Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis
Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis
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DOI：
10.1111/imb.12193
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发表时间：
2016-02-01
影响因子：
2.6
通讯作者：
Tanaka, T.
中科院分区：
文献类型：
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作者：
Kusakisako, K.;Masatani, T.;Tanaka, T.
蜱是专性吸血节肢动物，以含有高水平铁的脊椎动物血液为食。宿主来源的铁与蜱体内的氧气反应，然后可能产生高水平的活性氧，包括过氧化氢（H2 O2）。高水平的H2 O2会对需氧生物造成氧化应激。因此，抗氧化反应对于控制H2 O2是必要的。我们专注于过氧化物酶（Prxs），H2 O2-清除酶。从长角血蜱脂肪体cDNA文库中克隆了长角血蜱2-Cys Prx（HlPrx 2）基因，并利用大肠杆菌进行了重组。通过与其他生物体的2-Cys Prxs的比较，我们在HlPrx 2中发现了两个保守的半胱氨酸，Cys 51和Cys 172。我们研究了HlPrx 2和突变蛋白的抗氧化活性，所产生的一个单一的碱基取代，将这些半胱氨酸之一或两者转化为丝氨酸。实验结果表明，去除H2 O2后，Cys 51蛋白具有抗氧化活性。十二烷基硫酸钠聚丙烯酰胺凝胶电泳和尺寸排阻色谱法表明，只有野生型HlPrx 2形成同源二聚体，我们所做的所有蛋白质都有一个高分子量峰。这些结果表明，Cys 51和Cys 172是必不可少的HlPrx 2的二聚体，而只有Cys 51残基是必要的抗氧化活性。
Ticks are obligate haematophagous arthropods that feed on vertebrate blood containing high levels of iron. The host-derived iron reacts to oxygen in the tick's body, and then high levels of reactive oxygen species, including hydrogen peroxide (H2O2), may be generated. High levels of H2O2 cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H2O2. We focused on peroxiredoxins (Prxs), H2O2-scavenging enzymes. The sequence of Haemaphysalis longicornis 2-Cys Prx (HlPrx2) was identified from fat body cDNA libraries of this tick and recombinant HlPrx2 was then prepared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two conserved cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitution, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H2O2 was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatography demonstrated that only the wild-type HlPrx2 formed homodimers and that all of the proteins that we made had a high molecular weight peak. These results indicate that both Cys51 and Cys172 are essential for the dimerization of HlPrx2, whereas only the Cys51 residue is necessary for antioxidant activity.