A DM9-containing protein from oyster Crassostrea gigas (CgDM9CP-2) serves as a multipotent pattern recognition receptor

Abstract DM9 is a novel protein domain with unknown function originally discovered in Drosophila melanogaster . Recently, a protein harboring DM9 repeats was identified as mannose-specific lectin ( Cg CGL1, renamed as Cg DM9CP-1) from the Pacific oyster Crassostrea gigas . In the present study, another DM9 containing protein was identified from oyster C . gigas (designated as Cg DM9CP-2). The open reading frame of Cg DM9CP-2 gene was of 432 bp, encoding a polypeptide of 143 amino acids with two tandem DM9 repeats. The deduced amino acid sequence of Cg DM9CP-2 shared 60.8% identity with that of Cg DM9CP-1. In the unrooted phylogenetic tree, Cg DM9CP-2 was closely clustered with Cg DM9CP-1, and then assigned into the branch of invertebrate DM9CPs. The mRNA transcripts of Cg DM9CP-2 were expressed in all the tested tissues, including mantle, gonad, gills, adductor muscle, hemocytes, and hepatopancreas, with the highest expression level in gills. Cg DM9CP-2 protein was mainly distributed on the cytomembrane of oyster hemocytes. After mannose stimulation, the mRNA expression of Cg DM9CP-2 in gills was up-regulated to the peak level (5.90-fold of that in SSW group, p p p Vibrio splendidus stimulation, and then gradually decreased from 48 to 72 h ( p Cg DM9CP-2 protein (r Cg DM9CP-2) displayed higher binding affinity to D-(+)-mannose while lower binding affinity to lipopolysaccharide and peptidoglycan. r Cg DM9CP-2 also exhibited binding activity towards fungi ( Pichia pastoris and Yarrowia lipolytica ), gram-positive bacteria ( Staphylococcus aureus and Micrococcus luteus ), and gram-negative bacteria ( Escherichia coli , Vibrio anguillarum , Aeromonas hydrophila and V. splendidus ). It could agglutinate fungi P. pastoris and Y. lipolytica , and inhibit the growth of P. pastoris , S. aureus , V. anguillarum, and V. splendidus . These results collectively indicated that Cg DM9CP-2 not only served as a pattern recognition receptor with a broad range of recognition spectrum, but also involved in inhibiting the growth of invading microbe in the innate immune response of oyster, which would provide further evidence for the function of DM9 domain in the innate immune system.