Identification of a Novel Pattern Recognition Receptor DM9 Domain Containing Protein 4 as a Marker for Pro-Hemocyte of Pacific Oyster .

DM9 refers to an uncharacterized protein domain that is originally discovered in . Two proteins with DM9 repeats have been recently identified from Pacific oyster as mannose-specific binding pattern-recognition receptors (PRRs). In the present study, a novel member of DM9 domain containing protein (designated as DM9CP-4) was identified from . DM9CP-4, about 16 kDa with only two tandem DM9 domains, was highly enriched in hemocytes and gill. The transcripts level of DM9CP-4 in circulating hemocytes were decreased after LPS, PGN and stimulations. The recombinant protein of DM9CP-4 (rDM9CP-4) displayed a broad binding spectrum towards various pathogen-associated molecular patterns (PAMPs) (LPS, PGN, β-glucan and Mannose) and microorganisms (, , , , , and ). DM9CP-4 was mostly expressed in gill and some of the hemocytes. Flow cytometry analysis demonstrated that the DM9CP-4-positive hemocytes accounted for 7.3% of the total hemocytes, and they were small in size and less in granularity. DM9CP-4 was highly expressed in non-phagocytes (~82% of total hemocytes). The reactive oxygen species (ROS) and the expression levels of cytokines in DM9CP-4-positive hemocytes were much lower than that in DM9CP-4-negative hemocytes. The mRNA expression level of DM9CP-4 in hemocytes was decreased after RNAi of hematopoietic-related factors (GATA, Runt, SCL, and Notch). In addition, DM9CP-4-positive cells were found to be much more abundant in hemocytes from gill than that from hemolymph, with most of them located in the gill filament. All these results suggested that DM9CP-4 was a novel member of PRR that expressed in undifferentiated pro-hemocytes to mediate immune recognition of pathogens.