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

硫酸乙酰肝素（HS）在多种发育、生理和致病过程中起关键作用。这些功能是由于以结构依赖的方式与许多参与细胞信号传导的生长因子相互作用。我们用小鼠、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。硫酸肝素对肝素结合生长因子（HB-GF）诱导的信号传导的调节(1)我们发现，硫酸肝素中的6- o -硫酸盐可以增强vegf165依赖性管的形成和有丝分裂活性（2005，J.Biol.Chem.）。(2) HS6ST-1基因敲除小鼠胎盘迷路区血管生成异常。这些缺陷似乎是由于VEGF的表达减少（2007,j.b ol.chem .）。(3)在果蝇中，FGF信号调节气管系统的形成。39%的Hs6st胚胎存在气管缺陷（2006，J.Cell.Biol）。(4)肢芽的形成涉及多种HB-GFs。当siRNA抑制鸡胚前肢的HS2ST表达时，鸡胚前肢芽被截断，顶端外胚层嵴中FGF-8的表达减少（2007,j.b oll . chem .）。(5)我们利用HS6ST-1衍生的成纤维细胞检测了HS6 - o -硫酸化在HB-GFs信号传导中的作用；HS6ST-2双突变小鼠（dKO-MEF）。在dKO-MEFs中，fgf -4和fgf -2依赖性信号分别减少到WT-MEFs的30%和60%左右。与WT-MEFs相比，FGF-1依赖性信号传导适度减少，但仅在较低的FGF-1浓度下。结果表明，HS中的6- o -硫酸盐可能通过诱导配体与其受体之间的差异相互作用来调节一些HB-GFs（包括FGFs）的信号传导。肥大细胞有许多蛋白酶，它们与肝素颗粒结合，并通过抗原与细胞表面IgE的相互作用释放。肝素的磺化是与颗粒中的某些蛋白质相互作用所必需的。因此，阐明6- o -硫代肝素的生物合成途径具有重要意义。从敲除小鼠耳中提取的肝素结构分析表明，HS6ST1缺失小鼠产生正常的硫酸化肝素。另一方面，来自HS6ST-2缺失小鼠的肝素被减少到正常肝素的6- o -硫酸化的50%。我们利用培养的胚胎皮肤肥大细胞来研究双敲除小鼠产生的肝素的结构变化。少

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