Developmental and Comparative Immunology 35 (2011) 611–619 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci Localization of the serine protease homolog BmSPH-1 in nodules of E. coli-injected Bombyx mori larvae and functional analysis of its role in nodule melanization Maki Sakamoto a , Masayuki Ohta a , Asahi Suzuki a , Hinako Takase b , Yasutaka Yoshizawa a , Madoka Kitami a , Ryoichi Sato a,∗ a Graduate School of Bio-Applications and Systems Engineering, Tokyo University of Agriculture and Technology, Koganei 2-24-16, Tokyo 184-8588, Japan b Laboratory of Cell Growth and Differentiation Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan article info Article history: Received 27 October 2010 Received in revised form 9 January 2011 Accepted 9 January 2011 Available online 13 January 2011 Keywords: Bombyx mori Serine protease homolog Melanization Nodule Innate immunity C-type lectin abstract The molecular mechanisms underlying nodule formation and melanization, an important pathogen defense mechanism in insects, are poorly understood. In this study, we investigated the role of BmSPH-1, a catalytically inactive Bombyx mori serine protease homolog, in nodule melanization induced by injection of Escherichia coli cells into the B. mori larval hemocoel. Addition of the melanization substrate l-3,4- dihydroxyphenylalanine (DOPA) to newly formed nodules prompted nodule melanization, confirming that nodules contain activated prophenoloxidase needed for melanization. Immunoprecipitation and immunoblot studies demonstrated that BmSPH-1 interacts with BmLBP, a C-type lectin that binds Gram- negative bacteria, and that BmSPH-1 is present in a truncated, putatively activated form at the E. coli cell surface in nodules. Pretreatment of larvae with anti-BmSPH-1 serum inhibited nodule melanization in E. coli-injected larvae. These results suggest that BmSPH-1 regulates nodule melanization and is recruited into nodules from the hemolymph by BmLBP. © 2011 Elsevier Ltd. All rights reserved. 1. Introduction Innate immunity plays a key role in the defense against micro- bial infection in insects and other invertebrates which lack an acquired immune system. In insects, pathogens recognized as “non- self” are eliminated by both humoral and cellular responses (Vilmos and Kurucz, 1998). The humoral responses include the production of antimicrobial peptides and melanization, a process induced by rapid activation of the phenoloxidase cascade in which brown- black melanin pigments are deposited on invading pathogens and parasites. The cellular responses, which are mediated by hemo- cytes, include phagocytosis, encapsulation, and nodule formation (Lemaitre et al., 1996; Hoffmann and Reichhart, 2002; Cerenius and Söderhäll, 2004). Researchers studying the insect immune system have exam- ined antimicrobial peptide production in Drosophila melanogaster (Ferrandon et al., 2007) and melanization in the hemolymph of Abbreviations: SP, serine protease; SPH, serine protease homolog; proPO, prophenoloxidase; PO, phenoloxidase; PPAE, proPO-activating enzyme; ROS, reac- tive oxygen species; BmSPH-1, B. mori serine protease homolog-1; BmSco, B. mori scolexin; BmHP, B. mori hemocyte protease. ∗ Corresponding author. Tel.: +81 42 388 7277; fax: +81 42 388 7277. E-mail address: ryoichi@cc.tuat.ac.jp (R. Sato). Manduca sexta (Gorman et al., 2007; Ji et al., 2004; Wang and Jiang, 2006; Yu et al., 2003; An et al., 2009) in detail. In M. sexta, serine proteases (SPs) mediate a reaction cascade leading to the melanization of the hemolymph. These SPs are activated by pattern recognition receptors (PRRs), such as -1,3-glucan recog- nition protein, in response to bacterial infection (Wang and Jiang, 2006). Non-proteolytic serine protease homologs (SPHs), in which a glycine residue replaces the serine residue in the SP catalytic triad, are also involved in the melanization reaction cascade (Yu et al., 2003; Gupta et al., 2005). The terminal enzyme in the cascade is prophenoloxidase (proPO), which is converted by proPO-activating enzyme (PPAE) to phenoloxidase. The oxidation of phenolic com- pounds, such as tyrosine and 3,4-dihydroxyphenylalanine (DOPA), by phenoloxidase leads to the production of quinones (Cerenius and Söderhäll, 2004), which polymerize to form melanin, and microbi- cidal reactive oxygen species (ROS) (Nappi and Christensen, 2005). Nodule formation is an expeditious cellular response in which hemocytes surround and isolate invading pathogens (Ratcliffe and Gagen, 1977; Rowley and Ratcliffe, 1981). In the silkworm Bombyx mori, the hemolymph can be cleared of 10 5 bacterial cells within 30 min, a highly efficient process that depends on nodule forma- tion (Koizumi et al., 1997). The melanization response that follows nodule formation is thought to reinforce the nodules and provide microbicidal ROS that, together with antimicrobial peptides, con- tribute to immune defense. Its contribution is suggested by the 0145-305X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.01.003