A DM9-containing protein from oyster Crassostrea gigas (CgDM9CP-3) mediating immune recognition and encapsulation

DM9 domain containing protein (DM9CP) is a recently identified pattern recognition molecules exiting in most organisms except plants. In the present study, a novel DM9-containing protein (CgDM9CP-3) was identified from Pacific oyster Crassostrea gigas with an open reading frame of 438 bp, encoding a polypeptide of 145 amino acids containing two tandem DM9 repeats. The deduced amino acid sequence of CgDM9CP-3 shared 52.4% and 58.6% identity with CgDM9CP-1 and CgDM9CP-2, respectively. The mRNA transcripts of CgDM9CP-3 were highest expressed in oyster gills and its protein was mainly distributed in cytomembrane of haemocytes. After the stimulations with Vibrio splendidus and mannose, the mRNA expression of CgDM9CP-3 in oyster gills was significantly up-regulated and reached the peak level at 12 h and 24 h (p < 0.05), which was 7.80-fold (p < 0.05) and 42.82-fold (p < 0.05) of that in the control group, respectively. The recombinant CgDM9CP-3 protein (rCgDM9CP-3) was able to bind LPS, PGN and d-Mannose, fungi Pichia pastoris and Yarrowia lipolytica, as well as gram-negative bacteria Escherichia coli, Vibrio anguillarum and V. splendidus in a Ca²⁺-dependent manner. Moreover, it could enhance the encapsulation of haemocytes and exhibited agglutination activity towards fungi P. pastoris and Y. lipolytica in vitro with Ca²⁺. These results suggested that CgDM9CP-3 not only acted as a PRR involved in the pathogen recognition, but also enhanced cellular encapsulation in oyster C. gigas.