Analysis Of The Structure Of Immunomodulatory Molecule BTN2A2 And Expression In Lung Cancer And Type 1 Diabetes

Butyrophilin(BTN)molecule belongs to the immunoglobulin(Ig)superfamily and has a typical extracellular Ig-like domain.It has similarities with the B7 family members in sequence,structure and function.Therefore,the BTN molecule is considered to be an extended member of the B7 family.The BTN2A2 molecule is one of the members of the BTN family.Studies have shown that BTN2A2 can negatively regulate the immune response of T cells.However,the current research on the structure of BTN2A2 and its expression and function analysis in T cell immune-related diseases,such as tumors and autoimmune diseases(type 1 diabetes,etc.)are still limited.Therefore,on the one hand,the expression of h BTN2A2 in common tumor tissues(lung cancer,liver cancer,kidney cancer,esophageal cancer and ovarian cancer)and tumor cell lines(Ca CO2,Hela,MCF-7 and Hep G2 cells)were detected,and the relationship between h BTN2A2 and prognosis of lung cancer was analyzed with bioinformatics.On the other hand,the nucleic acid and protein expression of m BTN2A2 in pancreas and other tissues of type 1 diabetes(T1D)mice were detected.The research results illustrate the structure of BTN2A2 and its expression in T cell-related diseases,and further enrich the existing research on the understanding of BTN2A2 molecular immunomodulatory function.Objective:The aim of this study was to analyze the structure of the immunoregulatory molecule BTN2A2 and its expression in T cell immune-related diseases by using bioinformatics,immunohistochemistry and molecular biology techniques in combination with human tumor tissues,tumor cell lines and T1D mouse pancreatic tissue samples.The research results provided theoretical and experimental support for exploring the immunomodulatory function of BTN2A2 molecules.Methods:1)Sequence alignment analysis:The sequence alignment between BTN2A2 and known B7 family molecules was analyzed by Clustal W program and the phylogenetic tree was constructed.Signal peptide was predicted by Signal P 5.0,and TMHMM and Interpro were used to predict transmembrane domains and Ig-like domains.2)The 3D crystal structure model of BTN2A2 protein was constructed by I-TASSER.The domain and the amino acid residues involved in the ligand were displayed by Py Mol.3)A topology module of protein-protein interaction(PPI)with BTN2A2 was constructed using the String biological database and visualized by Cytoscape(version 3.8.0)software(National Academy of General Medical Sciences,US).The docking of BTN2A2 with other proteins was performed using ZDOCK server version 3.0.2.4)Clusterprofiler was used to investigate the functional enrichment analysis of BTN2A2 and its protein-interacting molecules,and gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathways were used to obtain relevant functional and signaling pathway annotation information.5)The fusion protein of BTN2A2-Fc was prepared,and the spleen cells of normal mice were collected and co-cultured with the fusion protein of BTN2A2-Fc for 72h in vitro.The control group was co-cultured with Fc protein.T cell proliferation(CD4~+Ki67~+,CD8~+Ki67~+)and activation(CD4~+CD69~+,CD8~+CD69~+)were detected by flow cytometry(FACS).Functionally verify whether there was an interaction between BTN2A2 protein and predicted T cells expressing its interacting molecules(CD4 and CD8).6)Human lung cancer,liver cancer,kidney cancer,esophagus cancer and ovarian cancer tissue sections were collected and immunohistochemical staining of anti-h BTN2A2 antibody was performed to observe the expression of h BTN2A2 in the above cancer tissues and corresponding normal tissues.Serum from the primary antibody source was used as negative/isotype control.Human cancer cells(Caco2,Hela,MCF-7 and Hep G2)cultured in vitro were collected,and the expression of h BTN2A2 in them was analyzed by FACS.7)Progno Scan database and Kaplan-Meier database were used to evaluate the association between BTN2A2expression and lung cancer prognosis,and TIMER and GEPIA database were used to evaluate the association between BTN2A2 expression and lung cancer immune cell infiltration.8)The spontaneous T1D model of NOD(non-obese diabetes)mice was used to detect the m RNA expression of m BTN2A2 in the tissues and organs of NOD mice before and after the onset of disease by real-time fluorescence quantitative PCR(RT-q PCR).The expression of m BTN2A2 in the pancreas of mice was detected by western blot(WB)and immunohistochemistry.Results:1)BTN2A2 shared sequence and structural homology and similarity with B7family molecules and other BTN family members,and has an IGV-like domain.The homology and similarity of h BTN2A2 and m BTN2A2 were 65%and 77%,respectively.Phylogenetic analysis showed that species with BTN2A2 and known B7family molecules were phylophilic.2)The 3D crystal structure model of BTN2A2protein was constructed by I-TASSER.Pymol showed that h BTN2A2 ligand was CYS,and the corresponding amino acid residues at the ligand binding sites were ARG-54,HIS-56,SER-111,VAL-112 and ALA-113,respectively.The m BTN2A2ligand was DHL,and the corresponding amino acid residues of the ligand binding site were ARG-345,GLU-381,LYS-383,GLY-384 and PHE-394.3)The protein-protein interaction network screened out 10 BTN2A2-related molecules,of which CD4 and CD8 were associated with T cell immunity.Molecular docking analysis of Zdock Server Ver 3.0.2 showed that BTN2A2 could indeed bind to CD4 and CD8 molecules on the surface of T cells.4)Cluster Profiler analysis showed that BTN2A2 and related molecules were mainly concentrated in primary immunodeficiency,antigen processing and presentation,T cell receptor signaling pathway and other immune regulation.5)FACS analysis further showed that BTN2A2 protein interacted with CD4 and CD8 molecules on the surface of T cells,and BTN2A2 protein inhibited the proliferation and activation of CD4~+and CD8~+T cells.6)Immunohistochemical staining showed that the expression of h BTN2A2 in human lung,liver,kidney and esophageal cancer tissues was higher than that in normal tissues,but no expression in ovarian cancer tissues.FACS analysis showed that h BTN2A2 was expressed in human Ca CO2,He LA,Hep G2 and MCF-7 cancer cells.7)Progno Scan database and Kaplan-Meier curve for lung cancer patient survival analysis showed that patients with high expression of h BTN2A2 in lung cancer had a good prognosis.TIMER database analysis showed that the expression level of h BTN2A2 in lung cancer was positively correlated with immune cell infiltration.GEPIA database analysis showed that the expression of h BTN2A2 in lung cancer was mainly related to the infiltration of immune cells such as CD8~+T,CD3~+T cells,dendritic cells(DC),and Th 1.8)RT-q PCT,WB and immunohistochemistry showed that after T1D in NOD mice,the expression of m BTN2A2 in pancreatic tissue was significantly higher than that before the onset,and the difference was statistically significant(P<0.001).Conclusions:1)The 3D crystal structure model of BTN2A2 protein was successfully constructed.The ligand of h BTN2A2 was CYS,and the ligand of m BTN2A2 was DHL.2)BTN2A2 could play an immunomodulatory function by interacting with T cell surface molecules such as CD4 and CD8.3)In T cell immune-related diseases such as lung cancer and T1D,BTN2A2 may enhance anti-tumor immunity by promoting immune cell infiltration in tumor tissues.Or adaptively increased the expression of BTN2A2 in the pancreatic tissue of the T1D damaged target organ,thereby inhibiting the over-activated T cell immune response and reducing autoimmune damage.