Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis
Molecular characterization of a novel Clonorchis sinensis secretory phospholipase A2 and investigation of its potential contribution to hepatic fibrosis
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DOI：
10.1016/j.molbiopara.2009.05.003
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发表时间：
2009-10-01
影响因子：
1.5
通讯作者：
Yu, Xinbing
中科院分区：
文献类型：
--
作者：
Hu, Fengyu;Hu, Xuchu;Yu, Xinbing
从华支睾吸虫成虫 cDNA 质粒文库中鉴定出编码磷脂酶 A(2) 同源物的基因。推导的氨基酸序列包括与其他物种的分泌性磷脂酶A(2)、III组(group III sPLA(2))具有28-46%同一性的信号肽。它还具有 III 族 sPLA(2)S 的典型特征，包括 10 个半胱氨酸、Ca2+ 环和催化位点的关键残基。在大肠杆菌中表达的该基因编码的重组蛋白在SDS-PAGE中显示出约34kDa的产物。信号肽预测和Western blot分析表明中华线虫III族分泌型磷脂酶A(2)(CsGIIIsPLA(2))是一种排泄-分泌产物(ES产物)。使用磷脂酰胆碱作为底物测定重组蛋白的酶活性。结果表明该蛋白是Ca2+依赖性PLA(2)。 LX-2的MTT测试和细胞周期分析均显示较高比例的细胞处于增殖期。半定量 RT-PCR 实验表明，与重组蛋白一起孵育后，这些细胞中 III 型胶原蛋白的表达上调。我们还通过免疫荧光鉴定重组CsGIIIsPLA(2)可以与LX-2细胞上的一些膜蛋白特异性结合，因此LX-2细胞膜上可能存在CsGIIIsPLA(2)受体。我们的结果表明CsGIIIsPLA(2)可能在中华线虫引起的肝纤维化的发生和发展中发挥重要作用。 (C) 2009 Elsevier B.V. 保留所有权利。
A gene encoding a homologue of phospholipase A(2) was identified from the Clonorchis sinensis adult cDNA plasmid library. The deduced amino acid sequence including a signal peptide that has 28-46% identity with secretory phospholipase A(2), group III (group III sPLA(2)) of other species. It also has typical features of group III sPLA(2)S including 10 cysteines, the key residues of the Ca2+ loop and catalytic site. The recombinant protein encoded by this gene expressed in Eschetichia coli showed a product of about 34 kDa in SDS-PAGE. Prediction of signal peptide and Western blot analysis indicated the group III secretory phospholipase A(2) of C. sinensis (CsGIIIsPLA(2)) was an excretory-secretory product (ES product). The enzyme activity of the recombinant protein was determined using phosphatidylcholine as substrates. The result revealed that the protein was a Ca2+-dependent PLA(2). Both MTT test and cell cycle analysis of LX-2 showed a higher percentage of cells are in proliferation phase. Semi-quantitative RT-PCR experiments demonstrated an up-regulated expression of collagen III in these cells after incubation with the recombinant protein. We also identified that the recombinant CsGIIIsPLA(2) could bind to some membrane proteins on LX-2 cells specifically by immunofluorescence, thus there might be receptors of CsGIIIsPLA(2) on the LX-2 cell membrane. Our results suggest that CsGIIIsPLA(2) Might play an important role in the initiation and development of hepatic fibrosis caused by C. sinensis. (C) 2009 Elsevier B.V. All rights reserved.