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A fibrinogen-related protein (FREP) is involved in the antibacterial immunity of Marsupenaeus japonicus

https://doi.org/10.1016/j.fsi.2014.05.005Get rights and content

Highlights

  • A fibrinogen-related protein (MjFREP2) was identified in Marsupenaeus japonicus.

  • MjFREP2 is upregulated in hemocytes after Vibrio anguillarum and Staphylococcus aureus challenge.

  • MjFREP2 agglutinates bacteria in a calcium-dependent manner.

  • MjFREP2 binds to bacteria in vitro and in vivo.

  • MjFREP2 can enhance bacterial clearance by inducing hemocyte phagocytosis.

Abstract

Fibrinogen-related proteins (FREPs) in invertebrates have important functions in innate immunity. In this study, the cDNA of FREP was identified from the kuruma shrimp Marsupenaeus japonicus (MjFREP2). The full-length cDNA of MjFREP2 is 1138 bp with an open reading frame of 954 bp that encodes a 317-amino acid protein comprising a signal peptide and a fibrinogen-like domain. MjFREP2 could be detected in hemocytes, heart, hepatopancreas, gills, stomach, and intestines. MjFREP2 could also be upregulated in hemocytes after Vibrio anguillarum and Staphylococcus aureus challenge. Agglutination and binding assay results revealed that the recombinant MjFREP2 bound to bacteria and polysaccharides. Immunocytochemical analysis results showed that MjFREP2 proteins were mainly distributed in the cytoplasm of hemocytes from unchallenged shrimp and transported to the membrane or secreted out of the cell after V. anguillarum or S. aureus challenge. The secreted MjFREP2 bound to the bacteria presented in shrimp hemolymph. The overexpression of MjFREP2 could enhance bacterial clearance by inducing the phagocytosis of hemocytes. This ability was impaired by knockdown of MjFREP2 with RNA interference. The cumulative mortality of MjFREP2-silenced shrimp was significantly higher than that of the control shrimp. These results suggested that MjFREP2 has an important function in the antibacterial immunity of M. japonicus.

Introduction

Invertebrates exhibit no antibody-driven adaptive immunity and rely on innate immunity to prevent pathogen invasion. Innate immunity is activated by pathogen sensors called pattern recognition receptors (PRRs). More than 10 kinds of PRRs are found in invertebrates [1], [2]. Among these PRRs, lectins are important in the recognition of microbe-associated molecular patterns (MAMPs) located on microbial surfaces; these lectins are also involved in the initiation of defense responses [2]. Fibrinogen-related proteins (FREPs), also known as fibrinogen-like domain immunolectins, FBNs, have a fibrinogen-related domain (FReD) consisting of about 200 amino acid residues in the domain with high sequence similarity to the C terminus of the fibrinogen β and γ chains. FREPs are composed of different kinds of proteins, such as ficolins, tenascins, tachylectins, angiopoietins, ixoderins, fibroleukin, and some other extracellular proteins [3], [4], [5], [6]. These proteins perform various functions, including agglutination and bacterial defense, developmental processes, and allorecognition [7], [8]. Ficolins, one kind of the best studied FREPs, are the oligomeric lectins that usually consist of an N-terminal collagen-like domain and a C-terminal FReD. Ficolins are also widely distributed among vertebrates and invertebrates. Furthermore, ficolins are mainly involved in innate immunity, particularly in pathogen recognition. Three different ficolins, designated as L-, M−, and H-, are found in human [7]. Ficolins from mammals can initiate complement activation via the lectin pathway [5], [9], [10], [11] and function as recognition molecules against pathogens and activate the associated serine proteases of the MBL-associated serine protease (MASP)/C1r/C1s family; the activated MASPs and C1s trigger complement activation [12], [13].

There are several FREPs identified in various species of invertebrates and almost all of them are implicated in innate immune responses. For example, 59 putative members of the FREPs were discovered in the mosquito Anopheles gambiae, and 37 members in Anopheles aegypti. The FREP family plays a central role in mosquito immune system [14]. In crustaceans, several FREPs were also discovered in several species. Tachylectins from the horseshoe crab Tachypleus tridentatus are well characterized in terms of structure and function. Tachylectins consist of a short N-terminal Cys-containing segment and a C-terminal FReD [15]. Tachylectins 5A and 5B bind to acetyl groups, such as N-acetylglucosamine (GlcNAc), forming dimers or tetramers that may agglutinate human erythrocytes or different bacteria [15], [16]. Four aromatic amino acids in FReD (namely, Tyr210, Tyr236, Tyr248, and His220 in TL-5A) are involved in mediating contact between proteins and carbohydrates, specifically via acetyl groups [16], [17]. Unlike ficolins found in vertebrates, ficolin-like proteins in invertebrates contain different primary structures that exhibit variations in the number of FReDs, lack of collagen-like domain, and presence of additional domains [18]. Ficolin-like proteins were found in the freshwater crayfish Pacifastacus leniusculus with repeated Gln-rich region in the N-terminal and function as PRRs against invading Gram-negative bacteria [19]. Two ficolin-like proteins were discovered in giant freshwater prawn Macrobrachium rosenbergii with the coiled coil region in N-terminal. These ficolin-like proteins can enhance the bacteria clearance in the prawn [20].

In the present study, a new FREP was identified from kumura shrimp Marsupenaeus japonicus, and designated as MjFREP2. Comparing with our previous studied MjFREP1 [21] and other reported members of crustacean FREP family, MjFREP2 shows difference in the N-terminal region. BLAST search showed that the protein with highest score is the predicted protein from sea anemone Nematostella vectensis (accession no. XP_001636335.1, 42% identity). MjFREP2 has no putative conserved domain at the N-terminal. The identity of the FReD in the C-terminal between MjFREP1 and 2 is only 29.6%. These differences prompted us to investigate the function of MjFREP2 in shrimp innate immunty.

Section snippets

Immune challenge and tissue collection

M. japonicus weighing approximately 9 g–12 g was purchased from a seafood market in Jinan, Shandong Province, China. The shrimp were temporarily kept in laboratory aquarium tanks containing aerated seawater. For the immune challenge, either Vibrio anguillarum or Staphylococcus aureus [obtained from Shandong University Organism Culture Collection (SDMCC)], (2 × 107 CFU) was injected into the abdominal segment of each shrimp. The hemolymph was collected from the ventral sinus at different time

Cloning and sequence analysis of MjFREP2

The full-length of MjFREP2 cDNA was 1138 bp with an open reading frame (ORF) of 954 bp (GenBank accession no. KJ158837). The ORF of MjFREP2 encoded a putative 317-amino acid protein with a calculated molecular weight of about 36 kDa. This putative protein consisted of a signal peptide, the N-terminal unknown region, and C-terminal FReD. It had four potential Asn-linked glycosylation sites at residue 29, 130, 136, and 287, and two potential calcium-binding sites in the C-terminal (Fig. S1). The

Discussion

FREPs are present in invertebrates and vertebrates and mainly function as PRRs [3], [10]. In the present study, a new FREP from M. japonicus (MjFREP2) was identified and functionally characterized. MjFREP2 was upregulated by bacterial challenge and could bind and agglutinate different bacteria. It could also enhance bacterial clearance by promoting the hemocyte phagocytosis.

In our previous study, we identified MjFREP1 in shrimp M. japonicus [21]. Similarities and differences were observed

Acknowledgments

This study was supported financially by the National Natural Science Foundation of China (Grant No. 31130056), National Basic Research Program of China (973 Program, Grant No. 2012CB114405), Ph.D. Programs Foundation of the Ministry of Education of China (Grant No. 20110131130003), and the Provincial Natural Science Foundation of Shandong, China (Grant No. ZR2011CM014).

References (30)

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