Preliminary Study On The Role Of CgAKT1 In The Synthesis Of Interferon-like Of Oysters Crassotrea Gigas

The RAC-alpha serine/threonine-protein kinase(RAC-alpha serine/threonine-protein kinase,AKT)is one of the most important and common protein kinases involved in a series of critical cellular pathways,such as cell survival,proliferation and immunoregulation.In the present study,a novel AKT protein homolog named CgAKT1 was identified from Pacific oyster Crassostrea gigas.Using molecular biology and immunological methods to study its sequence structure and functional characteristics,explore its role in the synthesis of interferon and the antiviral immunity involved in the interferon signaling pathway.The main results achieved are as follows:The open reading frame(ORF)of CgAKT1 was of 1482 bp encoding a polypeptide of 493 amino acids.Its molecular weight was of 57 k Da,and its isoelectric point(pI)was of 5.5.There were classical domains in the predicted CgAKT1 protein,including an N-terminal pleckstrin homology domain,a central catalytic domain and a C-terminal hydrophobic domain.According to the multiple alignment,CgAKT1 is highly conserved compared to AKT1 in other species,showing similarity of 62.76% with human AKT1,and 55.43-97.95% similarity with other species.In the phylogenic tree,all the selected AKT1 members were separated into vertebrate and invertebrate clades.CgAKT1 was firstly clustered with AKT1 from oyster C.virginica and Ch AKT1 from Crassostrea hongkongensis,and then clustered with other invertebrate AKT1 to form an invertebrate clade with the same ancestral origin.The mRNA transcripts of CgAKT1 were detected in all the examined tissues of C.gigas with highest level in gills(8.24-fold of that in mantle,p < 0.001),followed by haemocytes(3.62-fold of that in mantle,p < 0.001).CgAKT1 is distributed in all three types of haemocytes(granulocytes,semi-granulocytes and agranulocytes)of C.gigas,and the expression level is the highest in granulocytes.After poly(I:C)stimulation,CgAKT1 mRNA decreased significantly in haemocytes from 3 h(0.44-fold of that in the control group,p < 0.001)to 24 h(0.20-fold of that in the control group,p < 0.001),then increased significantly at 48h(3.65-fold of that in the control group,p <0.05).And after r CgIFNLP stimulation,the expression of CgAKT1 at the mRNA level increased significantly at 6 h which was 3.60-fold of that in the control group(p < 0.001).The subcellular location showed that the positive signals of CgAKT1 were distributed in the cytoplasm and cell membrane of haemocytes.After poly(I:C)stimulation,the positive signal indicating CgAKT1 in the cytoplasm was reduced.In CgAKT1-RNAi oysters,the mRNA level of cyclic GMP-AMP synthase(CgcGAS)and TANK-binding kinase 1(CgTBK1)did not change significantly,but the mRNA level of stimulator of interferon gene(CgSTING),interferon regulatory factor-1(CgIRF-1),interferon regulatory factor-8(CgIRF-8)and IFN-like protein(CgIFNLP)was significantly increased which was 1.40-fold,1.53-fold,1.72-fold and 1.99-fold of that in EGFP-RNAi oysters(p < 0.05),after poly(I:C)stimulation.In CgIFNLP-RNAi oysters,the mRNA level of CgAKT1 was decreased,which was 0.16-fold of that in EGFP-RNAi oysters(p < 0.01).Moreover,p-AKT1activator(SC-79)observably suppressed the expression of p-CgTBK1,CgSTING and CgIFNLP at the protein level after poly(I:C)stimulation.After transfection of CgAKT1 into HEK293 T cells,the expression of p-cGAS protein was significantly increased,while the cyclic GMP-AMP in the cells and the interferon(IFN-β)in the cell culture fluid was significantly decreased which was0.61-fold(p < 0.01)and 0.92-fold(p < 0.05)of that in the control group.These results indicated that CgAKT1 might play a negative regulatory role in antiviral immunity of oyster through the regulation of CgIFNLP mediated signal pathway.In summary,a AKT1 homologue CgAKT1 with conserved domain was identified from Pacific oyster C.gigas.The expression of CgAKT1 was inhibited by poly(I:C)but promoted by CgIFNLP.CgAKT1 regulated the production of CgIFNLP through the phosphorylation of CgTBK1 and CgcGAS.These results indicated that CgAKT1 played a negative regulatory role in CgIFNLP-mediated antiviral immunity.The above findings provide experimental evidence for further exploring the immune function of CgAKT1 molecules in invertebrates.