Kamimura K, Fujise M, Villa F, Izumi S, Habuchi H, Kimata K, Nakato H
Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-0397, Japan.
J Biol Chem. 2001 May 18;276(20):17014-21. doi: 10.1074/jbc.M011354200. Epub 2001 Mar 8.
Heparan sulfate, one of the most abundant components of the cell surface and the extracellular matrix, is involved in a variety of biological processes such as growth factor signaling, cell adhesion, and enzymatic catalysis. The heparan sulfate chains have markedly heterogeneous structures in which distinct sequences of sulfate groups determine specific binding properties. Sulfation at each different position of heparan sulfate is catalyzed by distinct enzymes, sulfotransferases. In this study, we identified and characterized Drosophila heparan sulfate 6-O-sulfotransferase (dHS6ST). The deduced primary structure of dHS6ST exhibited several common features found in those of mammalian HS6STs. We confirmed that, when the protein encoded by the cDNA was expressed in COS-7 cells, it showed HS6ST activity. Whole mount in situ hybridization revealed highly specific expression of dHS6ST mRNA in embryonic tracheal cells. The spatial and temporal pattern of dHS6ST expression in these cells clearly resembles that of the Drosophila fibroblast growth factor (FGF) receptor, breathless (btl). RNA interference experiments demonstrated that reduced dHS6ST activity caused embryonic lethality and disruption of the primary branching of the tracheal system. These phenotypes were reminiscent of the defects observed in mutants of FGF signaling components. We also show that FGF-dependent mitogen-activated protein kinase activation is significantly reduced in dHS6ST double-stranded RNA-injected embryos. These findings indicate that dHS6ST is required for tracheal development in Drosophila and suggest the evolutionally conserved roles of 6-O-sulfated heparan sulfate in FGF signaling.
硫酸乙酰肝素是细胞表面和细胞外基质中含量最丰富的成分之一，参与多种生物学过程，如生长因子信号传导、细胞粘附和酶催化作用。硫酸乙酰肝素链具有明显的异质性结构，其中硫酸基团的不同序列决定了特定的结合特性。硫酸乙酰肝素每个不同位置的硫酸化由不同的酶——硫酸转移酶催化。在本研究中，我们鉴定并表征了果蝇硫酸乙酰肝素6 - O -硫酸转移酶（dHS6ST）。dHS6ST推导的一级结构显示出在哺乳动物HS6ST中发现的几个共同特征。我们证实，当cDNA编码的蛋白质在COS - 7细胞中表达时，它表现出HS6ST活性。整体原位杂交显示dHSmRNA在胚胎气管细胞中高度特异性表达。dHS6ST在这些细胞中的时空表达模式明显类似于果蝇成纤维细胞生长因子（FGF）受体——呼吸缺陷（btl）的表达模式。RNA干扰实验表明，dHS6ST活性降低会导致胚胎致死以及气管系统初级分支的破坏。这些表型让人联想到在FGF信号成分突变体中观察到的缺陷。我们还表明，在注射了dHS6ST双链RNA的胚胎中，FGF依赖的丝裂原活化蛋白激酶激活显著降低。这些发现表明dHS6ST是果蝇气管发育所必需的，并提示6 - O -硫酸化硫酸乙酰肝素在FGF信号传导中具有进化保守作用。
