Bentley Geoffrey N, Jones Andrew K, Agnew Alison
The School of Biology, University of Leeds, West Yorkshire, LS2 9JT, Leeds, UK.
Gene. 2003 Sep 18;314:103-12. doi: 10.1016/s0378-1119(03)00709-1.
Acetylcholinesterase (AChE) on the surface of the parasitic blood fluke Schistosoma is the likely target for schistosomicidal anticholinesterases. Determination of the molecular structure of this drug target is key for the development of improved anticholinesterase drugs and potentially a novel vaccine. We have recently cloned the cDNA encoding the AChE from the human parasite Schistosoma haematobium and succeeded in expressing functional recombinant protein. We now describe the cloning and molecular characterisation of homologues from two other schistosome species-Schistosoma mansoni and Schistosoma bovis, which are important parasites of man and cattle, respectively, but which differ in their sensitivity to the therapeutic anticholinesterase metrifonate. Comparison of the deduced amino acid sequences revealed that the AChE from all three species posses a high degree of identity, with conservation of all of the residues known to be important for substrate binding and catalytic activity. Also conserved is a unique C-terminal domain which is unusual in that it lacks the consensus for GPI modification, even though the native protein is considered to be GPI-anchored. We have also established the AChE gene structures for all three species and cloned the complete gene for S. haematobium AChE. The gene structure is relatively complex, comprising nine coding exons; the location of the splice sites is identical in all three species, but the size of the introns varies considerably. The two C-terminal splicing sites that are conserved in all species are also present in Schistosoma, but a third C-terminal conserved splicing site which is located 11-13 amino acids upstream of the histidine of the catalytic triad in all invertebrate AChE genes characterised to date is absent. We discuss our findings in the context of the molecular phylogeny of the AChE genes and the potential application to the control of schistosomiasis.
寄生血吸虫表面的乙酰胆碱酯酶（AChE）可能是杀血吸虫抗胆碱酯酶的作用靶点。确定该药物靶点的分子结构是开发改良抗胆碱酯酶药物以及潜在新型疫苗的关键。我们最近从人类寄生虫埃及血吸虫中克隆了编码AChE的cDNA，并成功表达了功能性重组蛋白。我们现在描述来自另外两种血吸虫——曼氏血吸虫和牛血吸虫同源物的克隆及分子特征，这两种血吸虫分别是人和牛的重要寄生虫，但它们对治疗性抗胆碱酯酶敌百虫的敏感性不同。对推导的氨基酸序列进行比较发现，所有这三种物种的AChE具有高度同一性，所有已知对底物结合和催化活性重要的残基均得以保留。同样保守的还有一个独特的C末端结构域，其不同寻常之处在于，尽管天然蛋白被认为是糖基磷脂酰肌醇（GPI）锚定的，但它缺乏GPI修饰的共有序列。我们还确定了所有这三种物种的AChE基因结构，并克隆了埃及血吸虫AChE的完整基因。该基因结构相对复杂，由9个编码外显子组成；所有这三种物种的剪接位点位置相同，但内含子大小差异很大。所有物种中保守的两个C末端剪接位点在血吸虫中也存在，但迄今已鉴定的所有无脊椎动物AChE基因中位于催化三联体组氨酸上游11 - 13个氨基酸处的第三个C末端保守剪接位点不存在。我们在AChE基因的分子系统发育背景下讨论了我们的发现以及对血吸虫病控制的潜在应用。
