Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor
Characterisation of EmMPK1, an ERK-like MAP kinase from Echinococcus multilocularis which is activated in response to human epidermal growth factor
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DOI：
10.1016/j.ijpara.2006.05.008
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发表时间：
2006-09-01
影响因子：
4
通讯作者：
Brehm, Klaus
中科院分区：
文献类型：
--
作者：
Spiliotis, Markus;Konrad, Christian;Brehm, Klaus
丝裂原活化蛋白(MAP)激酶是细胞信号系统的关键调节者，该系统介导对多种细胞外刺激的反应，也应该在寄生虫的发育机制中发挥核心作用。然而，到目前为止，还没有MAP激酶同源基因在这个寄生虫组的成员中得到表征。在这里，我们报告了这样一个分子，EmMPK1，从人类寄生虫多房棘球绦虫中鉴定和特征。利用简并聚合酶链式反应的方法，我们分离了EmMPK1的1.2kb的cDNA，并对其进行了全序列测定，该序列与已知的不同系统起源的MAPK有显著的同源性。EmMPK1包含MAP激酶特有的所有氨基酸残基，其中包括一个保守的Tey基序，该基序将该蛋白鉴定为MAP激酶ERK亚家族的成员。相应的基因emmpk1(6.9kb)由10个内含子组成。Southern杂交研究表明，emmpk1在多房棘球藻中以单拷贝的形式存在。通过RT-PCR分析，我们证明emmpk1以三种不同的转录本的形式表达，这三种转录本来自内含子9处的选择性剪接受体位点。利用Western印迹研究和免疫组织化学中的EmMPK1特异性抗体，我们在体外培养过程中和在感染中间宿主的过程中，检测了棘球绦虫幼虫阶段的包虫病蛋白及其磷酸化形式。从棘球绦虫裂解液中免疫沉淀的EmMPK1在活性检测中能够磷酸化髓鞘碱性蛋白，表明它是一种具有功能活性的MAPK。最后，我们还表明，EmMPK1的磷酸化在体外培养的多房棘球绦虫囊泡中被特异性地诱导，以响应外源宿主血清和添加人表皮生长因子。这些数据表明，多房棘球绦虫能够感应寄主的表皮生长因子，从而激活寄生虫的MAPK级联反应。(C)2006澳大利亚寄生虫学协会。爱思唯尔有限公司出版。版权所有。
Mitogen-activated protein (MAP) kinases are key regulators of cellular signalling systems that mediate responses to a wide variety of extracellular stimuli and should also play a central role in developmental mechanisms of parasitic helminths. Until now, however, no MAP kinase orthologue has been characterised in a member of this parasite group. Here, we report the identification and characterisation of such a molecule, EmMPK1, from the human parasitic cestode Echinococcus multilocularis. Using a degenerative PCR approach, we isolated and completely sequenced the 1.2 kb cDNA for EmMPK1 which displays significant homologies to known MAP kinases of different phylogenetic origin. EmMPK1 contains all amino acid residues which are characteristic for MAP kinases, including a conserved TEY motif which identifies the protein as a member of the ERK subfamily of MAP kinases. The corresponding gene, emmpk1 (6.9 kb), was characterised and contained 10 introns. Southern blot hybridisation studies showed that emmpk1 is present as single copy locus in E. multilocularis. Using RT-PCR analyses we demonstrated that, emmpk1 is expressed in form of three different transcripts which derive from alternative splice acceptor site utilisation at intron 9. Using EmMPK1-specific antibodies in Western blot studies and immunohistochemistry, we detected the Echinococcus protein and its phosphorylated form in the larval stages metacestode and protoscolex during in vitro cultivation and during an infection of the intermediate host. EmMPK1, immunoprecipitated from Echinococcus lysate, was able to phosphorylate myelin basic protein in activity assays, indicating that it is a functionally active MAP kinase. Finally, we also show that phosphorylation of EmMPK1 is specifically induced in vitro-cultivated E. multilocularis metacestode vesicles in response to exogenous host serum and upon addition of human epidermal growth factor. These data indicate that the E. multilocularis metacestode is able to sense epidermal growth factor from the host which results in an activation of the parasite's MAP kinase cascade. (c) 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.