Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP) bind to seaweed polysaccharides and activate the prophenoloxidase system in white shrimp Litopenaeus vannamei

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Highlights

  • Recombinant white shrimp Litopenaeus vannamei LGBP (rLvLGBP) was constructed.

  • rLvLGBP binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE and SDE.

  • PO activity of haemocytes incubated with a mixture of rLvLGBP-polysaccharide increased.

  • LvLGBP, a PRP binds to alginate, carrageenan, fucoidan, laminarin, GTE and SDE.

  • Binding of LvLGBP with seaweed polysaccharides leads to activate proPO system in shrimp.

Abstract

Lipopolysaccharide and β-1,3-glucan-binding protein (LGBP), important pattern recognition proteins (PRPs), recognize lipopolysaccharide (LPS) and β-1,3-glucan (βG), known as pathogen-associated molecular patterns (PAMPs), and subsequently trigger innate immunity. Several seaweed polysaccharides and seaweed extracts increase immune parameters and resistance to pathogens. Here, we constructed the expression vector pET28b-LvLGBP and transferred it into Escherichia coli BL21 (DE3) for protein expression and to produce the recombinant protein LGBP (rLvLGBP) in white shrimp Litopenaeus vannamei. We examined the binding of rLvLGBP with seaweed-derived polysaccharides including alginate, carrageenan, fucoidan, laminarin, Gracilaria tenuistipitata extract (GTE), and Sargassum duplicatum extract (SDE), and examined the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and each polysaccharide. We also examined the binding of rLvLGBP with LPS and βG, and the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS (rLvLGBP-LPS) or a mixture of rLvLGBP and βG (rLvLGBP-βG). An ELISA binding assay indicated that rLvLGBP binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE with dissociation constants of 0.1138–0.1770 μM. Furthermore, our results also indicated that the phenoloxidase activity of shrimp haemocytes incubated with a mixture of rLvLGBP and LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE significantly increased by 328%, 172%, 200%, 213%, 197%, 194%, 191%, and 197%, respectively compared to controls (cacodylate buffer). We conclude that LvLGBP functions as a PRP, recognizes and binds to LPS, βG, alginate, carrageenan, fucoidan, laminarin, GTE, and SDE, and subsequently leads to activating innate immunity in shrimp.

Introduction

Shrimp rely on an innate immune response to defend against pathogens. Once foreign particles enter the haemocoel, shrimp engage an innate immune response that includes cellular and humoral reactions. This system is initiated via the recognition and binding of foreign molecules like lipopolysaccharide (LPS) and peptidoglycan (PG) derived from bacteria cell walls, and by β-1,3-glucan (βG) derived from fungi and yeast mycelia, which are known as pathogen-associated molecular patterns (PAMPs) of the host's recognition molecules and are named pattern recognition proteins (PRPs) or receptors (PRPs) (Janeway and Medzhitov, 2002). Several types of PRPs have been reported in crustaceans including lipopolysaccharide and β-1,3-glucan binding protein (LGBP), β-1,3-glucan binding protein (βGBP), C-type lectin, tachylectin, masquerade-like protein, and peptidoglycan recognition protein (PGRP) (Lee and Söderhäll, 2002, Tassanakajon et al., 2013, Wang and Wang, 2013). LGBP and βGBP have been identified in white shrimp Litopenaeus vannamei (AY723297, EU102286), tiger shrimp Penaeus monodon (AF368168; Amparyup et al., 2012), and kuruma shrimp Marsupenaeus japonicus (AB162766, EU267001). Sequence analysis of amino acids between LGBP and βGBP share high similarity (99%) suggesting they are allelic variants of the same gene (Lin et al., 2008, Amparyup et al., 2012). LGBP and βGBP have two potential polysaccharide recognition motifs, polysaccharide binding motif (PsBM) and β-glucan recognition motif (βGRM), and a glucanase motif (GM) (Lin et al., 2008).

In crustacean, three types of haemocytes are identified based on cell size and degree of granularity (Tsing et al., 1989). These are the hyaline cells (HCs), semi-granular cells (SGCs), and granular cells (GCs). Semi-granular cells are characterized by a number of small granules, whereas granular cells are filled with large granules (Kitikiew et al., 2013). Both SGCs and GCs can be induced by foreign particles like LPS, βG, and PG to degranulate, and undergo exocytosis of granules to release several proteins like prophenoloxidase (proPO), peroxinectin, and prophenoloxidase activating enzyme (proppA) etc (Barracco et al., 1991, Söderhäll et al., 1994, Jiravanichpaisal et al., 2006). The proPO system is carried out via the proteolytical conversion of proPO to phenoloxidase (PO) by active ppA in the presence of PAMP that subsequently results in the activation of innate immunity processes such as melanisation, releases of cytotoxic compounds, and encapsulation of pathogens (Cerenius et al., 2008).

In shrimp, once bacteria, fungi, or other foreign particles invade a host, βGBP or LGBP recognizes and then activates the serine proteinase cascade that activates the proPO system (Cerenius et al., 2008). For instance, purified βGBP from Brazilian shrimp Farfantepenaeus paulensis and Litopenaeus schmitti has agglutination ability toward yeast Saccharomyces cerevisiae (Goncalves et al., 2012). A study using SDS-PAGE and immunoblotting indicated tiger shrimp Penaeus monodon recombinant βGBP binds to curdlan and zymosan (Sritunyalucksana et al., 2002). In the freshwater crayfish Pacifastacus leninusculus, βGBP reacts with βG and the resulting βGBP-βG complex induces the degranulation of haemocytes, and subsequently activates the proPO system (Barracco et al., 1991, Duvic and Söderhäll, 1990, Duvic and Söderhäll, 1992).

In addition to βGBP, immunoblotting indicates that the purified LGBP of the crayfish Pacifastacus leniusculus binds to LPS, curdlan, and laminarin (Lee et al., 2000). A binding assay indicated that the recombinant LGBP of fleshy shrimp Fenneropenaeus chinensis strongly binds to Gram-negative bacteria Klebsiella penaeumoniae (Du et al., 2007). Furthermore, in vitro studies indicate that βG, curdlan, laminarin, LPS, and PG increase the PO activity of shrimp (Söderhäll et al., 1994, Huynh et al., 2011). Recombinant LGBP is able to recognize LPS and βG, and increase PO activity in tiger shrimp P. monodon (Amparyup et al., 2012). Therefore, shrimp haemcytes receiving LPS and βG trigger the activation of innate immunity.

Several polysaccharides like βG, PG, and LPS that are considered PAMPs are immunostimulants that effectively enhances resistance to shrimp pathogens. For instance, the βG variants derived from bacteria and fungi, including curdlan, schizophyllan, scleroglucan, VitaStim, PG, LPS, and β-1,3/1,6-glucan have been widely studied for their ability to increase the immune response of shrimp and for their resistance to pathogens (Sakai, 1999, Raa, 2002, Smith et al., 2003, Ringø et al., 2012). Administering PG increases the resistance of kuruma shrimp M. japonicus to Vibrio panaeicida and white spot syndrome virus (WSSV), and increase the phagocytic index (Itami et al., 1998). Administering LPS increases PO activity and resistance against virus, and improves the survival of shrimp at post Vibrio harveyi challenge (Takahashi et al., 2000, Rungrassamee et al., 2013). In addition, seaweed-derived polysaccharides, including alginate, carrageenan, fucoidan, and laminarin, and the extracts of seaweeds like Gracilaria tenuistipitata and Sargassium duplicatum, increase the immune responses in shrimp and their resistance against pathogens (Hou and Chen, 2005; Cheng et al., 2005a, Liu et al., 2006, Yeh et al., 2006, Yeh and Chen, 2008, Kitikiew et al., 2013; Chen et al., 2014). However, little is known about the mechanism underlying the activation of the innate immune response in shrimp receiving seaweed polysaccharides, so the recognition and binding functions of LGBP, βGBP, and other PRPs remains to be examined.

We assumed that white shrimp haemocytes incubated with alginate, carrageenan, fucoidan, laminarin, and seaweed extract can initiate the proPO activating system through the recognition and binding of LGBP, similar to the response of shrimp haemocytes receiving LPS and βG. Therefore, this study was undertaken to (1) construct the recombinant LGBP of white shrimp L. vannamei (rLvLGBP), (2) examine the binding ability of rLvLGBP, and (3) quantify the PO activity of shrimp haemocytes receiving a mixture of rLvLGBP and each seaweed polysaccharide substance, including alginate, crarageenan, fucoidan, laminarin, and extracts of Gracilaria tenuistipitata (GTE) and Sargassum duplicatum (SDE). Examinations of the binding ability of rLvLGBP with LPS and βG, as well as an increase of PO activity in shrimp haemocytes receiving a mixture of rLvLGBP with LPS (rLvLGBP-LPS) and a mixture of rLvLGBP with βG (rLvLGBP-βG), were concurrently conducted and served as reference groups.

Section snippets

Experimental animals

White shrimp L. vannamei with an average weight of ∼13 g obtained from the University Marine Station, Keelung, Taiwan were placed in aerated seawater in the laboratory for two weeks prior to the experiment. Only shrimp in the intermoult stage were used for the study. The moult stage was determined by examining the uropoda in which partial retraction of the epidermis could be distinguished (Chan et al., 1988).

Reagents

Alginate (A2158) derived from Macrocystis pyrifera, βG (G5011) derived from S.

Expression and enrichment of rLvLGBP

The recombinant plasmid pET28b-Lvlgbp was transferred to and expressed in E. coli BL21 (DE3). After IPTG induction for 3.5 h, whole cell lysate was analyzed by SDS-PAGE visualized by Coomassive Brilliant Blue R250 staining (data not shown). The rLvLGBP mature protein, after enrichment and refolding, appeared as a single band with an estimated molecular mass of ∼42 kDa after SDS-PAGE resolution of the purified recombinant LvLGBP (Fig. 1A). Western blotting with the rabbit anti-LGBP polyclonal

Discussion

The full-length sequence of white shrimp LGBP cDNA was characterized with an open reading frame of 1101 bp, encoding 367 amino acids (aa) that contains a signal peptide of 17 aa residues. The calculated molecular mass of the 350 aa mature peptide was 41.56 kDa whereas that of rLvLGBP in the present study was ∼42 kDa.

Scientists have studied the LGBP transcript level of shrimp receiving Vibrio or LPS. For instance, the LGBP transcript level of white shrimp L. vannamei is increased at 3 h after a

Acknowledgements

This research was supported by grants from the Ministry of Science and Technology (MOST 103-2313-B-019-008), Taiwan, and partially supported by the Center of Excellence for the Oceans, National Taiwan Ocean University.

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