Ticks and Tick-borne Diseases 1 (2010) 178–185 Contents lists available at ScienceDirect Ticks and Tick-borne Diseases journal homepage: www.elsevier.de/ttbdis Original article The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis Tetsuya Tanaka , Suguru Kawano , Sumihiro Nakao , Rika Umemiya-Shirafuji , Md. Morshedur Rahman , Damdinsuren Boldbaatar, Banzragch Battur, Min Liao, Kozo Fujisaki ∗ Laboratory of Emerging Infectious Diseases, Department of Frontier Veterinary Science, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan article info Article history: Received 15 June 2010 Received in revised form 3 September 2010 Accepted 11 September 2010 Keywords: Lysozyme Haemaphysalis longicornis Innate immunity Bacteria Blood sucking abstract A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41–79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 ◦ C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms. © 2010 Elsevier GmbH. All rights reserved. Introduction The bacteriolytic function of lysozyme is widely employed in the innate immunities of both animals and plants; therefore, this enzyme is probably one of the most studied defense molecules (Jolles and Jolles, 1984). Lysozyme hydrolyzes the 1,4-glycosidic bound between N-acetylmuramic acid and N-acetylglucosamine in cell-wall peptide glycans of Gram-positive bacteria. How- ever, it was recently demonstrated that the antibacterial activity of lysozyme operates independently of its muramidase activity (Ibrahim et al., 2001). Animal lysozymes have been categorized into the chicken (c)-, the goose (g)-, and the invertebrate (i)-type, according to their possession of type-specific amino acid sequence features and their species of origin (Nilsen and Myrnes, 2001). The expression of c-type multi-gene families of a number of ani- mal species is regulated in response to bacterial challenge on the innate immunity of humoral defense. In a recent study, seven c-type lysozymes have been characterized in Anopheles gambiae, which indicates the adaptation of lysozymes to play a role in immunity (Li et al., 2005). In the ruminant stomach, the function of lysozyme was adapted to digest symbiotic bacteria that provide nitrogen and phospho- rus molecules for milk production (Dobson et al., 1984). A similar ∗ Corresponding author. Tel.: +81 99 285 3569; fax: +81 99 285 3569. E-mail address: tick@ms.kagoshima-u.ac.jp (K. Fujisaki). adaptation of lysozyme to a digestive function has been reported from the midgut of Drosophila melanogaster (Regel et al., 1998) and Musca domestica (Lemos et al., 1993). In general, these digestive lysozymes are the c-type (Bachali et al., 2002) and are different from non-digestive lysozymes with regard to several features, namely, the lower number of basic amino acids (low pI values), resistance to proteases present in the alimentary tract (such as pepsin in the ver- tebrate stomach or cathepsin D in the insect midgut), and optimum pH in the acidic range. Previous reports characterizing lysozymes in ticks have been based on circumstantial evidence, such as the ability of lysozyme- like immunoreactivity of hemocytes of Ixodes ricinus (Kuhn and Haug, 1994). The outcomes of these reports revealed that purified lysates from various tick species display lysozyme-like activities in response to bacterial challenges (Kuhn and Haug, 1994; Alekseev et al., 1995). Kopácek et al. identified, cloned, and character- ized a c-type lysozyme from the digestive tissues of the soft tick Ornithodoros moubata (Kopácek et al., 1999; Grunclovà et al., 2003), in which they demonstrated that the c-type lysozyme possessed the combined features of both immune and digestive adaptive lysozymes. In hard ticks, Dermacentor variabilis and D. andersoni, the lysozyme expression level was increased after challenge with Escherichia coli but not after feeding (Simser et al., 2004). In this study, we report the molecular characterization of a c-type lysozyme (HILysozyme) isolated from Haemaphysalis longi- cornis and its temporal expression in response to blood feeding and immune activation with bacteria. 1877-959X/$ – see front matter © 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.ttbdis.2010.09.001