AUTHOR=Qin Yanping , Wan Weitao , Li Jiangwei , Wang Zhongyu , Yang Yue , Li Jun , Ma Haitao , Yu Ziniu , Xiang Zhiming , Zhang Yuehuan 

TITLE=A novel Fas ligand plays an important role in cell apoptosis of Crassostrea hongkongensis: molecular cloning, expression profiles and functional identification of ChFasL

JOURNAL=Frontiers in Immunology

VOLUME=14

YEAR=2023

URL=https://www.frontiersin.org/journals/immunology/articles/10.3389/fimmu.2023.1267772

DOI=10.3389/fimmu.2023.1267772

ISSN=1664-3224

ABSTRACT=<sec><title>Background</title><p>Apoptosis regulates normal development, homeostasis, immune tolerance and response to environmental stress by eliminating unwanted or diseased cells, and plays a key role in non-specific immunity of invertebrates. The exogenous pathway mediated by death receptors and death ligands is a very important pathway for cell apoptosis. Death ligands are mainly members of the tumour necrosis factor (TNF) family, of which FasL is an important member. The deep involvement of FasL in vertebrates cell apoptosis and immunity has been reported many times, but there is limited research on the FasL gene in shellfish, and its functional importance in oyster cell apoptosis and immunity remains unclear.</p></sec><sec><title>Methods</title><p>The full length of <italic>Ch</italic>FasL was identified and cloned based on the genome of <italic>Crassostrea hongkongensis</italic>. Quantitative PCR was used to detect the relative expression of <italic>Ch</italic>FasL in different developmental stages and tissues, as well as the changes of relative expression in hemocytes after bacterial infection. The expression position of <italic>Ch</italic>FasL in HEK293T cells was also located by subcellular localization, and the effect of increased recombinant protein content on the activity of reporter genes p53 and p21 was studied by dual-fluorescence reporter gene. Finally, the changes of apoptosis rate in hemocytes after <italic>Ch</italic>FasL silencing was identified by RNA interference technology.</p></sec><sec><title>Results</title><p>We identified a novel FasL gene from <italic>C. hongkongensis</italic> and named it <italic>Ch</italic>FasL. We found that <italic>Ch</italic>FasL has potential N-linked glycosylation site, a transmembrane domain and a TNF region, which was a typical characteristics of TNF family. <italic>Ch</italic>FasL was expressed in all developmental stages of larvae and in all tissues of oysters. After stimulation by <italic>V. alginolyticus</italic> or <italic>S. haemol</italic>yticus, its relative expression in hemocytes increased significantly, suggesting that <italic>Ch</italic>FasL was deeply engaged in the immune response process of <italic>C. hongkongensis</italic> to external microbial stimulation. The results of subcellular localization showed that <italic>Ch</italic>FasL was mainly distributed in the cytoplasm of HEK293T cells. With the overexpression of the recombinant protein pcDNA3 1- <italic>Ch</italic>FasL, the activity of p53 and p21 significantly increased, showing a positive regulatory effect. Moreover, after dsRNA successfully reduced the relative expression of <italic>Ch</italic>FasL, the apoptosis rate of hemocytes was significantly lower than that the dsGFP group.</p></sec><sec><title>Conclusion</title><p>These results comprehensively confirmed the important role of <italic>Ch</italic>FasL in the apoptosis process of <italic>C. hongkongensis</italic>, which provided the basis and premise for the in-depth understanding of the immune function of apoptosis in molluscs, and also contributed to the research on the pathogenic death mechanism and disease resistance breeding of marine bivalves.</p></sec>