Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis
Molecular cloning and functional characterization of an aspartic protease from the hard tick Haemaphysalis longicornis
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DOI：
10.1016/j.ibmb.2005.10.003
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发表时间：
2006-01-01
影响因子：
3.8
通讯作者：
Fujisaki, K
中科院分区：
文献类型：
--
作者：
Boldbaatar, D;Sikasunge, CS;Fujisaki, K
从中肠cDNA文库中鉴定了编码所有天冬氨酸蛋白酶（长肽酶）的长角血蜱cDNA。longepsin cDNA全长1176 bp，编码392个氨基酸残基，分子量为39.3kDa。cDNA具有与N-末端结构域相关的信号肽序列，结构域结构分析显示，推导的蛋白质具有两个天冬氨酸残基，这是天冬氨酸蛋白酶的单一活性位点的特征。该新的longepsin cDNA与埃及伊蚊的溶酶体天冬氨酸蛋白酶具有57%的同源性，与家蚕组织蛋白酶D具有52%的同源性，与犬钩虫具有38%的同源性，与曼氏血吸虫具有44%的同源性，与微小牛蜱天冬氨酸蛋白酶具有28%的同源性。将编码长胰蛋白酶的DNA片段克隆到pGFHX-4 T-3载体中，并在大肠杆菌中表达。重组longepsin，一旦激活，能够水解酪蛋白底物以及血红蛋白（Hb）在酸性条件下（pH 3.5）。RT-PCR分析表明，longepsin mRNA转录本在唾液腺和中肠中表达，而在卵巢中不表达。北方印迹分析表明，longepsin（1.5 kb）在未进食和部分进食的蜱中表达，并且在进食期间表达水平增加。长胰蛋白酶在中肠和唾液腺中表达，在酸性条件下蛋白水解活性降低，并且长胰蛋白酶可以被长胰蛋白酶基因沉默，这一发现为长胰蛋白酶在从宿主血粉获得的红细胞Hb的蛋白水解中发挥所有不可或缺的作用的假设提供了令人信服的支持。(C)2005爱思唯尔有限公司保留所有权利。
Haemaphlysalis longicornis cDNA encoding ail aspartic protease (longepsin) was identified from a midgut cDNA library. The longepsin cDNA contains 1176 bp that code for 392 amino acid residues With a predictable molecular weight of 39.3 kDa. The cDNA has a signal peptide sequence associated with the N-terminal domains and domain Structure analysis revealed that the deduced protein has two aspartic acid residues that are characteristic of a single active site for aspartic proteases. This novel longepsin cDNA exhibits 57% identity to the lysosomal aspartic protease of Aedes aegypti, 52% to Bombyx mori cathepsin D, 38% to Ancylostoma caninum, 44% to Schistosoma mansoni and 28% to Boophilus microplus aspartic proteases. The DNA fragment coding for longepsin was cloned into a pGFHX-4T-3 vector and expressed in Escherichia coli. The recombinant longepsin, once activated was able to hydrolyze casein substrate as well as hemoglobin (Hb) under acidic conditions (pH 3.5). RT-PCR analysis showed that the longepsin mRNA transcripts were expressed in salivary glands and midgut and not in the ovary. Northern blot analysis revealed that longepsin (1.5 kb) was expressed in unfed and partially fed ticks and expression levels increased during feeding. The finding that longepsin is expressed in the midgut and salivary glands, proteolytic activity Occurs under acidic conditions and longepsin can be gene silenced of longepsin provides compelling support for the hypothesis that longepsin plays ail integral role in the proteolysis of erythrocyte Hb obtained from a host blood meal. (C) 2005 Elsevier Ltd. All rights reserved.