Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis K. Kusakisako*†, T. Masatani‡, T. Miyata§, R. L. Galay*¶¶, H. Maeda*†, M. R. Talactac*†, N. Tsuji**, M. Mochizuki*†, K. Fujisaki†† and T. Tanaka*† *Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima, Japan; †Department of Pathological and Preventive Veterinary Science, The United Graduate School of Veterinary Science, Yamaguchi University, Yoshida, Yamaguchi, Japan; ‡Joint Faculty of Veterinary Medicine, Transboundary Animal Diseases Center, Kagoshima University, Korimoto, Kagoshima, Japan; §Laboratory of Food Chemistry, Department of Biochemistry and Biotechnology, Division of Molecular Function of Food, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima, Japan; ¶Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Ba~ nos, Los Ba~ nos, Laguna, The Philippines; **Department of Parasitology, Kitasato University School of Medicine, Minami, Sagamihara, Kanagawa, Japan; and ††Zen-Noh Institute of Animal Health, Ohja, Sakura, Chiba, Japan Abstract 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 (H 2 O 2 ), may be generated. High levels of H 2 O 2 cause oxidative stress to aerobic organisms. Therefore, antioxidant responses are necessary to control H 2 O 2 . We focused on peroxi- redoxins (Prxs), H 2 O 2 -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 pre- pared using Escherichia coli. By comparison with the 2-Cys Prxs of other organisms, we found two con- served cysteines in HlPrx2, Cys51 and Cys172. We examined the antioxidant activity of HlPrx2 and mutant proteins produced by a single base substitu- tion, converting one or both of these cysteines into serines. The assays revealed that proteins containing Cys51 showed antioxidant activity when H 2 O 2 was removed. Sodium dodecyl sulphate polyacrylamide gel electrophoresis and size-exclusion chromatogra- phy 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. Keywords: tick 2-Cys peroxiredoxin, antioxidant activity, dimer and oligomer, thioredoxin system. Introduction Ticks are obligate haematophagous arthropods, as they need blood feeding in all of their developmental stages. Blood feeding and the digestion of blood provide nutrition and energy for moulting, development and vitellogenesis in ticks (Grandjean, 1983). Ticks feed on vertebrate blood that contains high levels of iron, such as heme, ferrous iron and other pro-oxidants. Host-derived iron reacts with oxygen in the tick’s body, and then high levels of reactive oxygen species (ROS), including hydrogen peroxide (H 2 O 2 ), may be generated (Citelli et al., 2007; Galay et al., 2014). As a high concentration of H 2 O 2 causes serious damage to membrane lipids, nucleic acids and proteins (Robinson et al., 2010), this molecule is known to be a harmful chemical compound for aerobic organisms. Almost all aerobic organisms have developed defence systems to scavenge H 2 O 2 . Catalases, peroxidases and peroxiredoxins (Prxs) are all scavengers of H 2 O 2 (Rhee, 2006). Prxs are ubiquitous antioxidant enzymes that have been investigated in various organisms (Hall et al., 2011). In particular, Prxs are produced at high levels in Correspondence: Tetsuya Tanaka, PhD, Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan. Tel.: 1 81 99 285 3570; fax: 1 81 99 285 3570; e-mail: tetsuya@ms.kagoshima-u.ac.jp V C 2015 The Royal Entomological Society 1 Insect Molecular Biology (2015) 00(00), 00–00 doi: 10.1111/imb.12193