Lipopolysaccharide And Beta-1,3-Glucan Binding Protein (LGBP) Stimulates Prophenoloxidase Activating System In Chinese Mitten Crab (Eriocheir Sinensis)

Melanization mediated by prophenoloxidase (proPO) activivating system play an essential role in killing bacterial in invertebrates. Lipopolysaccharide and β-1, 3-glucan binding protein (LGBP) as a pattern recognition protein have been demonstrated to active the proPO cascade in insects and shrimp. In this study, we investigated the role of LGBP in prophenoloxidase cascade-induced melanization in Chinese mitten crab (Eriocheir sinensis).By RT-PCR analysis, LGBP was detected in all tested tissues, and showed highest expression in hemocytes, gill, intestine and brain. The expression of EsLGBP was up-regulated in the hemocytes following injections of LPS and β-1,3-glucan. The recombinant LGBP protein (rEsLGBP) was produced via prokaryotic expression system and affinity chromatography. By western blotting, rEsLGBP was discovered to exhibit the ability to bind to all tested microorganisms, including Gram-negative bacteria, Gram-positive bacteria and yeast (Pichia pastoris). Meanwhile we found rEsLGBP has a high binding activity towards microbial immune elicitors such as LPS and β-1,3-glucan whereas no binding activity is detected with peptidoglycan. The ability of rEsLGBP to promote cellular degranulation and encapsulation in vitro was detected by incubating rEsLGBP coated nickel agarose beads with hemocytes. Further experiments demonstrate that rEsLGBP can trigger the whole hemolymph dependent melanization and stimulate to proPO cascade in vitro. To investigate the role of LGBP on expression of proPO cascade-associated genes, EsLGBP was knocked down in hemocytes and the expression of the genes encoding EsPPAEl, PPAE2 and EsproPO were analyzed. Taken together, these results provide experimental evidence for role of LGBP in innate immunity, especially in the activation of prophenoloxidase activating system.