The Cloning And Characterization Of Immune Response Regulatory Pathway Of Scavenger Receptors Class B In Apostichopus Japonicus

Scavenger receptor(SR)is a class of typical endocytic pattern recognition receptors,located on the surface of macrophage membranes,which can recognize and bind a variety of ligands,including various modified or oxidized low-density lipoproteins,apoptotic cells and pathogens.Among them,the B-type scavenger receptor(SR-B)has a wide range of ligands,which can bind various lipoproteins and polyanionic phospholipids,regulate lipid metabolism and maintain the environmental balance in the host,and participate in a series of physiological and the pathological process,such as the formation or suppression of atherosclerosis and other cardiovascular diseases,the immune defense of the host,and the removal of apoptotic cells.At present,the research on scavenger receptors in lower invertebrates is limited.In addition,as an important economical breed,sea cucumber’s industrial development is severely restricted by disease problems caused by bacteria and viruses,while scavenger receptors are the important molecules in innate immunity.Therefore,it is particularly urgent to study the role of scavenger receptors in the immune response of sea cucumber Apostichopus japonicus and its immune regulation pathway as the first line of defense against invading pathogens.In this study,we obtained the full length of the sea cucumber A type B scavenger receptor(Aj SR-B)by gene clone,and analyzed molecular characteristic;studied the m RNA expression of Aj SR-B in vivo after Vibrio splendidus challenge.Analyzed subcellular localization and its ability to recognize and bind PAMPs and various pathogens.The main findings were as follows:(1)Aj SR-B gene was cloned by RACE technology to obtain a total length of2519 bp,including an open reading frame of 1581 bp,encoding 526 amino acids.SMART analysis showed that Aj SR-B contains a conserved CD36 domain,two transmembrane domains,and two intracellular domains at the N-terminus and C-terminus.Among them,there are nine potential N-glycosylation sites located in the CD36 domain of the extracellular region.BLAST and phylogenetic tree analysis showed that Aj SR-B was similar to Mesocentrotus nudus and Strongylocentrotus purpuratus,indicating Aj SR-B was closer to marine invertebrate species.(2)Spatial expression analysis of Aj SR-B m RNA showed that it was widely distributed in various tissues of sea cucumber,the m RNA transcript in intestine showed the highest level,which was increased by 181.6-fold(P < 0.01)compared with the control group(coelomocytes),and followed by tentacles(21.3-fold,P < 0.01)and respiratory tree(12.9-fold,P < 0.01).In addition,the m RNA expression level of Aj SR-B in coelomocytes and intestine reached to the highest level at 24 h after Vibrio splendidus challenge,and then the expression level in coelomocytes recovered to the control level at 48 h.However,its transcript in intestine still maintain at a higher level during the whole experiment.(3)The extracellular region of Aj SR-B was amplified and expressed in E.coli Rosetta(DE3)?cells and induced by IPTG.The His-Aj SR-B fusion protein was obtained with a molecular weight of approximately 52 k Da,and polyclonal antibody was prepared.Cellular localization indicated the Aj SR-B protein was mainly localized on the surface of cell membranes.(4)The renatured His-Aj SR-B fusion protein exhibited strong binding activities to lipopolysaccharide(LPS),peptidoglycan(PGN),and mannan(MAN).The maximum binding parameter for LPS was 2.6026.The maximum binding parameter of PGN was 2.6366,both of them showed higher binding activity than MAN 2.056.The apparent dissociation constant indicated that the recombinant Aj SR-B protein contained different affinity for different PAMPs.(5)The renatured His-Aj SR-B fusion protein has an obvious agglutination effect on V.splendidus,Vibrio harveyi,Vibrio parahaemolyticus and Micrococcus luteus under calcium ions.There is no agglutination effect on the above four bacteria without calcium ions.The agglutination activity with V.splendidus,V.harveyi and V.parahaemolyticus was disappeared after addition of free LPS,PGN and MAN,respectively,but still exhibited a low agglutination activity of M.luteus after adding with three PAMPs.(6)After interfering with the Aj SR-B gene in the individual sea cucumber,the phagocytic ability of the coelomocytes was greatly inhibited,which dropped to 31%of the control group.At the same time,the expression of clathrin and caveolin related to phagocytosis was also significant inhibition indicates that Aj SR-B plays an important role in the phagocytosis of sea cucumber.All our current results showed that Aj SR-B is a typical B-type scavenger receptor,which is located on the surface of the cell membrane and can recognize and bind a variety of Gram-negative bacteria,Gram-positive bacteria,and LPS,PGN and MAN derived from cell well of bacteria,and plays an important role in the phagocytosis of sea cucumber coelomocytes,indicating Aj SR-B involving in a variety of innate immunity of sea cucumber.The results of the study provide a basis for in-depth understanding of the immune function of sea cucumber scavenger receptors in the future.