6-O-硫酸盐在HS中的作用。1.硫酸乙酰肝素对肝素结合生长因子（HB-GF）诱导的信号传导的调节（1）我们表明，硫酸乙酰肝素中的6-O-硫酸盐增强了VEGF 165依赖性管形成和促有丝分裂活性（2005，J.Biol.Chem.）。(2)HS 6ST-1基因敲除小鼠胎盘绒毛膜区血管生成异常。这些缺陷似乎是由于VEGF的表达减少（2007，J.Biol.Chem.）(3)在果蝇中，FGF信号调节气管系统的形成。39%的Hs 6st缺失胚胎表现出气管缺陷（2006，J.Cell.Biol.）。(4)肢芽的形成涉及多种HB-GFs。当HS 2ST在前景中时， ...更多信息通过siRNA抑制鸡胚的主动肢，肢芽被截短，并且在顶端外胚层嵴中FGF-8的表达减少（2007，J.Biol.Chem.）。(5)我们使用来自HS 6ST-1 ; HS 6ST-2双突变小鼠（dKO-MEF）的成纤维细胞检测了HS 6-O-硫酸化在HB-GFs信号传导中的功能。在dKO-MEFs中，FGF-4-和FGF-2-依赖性信号传导分别降低至WT-MEFs的约30%和60%。与WT-MEFs相比，FGF-1依赖性信号传导中度降低，但仅在较低的FGF-1浓度下。结果表明，HS中6-O-硫酸酯可能通过诱导配体与受体之间的差异相互作用来调节某些HB-GFs（包括FGF）的信号传导（提交中）。2.肝素中6-O-硫酸酯的生物合成肥大细胞中有许多蛋白酶，它们与肝素结合成颗粒，并通过抗原与细胞表面IgE的相互作用而释放。肝素的硫酸盐是与颗粒中的某些蛋白质相互作用所必需的。因此，阐明肝素6-O-硫酸化的生物合成途径具有重要意义。来自基因敲除小鼠耳朵的肝素的结构分析表明，HS 6ST 1敲除小鼠产生正常的硫酸化肝素。另一方面，来源于HS 6ST-2缺失小鼠的肝素减少至正常肝素中6-O-硫酸化的50%。我们正在研究由双基因敲除小鼠利用培养的胚胎皮肤肥大细胞产生肝素的结构变化。少