Bioinformatic identification of cytochrome b 5 homologues from the parasitic nematode Ascaris suum and the free-living nematode Caenorhabditis elegans highlights the crucial role of A. suum adult-specific secretory cytochrome b 5 in parasitic adaptation Shinzaburo Takamiya a,f, ⁎, Muneaki Hashimoto a , Toshihiro Mita a , Takehiro Yokota b,1 , Yoshitaka Nakajima c , Fumiyuki Yamakura d , Shigetoshi Sugio b,2 , Tsutomu Fujimura e,3 , Takashi Ueno e , Hiroshi Yamasaki a,f a Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan b R & D Strategy Department, Yokohama Research Center, Mitsubishi Chemical Corporation, 1000 Kamoshida-cho, Aoba, Yokohama 227-8502, Japan c Department of Life Science, Faculty of Science and Engneering, Setsunan University, 17-8 Ikeda-naka-machi, Neyagawa, Osaka 572-8508, Japan d Department of Chemistry, Faculty of International Liberal Arts, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan e Laboratory of Proteomics and Biomolecular Science, Biomedical Research Center, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan f Department of Parasitology, National Institute of Infectious Diseases, Toyama 1-23-1, Shinjuku-ku, Tokyo 162-8640, Japan abstract article info Article history: Received 1 October 2015 Received in revised form 31 October 2015 Accepted 9 November 2015 Available online 10 November 2015 We previously reported that adult Ascaris suum possesses NADH-metmyoglobin and NADH-methaemoglobin reductase systems that are located in the cells of the body wall and in the extracellular perienteric fluid, respec- tively, which helps them adapt to environmental hypoxia by recovering the differential functions of myoglobin and haemoglobin. A. suum cytochrome b 5 , an adult-specific secretory protein and an essential component of the NADH-metmyo (haemo) globin reductase system, has been extensively studied, and its unique nature has been determined. However, the relationship between A. suum cytochrome b 5 and the canonical cytochrome b 5 proteins, from the free-living nematode Caenorhabditis elegans is unclear. Here, we have characterised four cyto- chrome b 5 -like proteins from C. elegans (accession numbers: CAB01732, CCD68984, CAJ58492, and CAA98498) and three from A. suum (accession numbers: ADY48796, ADY46277, and ADY48338) and compared them with A. suum cytochrome b 5 in silico. Bioinformatic and molecular analyses showed that CAA98498 from C. elegans is equivalent of A. suum cytochrome b 5 , which was not expressed as a mature mRNA. Further, the CAA98498 possessed no secretory signal peptide, which occurs in A. suum cytochrome b 5 precursor. These results suggest that this free-living nematode does not need a haemoprotein such as the A. suum cytochrome b 5 and highlight the crucial function of this A. suum adult-specific secretory cytochrome b 5 in parasitic adaptation. © 2015 Elsevier Ireland Ltd. All rights reserved. Keywords: NADH-metmyo (hemo) globin reductase sys- tem Secretory cytochrome b 5 NADH: ferricytochrome-b 5 oxidoreductase Ascaris suum Caenorhabditis elegans 1. Introduction Cytochrome b 5 (cyt b 5 ) 4 proteins are widely distributed haemoproteins that catalyse a variety of biological redox reactions [1,2]. Two forms of cyt b 5 have been isolated, a membrane-bound form and a soluble form. The former is localised in microsomes and the outer mem- branes of the mitochondria, whereas the latter is present in erythrocytes. Membrane-bound cyt b 5 is composed of two distinct domains, a hydrophilic N-terminal domain of approximately 100 amino acids and a hydrophobic C-terminal domain of approximately 30 amino acids. The N-terminal domain contains a protohaem that catalyses electron transfer, whereas the C-terminal domain consists of a hydrophobic membrane-anchoring portion (approximately 20 residues) and a ter- minal hydrophilic portion (approximately ten residues) that contains a sequence to target cyt b 5 to the endoplasmic reticulum and/or mito- chondrial outer membranes [3,4]. The terminal hydrophilic portion of Parasitology International 65 (2016) 113–120 Abbreviations: cyt b 5 , cytochrome b 5 ; PCR, polymerase chain reaction; EST, expressed sequence tag; CeCytb 5 1, CAB01732; CeCytb 5 2, CCD68984; CeCytb 5 3, CAJ58492; CeCytb 5 4, CAA98498; AsCytb 5 , BAA10888; AsCytb 5 1, ADY48796; AsCytb 5 2, ADY46277; AsCytb 5 3, ADY48338; BmCytb 5 1, CDP94089; BmCytb 5 2, CDP94357; BmCytb 5 3, CDP94090; WbCytb 5 1, EJW88614; WbCytb 5 2, EJW88222; OvCytb 5 1, OVP03947; OvCytb 5 2, OVP11713. ⁎ Corresponding author at: Department of Molecular and Cellular Parasitology, Graduate School of Medicine, Juntendo University, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113- 8421, Japan. E-mail address: stakamiy@juntendo.ac.jp (S. Takamiya). 1 Present address: Biology Research Laboratories, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida-cho, Aoba, Yokohama 227-0033, Japan. 2 Present address: Separation Materials Laboratories, R&D Center, Mitsubishi Chemical Corporation, 1-1 Shiroishi, Yahatanishi, Kitakyushu 806-0004, Japan. 3 Present address: Laboratory of Bioanalytical Chemistry, Tohoku Pharmaceutical University, 4-4-1 Komatsushima, Aoba-ku, Sendai, Miyagi 981-8558, Japan. http://dx.doi.org/10.1016/j.parint.2015.11.004 1383-5769/© 2015 Elsevier Ireland Ltd. All rights reserved. Contents lists available at ScienceDirect Parasitology International journal homepage: www.elsevier.com/locate/parint