| Eriocheir sinensis is an invertebrate crustacean and it is an important aquatic product.Because of rich in nutrients in the body,so it is popular with the public.But in recent years,the situation of Eriocheir sinensis’ s aquaculture is not optimistic,which has seriously affected the development of the aquaculture industry and the marine economy.Bacterial diseases are the main cause of Chinese mitten crabs’ diseases.These diseases are caused by different pathogenic bacteria in the water.Therefore,it is an important scientific problem that urgently needs to be solved that to explore the defense mechanism of Chinese mitten crabs against bacteria.It will provides theoretical support for the prevention and treatment of diseases and their ability to improve immunity and disease resistance.Phagocytosis is a very important cellular immunity of innate immunity in invertebrates.Removal of exogenous pathogens,cell debris and apoptotic cells needs to be completed by phagocytosis;however,phagocytosis of Eriocheir sinensis is currently studied Little,and in recent years,specific innate immunity and immune memory phenomena have been gradually found in invertebrates,but the molecular mechanism behind this is not clear.Downs Syndrome cell adhesion molecule Dscam is an one molecule of immunoglobulin superfamily members,which have been found to have superior molecular diversity in arthropods,but also participate in defense against different pathogens and phagocytosis,but at present,the causes of its supermolecular diversity and the mechanisms of its regulates in phagocytosis are not reported,therefore,it is important that solving the problem how EsDscam binds to different bacteria and regulates the phagocytosis of Chinese mitten crabs,and it will be of great significance in clarifying the innate immune specific molecular mechanism of invertebrates and further improving the immunology theory.In this study,genome cDNA of EsDscam was obtained through genome-wide sequencing.After comparing with cDNA,the gene structure was determined.It was found that the protein encoded by this gene is highly similar to the Dscam of arthropods such as Drosophila.It is theoretically possible to produce 30,600 isoforms;protein 3D structural analysis shows that EsDscam is very similar to Drosophila’s and has a horseshoe structure;its protein expression is detected by specific EsDscam antibody,the results showed that it mainly expressed in the intestine,heart,brain,stomach,hemocytes and hemolymph;after stimulation of hemocytes with two gram-positive bacteria and two gram-negative bacteria,EsDscam showed varying degrees of up-regulation in both transcriptional and translational levels;using TA cloning experiments,it was found that bacteria can induce EsDscam to produce different isoforms;the sequence of epitope I and epitope II of E.sinensis was determined by comparison with sequences of Drosophila and Daphnia;these results indicate that different bacteria can induce EsDscam’s upregulation at both protein and nucleic acid levels and can produce different kinds of isoforms.By categorizing the sequence of epitope II and combining with the experimental results of TA cloning,11 polypeptide sequences were synthesized in vitro;ELISA was used to detect its binding to different bacteria;in vitro,the expressed recombinant proteins were binding with different bacteria.The results showed that it can produce strong binding with the original bacteria,and the results of clearance experiments in vivo showed that the specific recombinant protein can promote the clearance of bacteria by hemocytes;in vitro,phagocytosis experiments showed that the specific recombinant protein can also promote the phagocytosis of hemocytes.Through Western blotting,a strong binding of the isoforms with original bacteria in the hemolymph were detected using the specific antibodies;truncated expression of the recombinant protein was detected to have no effect on phagocytosis.The reduced specific binding capacity and phagocytosis indicates that the presence of horseshoe-shaped structures is of great significance for the specific binding to bacteria and regulation of phagocytosis.To explore the role of transmembrane EsDscam and secreted EsDscam in the regulation of phagocytosis,we firstly established a system that transmembrane EsDscam could be silencing effectively,then its phagocytosis rate after bacterial challenge were detected.The experimental results showed that after transfection of dsEs-mDscam,the phagocytic ability of hemocytes to bacteria was reduced,and the phagocytic ability was still reduced using bacteria preincubated with recombinant proteins.Co-localization experiments revealed that co-localization of secreted EsDscam with transmembrane EsDscam could occur;after RNAi of the specific exons,the phagocytic ability of hemocytes to the original bacteria was obviously reduced in vitro.Furthermore,co-immunoprecipitation and Far-western were used to detect the interaction between homologous secreted and transmembrane EsDscam.The interaction of each other were comfirmed in vitro and in vivo.These results indicate that the secreted EsDscam promotes phagocytosis through transmembrane EsDscam,and this process depends on homophilic binding between the same EsDscams.In summary,our study demonstrated that EsDscam is upregulated after bacterial stimulation and produces specific Es Dscam isoforms,which can specifically bind to bacteria through epitope II;subsequently,bacteria-induced specific secreted EsDscam binds to original bacteria.The phagocytosis of hemocytes is increased by the interaction of transmembrane EsDscam and secreted EsDscam,and the precise regulation of this mechanism may also be related to the intracellular signaling pathway mediated by the cytoplasmic tail of EsDscam.This study was the first to elucidate the EsDscam’s function in Chinese mitten crabs,and the mechanism of regulation of phagocytosis has provided a solid foundation for further clarifying its role in the innate immune signaling pathway. |
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