A lipopolysaccharide- and beta-1,3-glucan-binding protein from hemocytes of the freshwater crayfish Pacifastacus leniusculus - Purification, characterization, and cDNA cloning

A lipopolysaccharide- and beta-1,3-glucan-binding protein (LGBP) was isolat ed and characterized from blood cells (hemocytes) of the freshwater crayfis h Pacifastacus leniusculus. The LGBP was purified by chromatography on Blue -Sepharose and phenyl-Sepharose, followed by Sephacryl S-200. The LGBP has a molecular mass of 36 kDa and 40 kDa on 10% SDS-polyacrylamide gel electro phoresis under reducing and nonreducing conditions, respectively. The calcu lated mass of LGBP is 39,492 Da, which corresponds to the native size of LG BP; the estimated pi of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to beta-1,3-glucans such as laminarin and curdlan, but peptidoglycan could not bind to LGBP. Cloning and sequencing of LGBP showed significant homology with several putative Gram-negative bac teria-binding proteins and beta-1,3-glucanases. Interestingly, LGBP also ha s a structure and functions similar to those of the coelomic cytolytic fact or-1, a lipopolysaccharide- and glucan-binding protein from the earthworm E isenia foetida. To evaluate the involvement of LGBP in the prophenoloxidase (proPO) activating system, a polyclonal antibody against LGBP was made and used for the inhibition of phenoloxidase (PO) activity triggered by the be ta-1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activit y could be recovered by the addition of purified LGBP. These results sugges t that the 36-kDa LGBP plays a role in the activation of the proPO activati ng system in crayfish and thus seems to play an important role in the innat e immune system of crayfish.