title: Functional analysis of recombinant 2-Cys peroxiredoxin from the hard tick Haemaphysalis longicornis.
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 peroxiredoxins (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 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 H(2)O(2) 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.
authors.affiliation: 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.
authors.forename: K
authors.initials: K
authors.lastname: Kusakisako
authors.affiliation: Joint Faculty of Veterinary Medicine, Transboundary Animal Diseases Center, Kagoshima University, Korimoto, Kagoshima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Masatani
authors.affiliation: Laboratory of Food Chemistry, Department of Biochemistry and Biotechnology, Division of Molecular Function of Food, Faculty of Agriculture, Kagoshima University, Korimoto, Kagoshima, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Miyata
authors.affiliation: Laboratory of Infectious Diseases, Joint Faculty of Veterinary Medicine, Kagoshima University, Korimoto, Kagoshima, Japan.|Department of Veterinary Paraclinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, Los Baños, Laguna, The Philippines.
authors.forename: R L
authors.initials: RL
authors.lastname: Galay
authors.affiliation: 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.
authors.forename: H
authors.initials: H
authors.lastname: Maeda
authors.affiliation: 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.
authors.forename: M R
authors.initials: MR
authors.lastname: Talactac
authors.affiliation: Department of Parasitology, Kitasato University School of Medicine, Minami, Sagamihara, Kanagawa, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Tsuji
authors.affiliation: 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.
authors.forename: M
authors.initials: M
authors.lastname: Mochizuki
authors.affiliation: Zen-Noh Institute of Animal Health, Ohja, Sakura, Chiba, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fujisaki
authors.affiliation: 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.
authors.forename: T
authors.initials: T
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000975:Antioxidants / Q000378:metabolism; D060829:Arthropod Proteins / Q000235:genetics / Q000378:metabolism; D002850:Chromatography, Gel; D019281:Dimerization; D006861:Hydrogen Peroxide; D026863:Ixodidae / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D054464:Peroxiredoxins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
chemical_list: D000975:Antioxidants; D060829:Arthropod Proteins; D011994:Recombinant Proteins; D006861:Hydrogen Peroxide; D054464:Peroxiredoxins
keywords: antioxidant activity; dimer and oligomer; thioredoxin system; tick 2-Cys peroxiredoxin