AUTHOR=Jia Zhihao , Jiang Shuai , Wang Mengqiang , Wang Xiudan , Liu Yu , Lv Zhao , Song Xiaorui , Li Yiqun , Wang Lingling , Song Linsheng 

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

JOURNAL=Frontiers in Immunology

VOLUME=Volume 11 - 2020

YEAR=2021

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2020.603270

DOI=10.3389/fimmu.2020.603270

ISSN=1664-3224

ABSTRACT=DM9 refers to an uncharacterized protein domain that originally discovered in Drosophila melanogaster. Two proteins with DM9 repeats have recently been identified in the Pacific oyster Crassostrea gigas as mannose-specific binding receptors. In the present study, a novel DM9 domain containing protein (designated as CgDM9CP-4) with two tandem DM9 domains was identified from C. gigas. The mRNA expression level of CgDM9CP-4 was highly enriched in hemocytes and gill tissue. After LPS, PGN and Vibrio splendidus stimulations, the expression levels of CgDM9CP-4 in hemocytes were all down-regulated. The recombinant protein of CgDM9CP-4 (rCgDM9CP-4) displayed a broad binding spectrum towards various PAMPs (LPS, PGN, β-glucan, and Mannose) and microorganisms (Staphylococcus aureus, Micrococcus luteus, V. splendidus, Vibro anguillarum, Escherichia coli, Pichia pastoris, and Yarrowia lipolytica). Immunohistochemistry revealed that CgDM9CP-4 was mostly expressed in gills and some hemocytes. Flow cytometry analysis demonstrated that the CgDM9CP-4-positive hemocytes, which accounted for 7.3% of the total hemocytes, were small in size and displayed less granularity. CgDM9CP-4 was also highly expressed in non-phagocytes (~82% of total hemocytes). Intracellular ROS levels and the expression levels of cytokines in CgDM9CP-4-positive hemocytes were much lower than that of CgDM9CP-4-negtive hemocytes. The mRNA expression levels of CgDM9CP-4 in hemocytes were decreased after RNAi of hematopoietic-related factors (CgGATA, CgRunt, CgSCL and CgNotch). In addition, CgDM9CP-4-positive cells were found to be more abundant in hemocytes from gills than those from hemolymph, with most of them located in the gill filament. These results suggest that CgDM9CP-4 is a novel member of PRR expressed in undifferentiated pro-hemocytes to mediate pro-hemocyte maturation in response to stimulation.