| Bony fishes are the lower vertebrates at the nodes of immune evolution.It is the first species that possess innate and adaptive immune systems.To date,there is still a lack of substantial research on the crystal structures and the related singnaling pathway of the bony fish key immune related moleculers.The CD8 molecule is a co-receptor molecule that presents antigens for T cells and plays an important role in adaptive immune responses.Interleukin-2 is a cytokine produced by T lymphocytes after stimulation with antigens or mitogens,it has the functions of promoting the activation and proliferation of lymphocytes and enhancing the killing effect of NK cells,and plays an important role in the innate immunity of the organism.In this paper,the bony fish grass carp was studied.The structure of grass carp CD8aa homodimer was resolved.The structure of grass carp CD8aa was analyzed and great diverse among grass carp CD8aa and the known warm-blooded animal CD8aa were found.The differences mainly reflected in the following three aspects.First,the topological structure of grass carp CD8aa is unique,and its P-sheet is relatively short.Second,the composition and position of its hydrophobic core amino acids are different,and thirdly,the key amino acid F32 has an S32 substitution in grass carp,resulting in the deletion of a "hole" in the CD8 molecule of the grass carp that can accommodate the MHC I a3.LOOP region.Despite the absence of a typical "hole" conformation,affinity experiments show that grass carp CD8 molecules can still specifically bind to MHC I.Therefore,this article believes.that during the course of immune evolution,the CD8 molecule undergoes drastic changes that may that affect its dimeric structure,and may use new strategy of interaction with p/MHCI.In order to study the related signaling pathways of CD8 moleculer,using purified CD8a protein as an immunogenic,successfully prepared monoclonal antibodies against fish CD8a molecules,and then the isolated grass carp head kidney lymphocytes were cultured at 27℃ for 18 h with Con A stimulation.The prepared CD8 monoclonal antibody was used to separate the CD8+ T lymphocytes from the control and Con A stimulation groups by flow cytometry.The total RNA of CD8+T lymphocytes and control group successfully constructed the CD8+T lymphocyte transcriptome database of grass carp head and kidney.Data analysis shows that after Con A stimulation,totally 1926 differentially expressed genes were identified,including 770 up-regulated genes and 1156 down-regulated genes.KEGG enrichment analysis showed their enrichment in different KEGG pathways,and the most significant four immune-related signaling pathways were studied in detail,include TLR signaling pathway,JAK-STAT signaling pathway and Proteasome signaling pathway.In this paper,the Ctid-IL-2 gene was obtained from the CD8+T lymphocyte transcriptome database of grass carp head kidney lymphocytes,;The Ctid-IL-2 shares low homology to mammalian IL-2(<23%).then the IL-2 gene was cloned and identified.After in vitro expression,renaturation,purification,crystallization and diffraction of the protein were performed,finally,the IL-2 structure of grass carp was resolved by single wavelength anomalous scattering method.The results showed that the full length of grass carp IL-2 gene coding area is 426 bp,encoded 141 amino acids with 4 exons and 3 introns.gene synteny analysis found that the IL-2 locus was well preserved during evolution,the neighbor genes orientation of the IL-2 is same to the humans and chickens;structural analysis showed that although the conformation of Ctid-IL-2 is similar to other species,Ctid-IL-2 structure exhibits obvious species specificity:Ctid-IL-2 is more flexible in the region that binding to the receptor,in this region,the A’ helix was substituted by S1 and the B helix is straight,these lead to a significant change in the receptor binding site area,and this change disrupts the distribution of the amino acids at the receptor binding aera,and the hydrophobic core amino acid that interacts with the receptor in grass carp IL-2 also changed,which suggest that the way IL-2 binds to its alpha receptor in grass carp differs from the known way that IL-2 binds to its receptor.The transcriptome databases of head kidney lymphocytes which were dealed with or without Ctid-IL-2 was established,after stimulation,totally 474 differentially expressed genes were identified,including 119 up-regulated genes and 355 down-regulated genes.KEGG enrichment analysis showed their enrichment in different KEGG pathways,and the most significant immune-related signaling pathways,the resoults showed that IL-2 can mediates signal transduction through JAK-STAT signaling,stimulates lymphocyte differentiation and proliferation,and promotes transcription and expression of itself through AP-1 and NF-κB pathways.In summary,the crystal structures of two key immune molecules,CD8α and IL-2,of grass carp were resolved,demonstrating the conservation and diversity of key immune molecules in bony fish at the early stages of evolution;the Illumina/Hiseq transcriptome Sequencing platform was used to study the metabolic pathways related to the proliferation and activation of CD8+ T lymphocyte subsets and total lymphocyte subsets of grass carp head kidney,and the results provided important cell surface markers for studying the activation and classification of lymphocytes in grass carp. |
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