Identification and characterization of a QM protein as a possible peptidoglycan recognition protein (PGRP) from the giant tiger shrimp Penaeus monodon Attasit Udompetcharaporn a,b , Kingkamon Junkunlo c , Saengchan Senapin a,b,d , Sittiruk Roytrakul e , Timothy W. Flegel a,b,d , Kallaya Sritunyalucksana a,b,f,⇑ a Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand b Center of Excellence for Shrimp Molecular Biology and Biotechnology, Faculty of Science, Mahidol University, Rama VI Rd., Bangkok 10400, Thailand c Department of Comparative Physiology, Evolutionary Biology Center (EBC), Uppsala University, Norbyvägen 18A, Uppsala, Sweden d National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani 12120, Thailand e Proteomics Research Laboratory, Genome Institute, National Center for Genetic Engineering and Biotechnology (BIOTEC), Thailand Science Park, Pathumthani 12120, Thailand f Shrimp-Virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Rama VI Rd., Bangkok 10400, Thailand article info Article history: Received 9 January 2014 Revised 3 April 2014 Accepted 6 April 2014 Available online 13 April 2014 Keywords: Peptidoglycan recognition proteins Penaeus monodon QM protein PmQM C-type lectin PmLec abstract In an attempt to identify a peptidoglycan recognition protein (PGRP) in Penaeus (Penaeus) monodon, in vitro pull-down binding assays were used between shrimp proteins and purified peptidoglycan (PG). By gel electrophoresis and mass spectrometry followed by Mascot program analysis, proteins from shrimp hemocyte peripheral membrane proteins showed significant homology to records for a QM protein, actin and prophenoloxidase 2 precursor (proPO2), while proteins from cell-free plasma showed significant homology to records for a vitellogenin, a fibrinogen related protein (FREP) and a C-type lectin. Due to time and resource limitations, specific binding to PG was examined only for recombinant PmQM protein and PmLec that were synthesized based on sequences reported in the Genbank database (acces- sion numbers FJ766846 and DQ078266, respectively). An in vitro assay revealed that hemocytes would bind with and encapsulate agarose beads coated with recombinant PmQM (rPmQM) or rPmLec and that melanization followed 2 h post-encapsulation. ELISA tests confirmed specific binding of rPmQM protein to PG. This is the first time that PmQM has been reported as a potential PGRP in shrimp or any other crustacean. The two other potential PGRP identified (FREP and the vitellin-like protein present in male P. monodon, unlike other vitellin subunits) should also be expressed heterologously and tested for their ability to activate shrimp hemocytes. Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction Innate immunity plays an important role in the defence mech- anism of both vertebrates and invertebrates. The main functions are to differentiate self from non-self and initiate highly regulated immune cascades such as the prophenoloxidase (proPO) activating system that finally eliminate pathogens (Cerenius and Soderhall, 2004). The non-self recognition process involves the binding of pathogen-associated molecular patterns (PAMPs) with specific shrimp humoral pattern recognition proteins (PRPs). Examples of PAMPs are b-1,3-glucan (BG), peptidoglycan (PG) and lipopolysac- charide (LPS) (i.e., microbial cell wall components that are con- served in pathogens but absent in the host). PRP–PAMP complexes trigger the proPO system resulting in melanogenesis accompanied by the production of antimicrobial substances such as quinones and melanin (Cerenius and Soderhall, 2004; Johansson and Soderhall, 1996). PG is a bacterial cell wall component consisting of a glycan strand of alternating b(1-4) linked N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc). The pattern recognition pro- teins responsible for binding to PG and activating an immune response are called peptidoglycan recognition proteins (PGRPs) and they can be found in a wide range of organisms such as insects, mollusks, echinoderms and vertebrates, but not in nematodes or http://dx.doi.org/10.1016/j.dci.2014.04.003 0145-305X/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author at: Shrimp-Virus Interaction Laboratory (ASVI), National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency (NSTDA), Rama VI Rd., Bangkok 10400, Thailand. Tel.: +66 2 2015869; fax: +66 2 3547344. E-mail address: kallaya@biotec.or.th (K. Sritunyalucksana). Developmental and Comparative Immunology 46 (2014) 146–154 Contents lists available at ScienceDirect Developmental and Comparative Immunology journal homepage: www.elsevier.com/locate/dci