Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis
Identification and characterisation of a leucine aminopeptidase from the hard tick Haemaphysalis longicornis
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DOI：
10.1016/j.ijpara.2006.05.010
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发表时间：
2006-09-01
影响因子：
4
通讯作者：
Fujisaki, Kozo
中科院分区：
文献类型：
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作者：
Hatta, Takeshi;Kazama, Kouji;Fujisaki, Kozo
尚未在吸血蜱中鉴定出负责血液消化的氨肽酶。我们在此报告编码亮氨酸氨肽酶的 cDNA 的克隆和分子特征，亮氨酸氨肽酶是来自硬蜱长角血蜱 (HILAP) 的 M17 胞质氨肽酶家族的成员。通过免疫印迹法在成年蜱提取物的可溶部分中检测到内源性 HILAP。免疫组织化学研究表明，内源性HILAP表达主要发生在中肠上皮细胞的胞浆中。此外，HILAP的表达是由血液喂养过程诱导的。在大肠杆菌中表达的功能性重组 HILAP 可有效水解氨肽酶的合成底物、亮氨酰底物（K-m 值为 0.19 +/- 0.011 mM 和 V-max，值为 157.2 +/- 3.17 nmol/min/mg 蛋白质）和甲硫氨酰底物（K-m 值为 0.12 +/- 0.0052 mM 和 Vmax 值） 171.9 +/- 2.31 nmol/分钟/毫克蛋白质）。酶活性在 pH 8 和 35°C 时达到最佳。重组 HILAP 酶活性强烈依赖于金属二价阳离子 Mn2+，并被贝他汀抑制。这些结果表明HILAP在宿主的血液消化过程中发挥重要作用。 (c) 2006 年澳大利亚寄生虫学协会。由 Elsevier Ltd 出版。保留所有权利。
Aminopeptidases responsible for blood digestion have yet to be identified in haematophagous ticks. We report here the cloning and molecular characterisation of a cDNA encoding leucine aminopeptidase, a member of the M17 cytosolic aminopeptidase family, from the hard tick Haemaphysalis longicornis (HILAP). Endogenous HILAP was detected in the soluble fraction of adult tick extracts by immunoblotting. Immunohistochemical studies demonstrated that endogenous HILAP expression mainly took place in the cytosol of midgut epithelial cells. Furthermore, expression of HILAP was induced by a blood-feeding process. A functional recombinant HILAP expressed in Escherichia coli efficiently hydrolyses synthetic substrates for aminopeptidase, a leucyl (with the K-m value 0.19 +/- 0.011 mM and V-max, value 157.2 +/- 3.17 nmol/min/mg protein) and a methionyl substrate (with the K-m value 0.12 +/- 0.0052 mM and Vmax value 171.9 +/- 2.31 nmol/min/mg protein). Enzyme activity was found to be optimum at pH 8 and 35 degrees C. The recombinant HILAP enzyme activity was strongly dependent on metal divalent cations, Mn2+, and was inhibited by bestatin. These results indicate that HILAP play an important role for host's blood digestion process. (c) 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.