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J Biol Chem, Vol. 275, Issue 2, 1337-1343, January 14, 2000

A Lipopolysaccharide- and -1,3-Glucan-binding Protein from Hemocytes of the Freshwater Crayfish Pacifastacus leniusculus
PURIFICATION, CHARACTERIZATION, AND cDNA CLONING^*

So Young Lee, Ruigong Wang, and Kenneth Söderhäll

From the Department of Comparative Physiology, Evolutionary Biology Center, Uppsala University, Norbyvägen 18A, S-75236, Uppsala, Sweden

A lipopolysaccharide- and -1,3-glucan-binding protein (LGBP) was isolated and characterized from blood cells (hemocytes) of the freshwater crayfish 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 electrophoresis under reducing and nonreducing conditions, respectively. The calculated mass of LGBP is 39,492 Da, which corresponds to the native size of LGBP; the estimated pI of the mature LGBP is 5.80. LGBP has binding activity to lipopolysaccharides as well as to -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 bacteria-binding proteins and -1,3-glucanases. Interestingly, LGBP also has a structure and functions similar to those of the coelomic cytolytic factor-1, a lipopolysaccharide- and glucan-binding protein from the earthworm Eisenia 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 -1,3-glucan laminarin in the hemocyte lysate of crayfish. The PO activity was blocked completely by the anti-LGBP antibody. Moreover, the PO activity could be recovered by the addition of purified LGBP. These results suggest that the 36-kDa LGBP plays a role in the activation of the proPO activating system in crayfish and thus seems to play an important role in the innate immune system of crayfish.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EMBL Data Bank with accession number(s) AJ250128.

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