Studies On Transcriptome Of Fish Lymphocytes And CD4+T Lymphocytes And Crystal Structure Of Complement C1qAgD Protein

Fish as the lowest vertebrate,is the node of the immunological phylogenetic tree that possesses both the innate immune system and the acquired immune system with complete humoral and cellular immune responses.Studies have shown that T lymphocytes and B lymphocytes are also present in fish,and T lymphocytes can also be divided into CD4 + T cells and CD8+ T cells;Moreover,B cells in bony fish have been proved to have phagocytosis.However,there is still a lack of substantive studies on metabolic pathways related to the proliferation and activation of T and B lymphocytes in fish.Bony fish including grass carp and zebrafish was used in this study.Firstly,the mechanism of Con A and LPS stimulation of grass carp lymphocytes was studies using the Illumina/Hiseq transcriptome sequencing platform.Finally,the crystal structure of zebrafish C1qA globular domain(Dare-C1qAgD)was solved,and the recognition mechanism of Dare-C1 qAgD was elucidated from the structural level.In this paper,the peripheral blood lymphocytes of grass carp were divided into three groups:the control group,ConA stimulation group and LPS stimulation group.After stimulation with Con A and LPS for 12 hours,the total RNA was extracted from each group,and the three transcriptome data of peripheral blood lymphocytes of grass carp were obtained.A total of 442 grass carp immune related genes were annotated by data analysis.After ConA stimulation,totally 77 differentially expressed genes were identified,including 71 up-regulated genes and 6 down-regulated genes.KEGG enrichment analysis showed their enrichment in different KEGG pathways,and the most significant three immune-related signaling pathways were studied in detail,include JAK-STAT signaling pathway,MAPK signaling pathway and TNF signaling pathway,which are involved in the process of cell metabolism,proliferation and differentiation.After LPS stimulation,totally 557 differentially expressed genes were identified,including 433 up-regulated genes and 124 down-regulated genes.KEGG enrichment analysis showed that the most important immune-related signaling pathways included Phagesome,Lysosome,NLR signaling pathway,TLR signaling pathway and JAK-STAT signaling pathways,which play an important role in the inflammatory responses,resistance to pathogen infections and cell metabolism,proliferation and differentiation.Furthermore,by the expression and purification of grass carp CD4 functional domains,six monoclonal antibodies against grass carp CD4 molecule were successfully prepared.Western blot analysis showed that the six monoclonal antibodies could specifically recognize the CD4 protein of grass carp.Subsequently,the lymphocytes of the grass carp head kidney were isolated,after Con A stimulation and cultured for 18 hours,purified CD4 monoclonal antibody was used to isolate CD4+ T cells of the control and Con A stimulatd groups by the flow cytometry technique.By extracting the RNA from the two groups respectively,the transcriptomic database of grass carp head kidney CD4+ T cells was constructed.The data showed that,1718 differentially expressed genes were identified after Con A stimulation,which included 881 up-regulated genes and 837 down-regulated genes.KEGG enrichment analysis showed that the most important immune-related signaling pathways including Proteasome,TLR signaling pathway,p53 signaling pathway,JAK-STAT signaling pathway and MAPK signaling pathway.Compared with the peripheral blood lymphocytes,the effect of Con A on signaling pathways of head kidney CD4+ T cells is more extensive due to prolonged stimulation time.Recently,C1q molecules have also been found in lower vertebrates such as bony fish,but their study is still at the level of gene structure and function,and studies on its protein structure remains to be done.In this paper,the crystal structure of zebrafish ClqAgD(Dare-C1qAgD)molecule of was solved by protein in vitro expression,renaturation,purification and crystallization techniques.The results showed that Dare-C1qAgD was existed as a homotrimer,although it has low amino acid homology with other Clq family molecules,they possessed similar topologies.By structural analysis,we believed that the hydrophobic force was the key factor to maintain the constant structure of Clq molecules from low to high biological evolution.Further structure and sequence alignments with hC1qAgD revealed that their sequence homology was 38%,and their structural differences were mainly present in the loop.The analysis showed that the CRP binding sites on hC1qAgD were not conservative in Dare-C1qAgD,and Dare-C1qAgD lacked the IgG binding sites,which may due to that no IgG molecules were identified in fish.Then the structure of Dare-C1qgD was successfully modeled,by analyzing the surface charge differences between Dare-C1qgD and hC1qgD,we proposed that this surface chargeability may be related to the recognition mechanism of Clq molecules.In summary,the Illumina/Hiseq transcriptome sequencing platform was performed to study the metabolic pathways associated with the proliferation and activation of T and B lymphocytes in fish,which provided many important surface markers for the study of lymphocytes activation in lower vertebrates.The analysis of the crystal structure of Dare-C1qAgP protein showed that the conservatism and variability of the Clq structure in the lower vertebrates,indicating that the subtle changes of Dare-C1qAgD in the evolutionary process were closely related to the evolution of the diversity ligands.