Molecular Mechanism Study Of Membrane-bound Dscam Regulating Immune Response Through Membrane-tonucleus Signaling In Chinese Mitten Crab Eriocheir Sinensis

Chinese mitten crabs（Eriocheir sinensis）belongs to crustaceans of Arthropoda,Decapoda,and Varunidae.It is widely distributed in different coastal and inland freshwater environments in China,and is one of the most valuable freshwater fishery products in China.In recent years,the continuous emergence of various diseases has led to huge economic losses of aquaculture in China.Therefore,the basic theoretical research on the immune system is not only helpful to understand the immune defense mechanism of E.sinensis and other crustaceans,but also has important significance for the sustainable development of E.sinensis and the whole aquaculture industry and the improvement of the theory of invertebrate immune defense mechanism.The innate immune response of invertebrates is associated with many immune receptors responsible for recognizing foreign microorganisms,and a key feature of immune receptors is the need for huge libraries to accommodate a large number of rapidly evolving potential pathogens.Due to the molecular diversity caused by alternative splicing,Dscam is considered to be a hypervariable pattern recognition receptor in insect and crustacean,and has been proved to be closely related to the innate immunity of invertebrates.Therefore,after it was found to have immune function,clarifying its specific function and mechanism in the immune system has always been the driving force of researchers.In this study,we first found that EsDscam-ICD can be cleaved by γ-secretase and enter the cytoplasm through Western blot and inhibitor experiments.Through the bioinformatics analysis and comparison of Dscam-ICDs in different arthropods,it was found that there were conserved nuclear localization signal（NLS）motifs.Different EsDscam-ICD plasmids were constructed and overexpressed in HEK 293 T and Drosophila S2 cells,respectively.Immunofluorescence and separation of nuclear and cytoplasmic proteins experiments confirmed that only the three isoforms containing exon 33 had significant nuclear import ability.In order to further clarify the molecular mechanism of nuclear import of EsDscam-ICDs,the nuclear import protein Importin-5 of E.sinensis（EsIPO5）was cloned through screening.The tissue-specific expression showed that EsIPO5 was expressed in various tissues,and the expression level of EsIPO5 in haemocytes was significantly increased after bacterial stimulation.However,after knocking down EsIPO5 by RNAi experiments,the nuclear import of EsDscamICDs was significantly weakened,indicating that EsIPO5 mediated the nuclear import of EsDscam-ICDs.Co-IP and laser scanning confocal microscopy technology confirmed that EsIPO5 only strongly binds to ICDs containing exon 33,and this result showed that the nuclear import of specific EsDscam-ICDs was due to the strong binding ability with EsIPO5.In order to further understand the gene expression that may be regulated by EsDscam-ICDs after nuclear import,the above three ICDs plasmids containing exon33 were constructed and transferred into Drosophila S2 cells.RNA-Seq showed that the nuclear import of EsDscam-ICDs regulated the transcription of important functional genes related to immunity,metabolism and cell proliferation.Experiments such as qRTPCR,cell proliferation detection（CFSE and EDU staining）and bacterial clearance test confirmed that the nuclear translocation of EsDscam-ICDs had the ability to promote the proliferation of primary haemocytes and the ability of the host to clear bacteria.These results suggest that the nuclear import of EsDscam-ICDs plays an important role in host innate immunity and haemocytes proliferation induced by pathogen stimulation.The nucleus import of the intracellular domain of transmembrane proteins usually occurs after the shedding of the extracellular domain.In order to further verify the shedding of extracellular region abscission of Dscam and its related mechanism,two target proteins ADAM10 and ADAM17 were screened by T7 phage display library and related literature reports.ADAM10 was identified as a protease that mediates the shedding of extracellular domain of EsDscam by using inhibitor inhibitory protein activity and overexpression methods combined with fluorescence detection by flow cytometry.By interfering with the main components of the previously discovered Dscam signaling cascade that regulates the expression of antimicrobial peptides,we determined that ADAM10 was regulated by this signaling pathway,but there was a significant time difference between ADAM10 and the expression regulation of antimicrobial peptides,that is,the regulation of ADAM10 was significantly lagged,which fully explained the complexity of Dscam ’ s immune regulation function.In summary,this study took E.sinensis as the research object,and took Dscam intracellular domain regulation of immune response as the breakthrough point to deeply analyze the molecular regulation mechanism of EsDscam-ICD into the nucleus and its regulation of gene expression after entering the nucleus.This finding not only enriches the function of Dscam intracellular domain involved in immune regulation,but also provides a new perspective for further in-depth and comprehensive analysis of the immune regulation function of Dscam.