Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni
Praziquantel Affects the Regulatory Myosin Light Chain of Schistosoma mansoni
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DOI：
10.1128/aac.01222-08
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发表时间：
2009-03-01
影响因子：
4.9
通讯作者：
Kalyanasundaram, Ramaswamy
中科院分区：
文献类型：
--
作者：
Gnanasekar, Munirathinam;Salunkhe, Ashok M.;Kalyanasundaram, Ramaswamy
吡喹酮（PZQ）是治疗血吸虫病的首选药物，可能是目前治疗血吸虫病感染者的唯一高效药物。PZQ的作用模式涉及增加寄生虫的钙摄取，导致寄生虫的体表损伤和死亡。尽管PZQ具有显著的功能，但其作用靶点尚未明确。为了开始了解PZQ的作用机制，本研究在T7噬菌体表面表达了曼氏血吸虫的cDNA文库，并用标记的PZQ对该文库进行了筛选。该程序鉴定了与PZQ强结合的克隆。随后对插入片段的DNA分析表明，该克隆编码调节性肌球蛋白轻链蛋白。然后克隆了该基因，重组S。表达了曼氏肌球蛋白轻链（SmMLC）。使用针对重组SmMLC（rSmMLC）产生的抗体进行的免疫印迹分析表明，与丝虫期的表达量相比，SmMLC在血吸虫童虫和成虫期大量表达。体外分析也证实了PZQ与rSmMLC的强结合。此外，肽图谱研究表明，PZQ结合SmMLC的氨基酸46至76。免疫沉淀分析证实，SmMLC是磷酸化在体内暴露于PZQ。有趣的是，与感染小鼠中的量相比，免疫小鼠中存在显著水平的抗SmMLC抗体，这表明SmMLC可能是血吸虫病保护性免疫的潜在靶点。这些结果表明，PZQ影响SmMLC功能，这可能在PZQ行动中发挥作用。
Praziquantel (PZQ) is the drug of choice for schistosomiasis and probably is the only highly effective drug currently available for treating schistosomiasis-infected individuals. The mode of action of PZQ involves increasing the calcium uptake of the parasite, resulting in tegumental damage and death of the parasite. Despite its remarkable function, the target of PZQ has not been identified yet. To begin to understand where PZQ acts, in this study we expressed the cDNA library of Schistosoma mansoni on the surface of T7 bacteriophages and screened this library with labeled PZQ. This procedure identified a clone that strongly bound to PZQ. Subsequent DNA analysis of inserts showed that the clone coded for regulatory myosin light chain protein. The gene was then cloned, and recombinant S. mansoni myosin light chain (SmMLC) was expressed. Immunoblot analysis using antibodies raised to recombinant SmMLC (rSmMLC) showed that SmMLC is abundantly expressed in schistosomula and adult stages compared to the amount in cercarial stages. In vitro analyses also confirmed that PZQ strongly binds to rSmMLC. Further, peptide mapping studies showed that PZQ binds to amino acids 46 to 76 of SmMLC. Immunoprecipitation analysis confirmed that SmMLC is phosphorylated in vivo upon exposure to PZQ. Interestingly, significant levels of anti-SmMLC antibodies were present in vaccinated mice compared to the amount in infected mice, suggesting that SmMLC may be a potential target for protective immunity in schistosomiasis. These findings suggest that PZQ affects SmMLC function, and this may have a role in PZQ action.