Abstract
The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and β-1,3-glucan-binding protein (LGBP). However, shrimp LGBPs that are involved in the proPO system have yet to be clarified. Here, we focus on characterizing the role of a Penaeus monodon LGBP (PmLGBP) in the proPO system. We found that PmLGBP transcripts are expressed primarily in the hemocytes and are increased at 24 h after pathogenic bacterium Vibrio harveyi challenge. The binding studies carried out using ELISA indicated that recombinant (r)PmLGBP binds to β-1,3-glucan and LPS with a dissociation constant of 6.86 × 10(-7) M and 3.55 × 10(-7) M, respectively. Furthermore, we found that rPmLGBP could enhance the phenoloxidase (PO) activity of hemocyte suspensions in the presence of LPS or β-1,3-glucan. Using dsRNA interference-mediated gene silencing assay, we further demonstrated that knockdown of PmLGBP in shrimp in vivo significantly decreased the PmLGBP transcript level but had no effect on the expression of the other immune genes tested, including shrimp antimicrobial peptides (AMPs). However, suppression of proPO expression down-regulated PmLGBP, proPO-activating enzyme (PmPPAE2), and AMPs (penaeidin and crustin). Such PmLGBP down-regulated shrimp showed significantly decreased total PO activity. We conclude that PmLGBP functions as a pattern recognition protein for LPS and β-1,3-glucan in the shrimp proPO activating system.
Highlights
LGBP is an important pattern recognition protein (PRP)
The BLASTX results showed that Penaeus monodon LGBP (PmLGBP) significantly matched with the bGBP cDNA from P. monodon (AF368168; 99% amino acid sequence identity), suggesting that PmLGBP is the same gene as that reported previously [33]
Amino acid sequence comparison of PmLGBP with kuruma shrimp M. japonicus LGBP [32] indicated that PmLGBP contained the conserved motifs for a potential polysaccharide binding, glucanase, and -glucan recognition motifs, and so PmLGBP likely belongs to the family of crustacean LGBPs
Summary
LGBP is an important pattern recognition protein (PRP). Results: PmLGBP binds to -1,3-glucan and LPS and could enhance the phenoloxidase (PO) activity. The prophenoloxidase (proPO) system is activated upon recognition of pathogens by pattern recognition proteins (PRPs), including a lipopolysaccharide- and -1,3-glucan-binding protein (LGBP). The innate immune system is important to invertebrates [1], where cellular immune responses, such as phagocytosis, nodule formation, and encapsulation, are important in arthropods, whereas the prophenoloxidase (proPO) activation system, the clotting system, and synthesis of antimicrobial peptides are important humoral defense mechanisms [1,2,3,4,5] These immune responses are triggered by the specific recognition of microorganisms by host proteins referred to as pattern recognition proteins (PRPs), which are capable of binding to a variety of microbial cell wall components, referred to as pathogen-associated molecular patterns (PAMPs). The protein was named PmLGBP and its involvement in the proPO system was elucidated
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