Molecular cloning and characterization of leucine aminopeptidase gene from Taenia pisiformis

文献类型： 外文期刊

第一作者： Zhang, Shaohua

作者： Zhang, Shaohua;Wu, Run;Zhang, Shaohua;Cai, Xuepeng;Luo, Xuenong;Wang, Shuai;Guo, Aijiang;Hou, Junling

作者机构：

关键词： Taenia pisiformis; Leucine aminopeptidase; Enzymatic activity; Expression level; Immunolocalization

期刊名称：EXPERIMENTAL PARASITOLOGY （ 影响因子：2.011； 五年影响因子：2.132 ）

ISSN： 0014-4894

年卷期： 2018 年 186 卷

页码：

收录情况： SCI

摘要： Leucine aminopeptidase (LAP, EC: 3.4.11.1) is an important metalloexopeptidase that catalyze the hydrolysis of amino-terminal leucine residues from polypeptides and proteins. In this study, a full length of cDNA encoding leucine aminopeptidase of Taenia pisiformis (TpLAP) was cloned by rapid amplification of cDNA-ends using the polymerase chain reaction (RACE-PCR) method. The full-length cDNA of the TpLAP gene is 1823 bp and contains a 1569 bp ORF encoding 533 amino acids with a putative mass of 56.4 kDa. TpLAP contains two characteristic motifs of the MI7LAP family in the C-terminal sequence: the metal binding site 265-[VGKG]-271 and the catalytic domain motif 351-[NTDAEGRL]-357. The soluble GST-TpLAP protein was expressed in Escherichia coli Transetta (DE3) and four specific anti-TpLAP monoclonal antibodies (mAbs) were prepared. In enzymatic assays, the optimal activity was observed at pH 9.5 at 45 degrees C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/mI. The enzymatic activity was significantly enhanced by Mn2+ and completely inhibited by 20 nM bestatin and 0.15 mM EDTA. The native TpLAP was detected specifically in ES components of adult T. pisiformis by western blotting using anti-TpLAP mAb as a probe. Quantitative real-time PCR revealed that the TpLAP gene was expressed at a high level in adult worm tissues, especially in the gravid proglottids (50.71-fold). Immunolocalization analysis showed that TpLAP was located primarily in the subtegumental parenchyma zone and the uterine wall of adult worms. Our results indicate that TpLAP is a new member of the MI7LAP family and can be considered as a stage-differentially expressed protein. These findings might provide new insights into the study of the mechanisms of growth, development and survival of T pisiformis in the final host and have potential value as an attractive target for drug therapy or vaccine intervention. (C) 2018 Elsevier Inc. All rights reserved.

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