The Involvement Of CgAstakine And Its Receptor In Regulating Proliferation Of Haemocytes In Oyster (Crassostrea Gigas)

Circulating haemocytes are the main immune cells in invertebrates to resist environmental stress or pathogen infection.They involved in phagocytosis,encapsulation and other immune processes,and play important roles in innate immunity of invertebrates.The proliferation and differentiation of haemocytes is an important process of haematopoiesis,as well as a vital mechanism in maintaining immune homeostasis,which is regulated by haematopoietic transcription factors,cytokines,other factors and signal pathways.Studies have shown that cytokines play important role in the regulation of immune cell proliferation and differentiation in vertebrates.At present,the research of invertebrate cytokines and their regulation role in is still at the initial stage.Astakine is a class of cytokines unique in invertebrates,which have been identified mainly in Arthropod,such as in freshwater crayfish,Chinese mitten crabs and white shrimp.And an Astakine（CgAstakine）was identified previously from oyster Crassostrea gigas in our laboratory.In the present study,an ATP synthaseβsubunit of CgAstakine（named CgATP synthaseβsubunit）was identified from C.gigas,and the interaction between CgATP synthaseβsubunit and CgAstakine and the regulation of the two genes on haemocytes proliferation was explored by using bioinformatics methods,real time fluorescence quantitative PCR,western blot and flow cytometry.The results obtained are as follows:1.Molecular structure and expression characteristics of CgATP synthaseβsubunitAn ATP synthaseβsubunit was cloned from C.gigas.The opening reading frame（ORF）of CgATP synthaseβsubunit was of 1575 bp,which encoded 524 amino acids residues with a predicted relative molecular weight of 56.57 k Da.CgATP synthaseβsubunit protein contains one conserved ATP-synt＿ab＿N domain and one conserved AAA domain.CgATP synthaseβsubunit was expressed in adductor muscle,mantle,labial palp,hepatopancreas,gills,gonad and haemocytes of C.gigas,and the highest expression level was observed in hepatopancreas and gills,which was 109.11-fold（p<0.01）and 97.21-fold（p<0.01）of that in labial palps,respectively.Immunofluorescence results showed that CgATP synthaseβsubunit protein was mainly distributed on the membrane of haemocytes.2.Regulation of CgAstakine on proliferation of haemocytesAfter injection of recombinant CgAstakine protein（rCgAstakine）,the proportions of EdU positive cells in haemocytes ware significantly increased,which was 2.24-fold（p<0.01）and1.28-fold（p<0.05）of that in seawater（SW）group at 6 h and 12 h,respectively.The mRNA expression levels of potential granulocytes markers CgSOX11 and CgAATase and agranulocytes markers CgCD9 were significantly increased,and the expression levels of CgSOX11 were 2.86-fold（p<0.01）and 1.78-fold（p<0.05）of that in SW group at 12 h and 24h,respectively.The expression level of CgAATase was 4.76-fold（p<0.01）and 5.05-fold（p<0.01）of that in SW group at 12 h and 24 h,respectively.The expression level of CgCD9 was significantly increased at 6 h and 12 h,which was 1.62-fold（p<0.05）and 2.06-fold（p<0.01）of that in SW group,respectively.The protein expression level of CgAATase was also significantly increased after injection of rCgAstakine.3.Interaction between CgATP synthaseβsubunit and CgAstakineCgAstakine and rCgATP synthaseβsubunit proteins were obtained by prokaryotic expression,and they showed high affinity with Kd value of 8.21×10^-7 by Bio-layer interferometry（BLI）assay.After rCgAstakine stimulation,the mRNA transcripts of CgATP synthaseβsubunit in agranulocytes and semi-granulocytes were significantly increased at 24 h（2.44-fold,and 9.01-fold of that in SW group,p<0.01）,and those in granulocytes were significantly increased at 6 h（1.83-fold,p<0.01）,12 h（1.92-fold,p<0.01）and 24 h（3.47-fold,p<0.01）.The expression level of CgATP synthaseβsubunit protein in agranulocytes and granulocytes was also significantly increased after rCgAstakine stimulation,which was 1.64-fold（p<0.05）and 1.85-fold（p<0.05）of that in SW group,respectively.The expression level of CgATP synthaseβsubunit protein in semi-granulocytes did not change significantly compared with the SW group.4.Regulation of CgATP synthaseβsubunit and CgJAK2 on proliferation of haemocytesAfter interference with the expression of CgATP synthaseβsubunit by RNAi technology,the proportions of EdU positive cells in haemocytes in CgATP synthaseβsubunit-RNAi oysters were significantly reduced by flow cytometry,which was 0.28-fold of that in Negative control（NC）group（p<0.01）.The proportions of EdU positive cells in granulocytes were significantly declined compared with NC group（0.89-fold,p<0.01）,while the proportions of EdU positive cells in agranulocytes and semi-granulocytes did not change significantly.There results suggested that CgATP synthaseβsubunit acts as the receptor of CgAstakine and plays important roles in CgAstakine induced renewal of haemocytes in C.gigas.After treatment with CgJAK2 inhibitor and rCgAstakine,the proportions of EdU positive cells in haemocytes significantly reduced.The proportions of EdU positive cells in agranulocytes,semi-granulocytes and granulocytes decreased significantly,which was0.40-fold（p<0.01）,0.40-fold（p<0.05）and 0.35-fold（p<0.01）of that in control group,respectively.In conclusion,CgAstakine and CgATP synthaseβsubunit were interacted and participated in the regulation of proliferation of haemocytes,and CgJAK2 was also involved in the regulation of proliferation of haemocytes by CgAstakine.These results will provide an important reference for the regulation mechanism of haemocytes proliferation in invertebrates.