Original Contribution Cloning and characterization of Arenicola marina peroxiredoxin 6, an annelid two-cysteine peroxiredoxin highly homologous to mammalian one-cysteine peroxiredoxins ☆ Eléonore Loumaye a , Ann C. Andersen b , André Clippe a , Hervé Degand c , Marlène Dubuisson a , Franck Zal b , Pierre Morsomme c , Jean-François Rees a , Bernard Knoops a, ⁎ a Laboratoire de Biologie Cellulaire, Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium b Equipe Ecophysiologie, Adaptation et Evolution Moléculaires, UMR 7144 CNRS-UPMC, Station Biologique, 29682 Roscoff, CEDEX, France c Unité de Biochimie Physiologique, Institut des Sciences de la Vie, Université catholique de Louvain, B-1348 Louvain-la-Neuve, Belgium ABSTRACT ARTICLE INFO Article history: Received 6 January 2008 Revised 14 April 2008 Accepted 23 April 2008 Available online 2 May 2008 Keywords: Annelid Arenicola marina Peroxidase Peroxiredoxin Antioxidant Peroxiredoxins (PRDXs) are a superfamily of thiol-dependent peroxidases found in all phyla. PRDXs are mechanistically divided into three subfamilies, namely typical 2-Cys, atypical 2-Cys, and 1-Cys PRDXs. To reduce peroxides, the N-terminal peroxidatic Cys of PRDXs is first oxidized into sulfenic acid. This intermediate is reduced by forming a disulfide bond either with a resolving Cys of another monomeric entity (typical 2-Cys) or of the same molecule (atypical 2-Cys). In 1-Cys PRDXs, the resolving Cys is missing and the sulfenic acid of the peroxidatic Cys is reduced by a heterologous thiol-containing reductant. In search of a homolog of human 1-Cys PRDX6 in Arenicola marina, an annelid worm living in intertidal sediments, we have cloned and characterized a PRDX exhibiting high sequence homology with its mammalian counterpart. However, A. marina PRDX6 possesses five Cys among which two Cys function as peroxidatic and resolving Cys of typical 2-Cys PRDXs. Thus, A. marina PRDX6 belongs to a transient group exhibiting sequence homologies with mammalian 1-Cys PRDX6 but must be mechanistically classified into typical 2-Cys PRDXs. Moreover, PRDX6 is highly expressed in tissues directly exposed to the external environment, suggesting that this PRDX may be of particular importance for protection against exogenous oxidative attacks. © 2008 Elsevier Inc. All rights reserved. Introduction Peroxiredoxins (PRDXs) are an ubiquitous family of peroxidases able to reduce H 2 O 2 , alkyl hydroperoxides (ROOH), and peroxynitrite (ONOO - ) [1–3]. These enzymes are highly conserved throughout evolution and widely distributed among bacteria, archaea, and eukary- otes [3,4]. Unlike most peroxidases that exhibit high catalytic efficiency with a selenocysteine, a flavin, or a heme ring in their active site [3], PRDXs lack cofactors or prosthetic groups. Their activity is depending on cysteines (Cys). PRDXs were initially classified into two subfamilies (2-Cys and 1-Cys PRDXs) according to the number of catalytically active Cys residues in their sequence [2]. Then, 2-Cys PRDXs were yet divided into two different subfamilies, namely typical and atypical 2-Cys PRDXs based on their catalytic mechanism [5]. All PRDXs share a peroxidatic Cys in their N-terminal domain which is oxidized into sulfenic acid (-SOH) in a first catalytic step during reaction with peroxides [1]. The second step of the reaction distinguishes the three PRDX subfamilies and consists in the reduction of sulfenic acid. For all 2-Cys PRDXs, this reactivation step requires an additional Cys called resolving Cys [1]. Typical 2-Cys PRDXs form homodimers with two identical active sites through intersubunit disulfide bonds reduced most frequently by thioredoxins (TXNs) [1]. In atypical 2-Cys PRDXs, the resolving Cys directly attacks the peroxidatic Cys by forming an intramolecular disulfide bond. Their classical physiological reductants are also TXNs [5]. The last subfamily, 1-Cys PRDXs, only contains the single peroxidatic Cys [6,7]. In this case, the sulfenic acid is reduced by an external thiol. Recently, it has been shown that one physiological Free Radical Biology & Medicine 45 (2008) 482–493 Abbreviations: Bt, Bos taurus; CHP, cumene hydroperoxide; C p , peroxidatic cysteine; C r , resolving cysteine; DAPI, 4′,6-diamidino-2-phenylindole; Dox, oxidized dimer; DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; FITC, fluoresceine isothiocya- nate; GPX, glutathione peroxidase; GR, glutathione reductase; GRX, glutaredoxin; GSH, glutathione; HMW, high molecular weight; H 2 O 2 , hydrogen peroxide; H 2 S, hydrogen sulfide; Hs, Homo sapiens; HSP, heat shock protein; M, monomer; MALDI, matrix- assisted laser desorption/ionization; Mm, Mus musculus; Mox, oxidized monomer; Mred, reduced monomer; NADPH, nicotinamide adenine dinucleotide phosphate; ONOO - , peroxynitrite; ORF, open reading frame; PBS, phosphate buffer saline; PCR, polymerase chain reaction; πGST, glutathione S-transferase π; PLOOH, phospholipid hydroperoxide; RACE, rapid amplification of cDNA ends; Rn, Rattus novergicus; ROOH, organic hydroperoxide; ROS, reactive oxygen species; SDS-PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; -SOH, sulfenic acid; TBHP, tert-butyl hydroperoxide; TOF, time of flight; TXN, thioredoxin; TXNRD, thioredoxin reductase. ☆ The nucleotide sequence reported in this paper has been submitted to GenBank with Accession Number DQ059567. ⁎ Corresponding author. Fax: +32 10 47 3515. E-mail address: bernard.knoops@uclouvain.be (B. Knoops). 0891-5849/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.freeradbiomed.2008.04.033 Contents lists available at ScienceDirect Free Radical Biology & Medicine journal homepage: www.elsevier.com/locate/freeradbiomed