Molecular characterization and expression analysis of chitinase from the Pacific oyster Crassostrea gigas

Abstract

Chitinases are necessary enzymes supporting functions as a host defense factor against chitin-coated pathogens. They also function as a digestive enzyme for the hydrolysis of dietary chitin. We conducted characterization and assessed the tissue expression of the encoding gene of a chitinase (EC 3.2.1.14), Cg-Chit1, and the production of recombinant protein of Cg-Chit1, from the Pacific oyster, Crassostrea gigas. Chitinase activity in mantle extracts was detected to a marked degree in samples collected in July and August. Mantle chitinase worked well at pH 5.5, 7.0, and 8.5 tested in this study. RT-PCR showed that Cg-Chit1 expression is highly tissue-specific in the hemocytes and mantle. We then determined the distribution of Cg-Chit1 mRNA in C. gigas hemocytes and mantle histologically using in situ hybridization. Of the two subgroups of oyster hemocytes, granulocytes (main phagocytes) and hyalinocytes, only the former were found to express Cg-Chit1. In the mantle, chitinase-2 was expressed at the inner lobe of the mantle edge. Recombinant Cg-Chit1 clearly showed chitinase activity in a wide range of neutral/basic pH. These findings suggest that Cg-Chit1 functions as a host defense factor to hydrolyze chitin-coated organisms after phagocytosis by granulocytes and to exclude foreign substances from the mantle cavity.