Functional Investigation Of Cation-dependent Mannose-6-phosphate Receptor(CD-MPR) In The Innate Immunity Of Marsupenaeus Japonicus

Marsupenaeus japonicus is one of the most important species of aquaculture in China.However,shrimps were easily infected by various viruses and bacteria,causing huge losses to the shrimp aquaculture industry.Shrimps belong to crustaceans of invertebrates,which mainly rely on innate immunity against the various pathogens.Therefore,the study of the innate immunity of shrimps can provide theoretical basis and guidance for the prevention and control of shrimp disease.The cation-dependent mannose-6-phosphate receptor(CD-MPR)is a kind of type I transmembrane glycoproteins,which is mainly distributed in the trans-Golgi network(TGN)and plasma membrane.CD-MPR could specifically recognize and bind mannose 6-phosphate(M6P)ligand,so it could direct new lysosomal enzymes on TGN to enter lysosomes and functions in the transportation of lysosomal enzymes and lysosome formation in higher eukaryotes.In addition,The CD-MPR in the plasma membrane could capture extracellular ligands containing M6P into cells.However,the role of CD-MPR in immunity remains to be studied.This study selected Marsupenaeus japonicus as experimental materials,research the function of CD-MPR in the invasion of white spot syndrome virus(WSSV)and Vibrio anguillarum.1.Cation-dependent 6-phosphoric mannose receptor(CD-MPR)is one of the receptors of WSSV and participated in intracellular transportation of WSSV.We identified a CD-MPR in Marsupenaeus japonicus and designed as MjCD-MPR.The MjCD-MPR was widely distributed in hemocytes and five organs:heart,hepatopancreas,gills,stomach,and intestine.It was significantly upregulated after challenged by WSSV.Knockdown of the MjCD-MPR,the expression level of WSSV vp28 and iel were decreased.MjCD-MPR antibody can block native MjCD-MPR in plasma membrane and block WSSV replication by decreasing the expression of WSSV vp28 and iel.The extracytoplasmic region of MjCD-MPR could bind WSSV VP19,24,26.The above results indicate that WSSV can enter host cell by binding MjCD-MPR."Overexpression" of MjCD-MPR by injection of rMjCD-MPR protein increase WSSV infection and decrease the survival rate of shrimps.MjCD-MPR move to plasma membrane at early stage of WSSV infection and return to the cytoplasm at late stage of WSSV infection by immunocytochemistry,and the colocalization of MjCD-MPR and WSSV particles can be found in the early and late stages of WSSV injection.These results showed that MjCD-MPR was utilized as a kind of receptors by WSSV for its internalization and intracellular MjCD-MPR was uesd for WSSV traffic process in the host cells.This is the first discovery of a molecule in virus infection which have double function:it can act as a viral receptor and participate in the intracellular transmission of virus.2.Cation-dependent 6-phosphoric mannose receptor(CD-MPR)plays an important role in anti-bacteria immunity of Marsupenaeus japonicus by promoting the expression of antimicrobial peptides.MjCD-MPR was significantly upregulated in shrimp after challenged by Vibrio anguillarum.Knockdown of the MjCD-MPR,the amount of Vibrio anguillarum in hemolymph of shrimp were increased.The extracytoplasmic region of MjCD-MPR could bind four Gram-positive bacteria(Staphylococcus aureus,Bacillus megaterium,Bacillus thuringiensis,Bacillus subtilis)and four Gram-negative bacteria(Escherichia coli,Vibrio anguillarum,Klebsiella pneumoniae,Pseudomona.s aeruginosa).MjCD-MPR can also bind peptidoglycan(PG),lipopolysaccharide(LPS)and lipoteichoic acid(LTA).Knockdown of the MjCD-MPR,the expression of various antimicrobial peptides decreased significantly.These results indicated that MjCD-MPR plays an important role in anti-bacteria immunity.MjCD-MPR works as one of the pattern recognition receptors of bacteria,which can activate downstream signaling pathway to promote the expression of antimicrobial peptides.