Zhang Shaohua, Cai Xuepeng, Luo Xuenong, Wang Shuai, Guo Aijiang, Hou Junling, Wu Run
College of Veterinary Medicine, Gansu Agricultural University, Lanzhou 730070, PR China; State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China.
State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, PR China.
Exp Parasitol. 2018 Mar;186:1-9. doi: 10.1016/j.exppara.2018.01.012. Epub 2018 Jan 10.
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 M17LAP 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 °C. GST-TpLAP displayed a hydrolyzing activity for the Leu-pNA substrate with a maximum activity of 46 U/ml. The enzymatic activity was significantly enhanced by Mn 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 M17LAP 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.
亮氨酸氨肽酶（LAP，EC：3.4.11.1）是一种重要的金属外肽酶，可催化从多肽和蛋白质的氨基末端亮氨酸残基处进行水解。在本研究中，采用聚合酶链反应（RACE-PCR）方法通过cDNA末端快速扩增克隆了编码豆状带绦虫亮氨酸氨肽酶（TpLAP）的全长cDNA。TpLAP基因的全长cDNA为1823 bp，包含一个1569 bp的开放阅读框，编码533个氨基酸，推测分子量为56.4 kDa。TpLAP在C末端序列中包含M17LAP家族的两个特征基序：金属结合位点265-[VGKG]-271和催化结构域基序351-[NTDAEGRL]-357。可溶性GST-TpLAP蛋白在大肠杆菌Transetta（DE3）中表达，并制备了四种特异性抗TpLAP单克隆抗体（mAb）。在酶活性测定中，在45°C、pH 9.5时观察到最佳活性。GST-TpLAP对Leu-pNA底物具有水解活性，最大活性为46 U/ml。Mn可显著增强酶活性，20 nM苯丁抑制素和0.15 mM EDTA可完全抑制酶活性。以抗TpLAP mAb为探针，通过蛋白质免疫印迹法在豆状带绦虫成虫的排泄分泌（ES）成分中特异性检测到天然TpLAP。实时定量PCR显示，TpLAP基因在成虫组织中高水平表达，尤其是在孕节片中（50.71倍）。免疫定位分析表明，TpLAP主要位于成虫的皮下实质区和子宫壁。我们的结果表明，TpLAP是M17LAP家族的一个新成员，可被视为一种阶段差异表达蛋白。这些发现可能为研究豆状带绦虫在终末宿主体内的生长、发育和存活机制提供新的见解，并具有作为药物治疗或疫苗干预的有吸引力靶点的潜在价值。
