Regulation Mechanism Of Protein 4.1R On B Cell Activation

Background:B cells are the dominant cells of humoral immunity.Mature B cells activation after being stimulated by antigens and then exert apply immune response functions.This process is involved in multiple molecules and each step is precisely regulate.Under normal physiological conditions,B cells participate in a series of immune responses to maintain physical health status.While in pathological conditions,abnormal B cell activation has closely related to the pathological damage of many diseases.The research on the regulation and mechanism of B cell activation is ongoing.Discovering and identifying new B cell activation regulators and explaining their mechanism,is the basis of immunological research,and it is also an urgent need to understand the pathogenesis of related diseases that caused by abnormal B cell activation.The first step in B cell activation is the recognition of the antigen by receptors on the cell membrane,then B cells are activated through membrane receptor-mediated signals to exert immune functions.Membrane receptors,such as Toll-Like Receptor 4,(TLR4),B Cell Receptor(BCR),and the costimulatory molecule CD40 play a significant role in the process of B cell activation.They are independent of each other but also coordinated to promote the complete activation of B cells.When lipopolysaccharide(LPS)stimulates B cells,it simultaneously binds to TLR4 and BCR to activate TLR4 and BCR signaling pathways,leading to B cell activation.Bacterial capsular polysaccharides can cross-link multiple BCR and continuously activate the BCR signaling pathway,then lead to B cell activation.Soluble protein antigens require BCR and CD40 receptors to provide activation signals to activate B cells.Recently,more and more studies have found that in B cell activation,membrane skeleton proteins play important role in B cell activation and immune response by regulating the transduction of membrane receptor signals.Protein 4.1R was the first to be discovered in the mature erythrocyte.In mammals,4.1R has two spliceosomes of 135 kDa and 80 kDa,they are extensively colocalized with transmembrane proteins and affect disease progression by regulating the activation of immune cells such as T cells and mast cells.However,the role of 4.1R in B cell activation and its effect on B cell immune response are still unclear.Method:(1)Expression and localization of protein 4.1R in B cellsCD 19 microbeads were used to obtain high-purity mature B cells.Then,amplifying the protein 4.1R gene sequence by RT-PCR and connecting it to the pMDTM19-T vector.Next,sequencing and analyzing its exon composition.Immunofluorescence(IF)and Western bolt(WB)are used to detect the localization and expression of protein 4.1R in B cells,respectively.(2)Proteomic analysis of 4.1R+/+and 4.1R-/-B cellsWe use proteomics sequencing technology to detect the difference proteins between 4.1R+/+and 4.1R-/-B cells.And bioinformatics is used to analyze the role of 4.1R in B cells,which provide significant clues to the function of protein 4.1R in B cell activation and response.(3)The influence of protein 4.1R on TLR4-mediate B cell activationFlow cytometry,ELISA,WB and qRT-PCR were performed to detect B cell activation,proliferation,Ig class switch recombination(CSR),antibody secretion,and apoptosis when stimulated by LPS.IF,Co-Immunoprecipitation(co-IP)and WB to assess the regulatory mechanism of 4.1R on TLR4-mediate B cell activation.(4)The influence of protein 4.1R on BCR-mediate B cell activationThe phosphoproteomics analysis was used to explore the influence of 4.1R on phosphorylation of BCR signaling pathway with the stimulation of anti-IgM.Then,flow cytometry,ELISA,WB was used to verify the phosphoproteomics results.GPS Kinase was used as prediction software to predict the key kinases.WB was used to verify the results of kinase prediction.IF was used to detect the interaction of protein 4.1R with B cell-related membrane receptors.(5)The influence of protein 4.1R on CD40-mediate B cell activationWe use CD4+T cell exosomes as stimulator to assess B cells activation,proliferation,CSR,and antibody secretion.Flow cytometry was used to detect B cell endocytosis and CD40 expression level;WB to investigate CD40 downstream signaling pathways.Results:(1)Expression and localization of protein 4.1R in B cellsThis study found that two spliceosomes of 135 kDa and 80 kDa were highly expressed in B cells,mainly distributed on the cell membrane.By analyzing the composition of exons,we found that the number of 3,14,15,and 16 of the two isoforms were missing.(2)Proteomic analysis of 4.1R+/+ and 4.1R-/-B cellsThrough the analysis of the difference in B cell protein expression,compared with 4.1+/+ B cells,there were 100 proteins up-regulated and 47 proteins down-regulated in 4.1R-/-B cells.Differential proteins mainly regulated B cell activation,proliferation,vesicle transport,immune response,and other biological processes,which also played a regulatory role in activation,proliferation,apoptosis,endocytosis,and B cell immune response.This part of the results provided important clues for studying the role of protein 4.1R in B cell activation and signal transduction.(3)The influence of protein 4.1R on TLR4-mediate B cell activationUnder LPS-stimulated conditions,B cells can be activated by inducing the TLR4 signaling pathway.Our results showed that compared with 4.1R+/+ B cells,4.1R-/-B cells have significantly enhanced the capabilities of activation,proliferation and IgGl class switch recombination.But the plasma cell differentiation and antibody secretion level have been decreased.Then we found that the activation of the Canonical NF-κB signaling pathway in 4.1R-/-B cells was enhanced.And the over-activated Canonical NF-κB broke the balance between pro-apoptotic and anti-apoptotic proteins,causing excessive cell apoptosis.Further study showed that protein 4.1R could regulate the TLR4-mediated canonical NF-κB pathway,thereby influence activated B cell fate.(4)The influence of protein 4.1R on BCR-mediate B cell activationBCR is one of the most abundant receptors on the surface of B cells.To understand the effect of protein 4.1R on BCR-mediated B cell activation,we used anti-IgM to stimulate B cells to induce the activation of the BCR signaling pathway.And we predicted the effect of protein 4.1R on the BCR signaling pathway through phosphoproteomics analyzing.We identified 174 differentially modified proteins were up-regulated,and 33 were down-regulated.There were 225 up-regulated and 39 downregulated in differentially modified sites.The proteins corresponding to differentially modified sites play a role in B cell activation,proliferation,and constructing immune synapses,which relate to its functions such as regulating BCR signaling pathways and immune responses.Our results showed that compared with 4.1R+/+B cells,4.1R-/-B cells have more powerful activation and proliferation ability.Then we found that 4.1R could inhibit BLNK phosphorylation and negatively regulate B cell activation by colocalized with the intracellular adaptor protein BLNK.Through GPS kinase prediction analysis,the results showed that protein 4.1R could inhibit the phosphorylation of downstream AKT and reduce BCR-mediated B cell proliferation by colocalized with CD 19.(5)The influence of protein 4.1R on CD40-mediate B cell activationWe used exosomes secreted by activated CD4+ T cells to stimulate B cells.The results showed that the ability of 4.1R-/-B cells to phagocytose exosomes was weaker than that of 4.1R+/+ B cells,but the abilities of activation,proliferation,antibody type conversion and antibody secretion were significantly enhanced.Then we found that the expression level of CD40 receptor on the membrane surface of 4.1R-/-B cells was higher than that of 4.1R+/+ B cells.And the phosphorylation of P38 and AKT in the downstream signaling pathway of CD40 in 4.1R-/-B cells was also significantly enhanced.Conclusion:(1)Protein 4.1R regulates the canonical NF-κB pathway through interacting with TLR4,that affects in fate determination of activated B cells.(2)Protein 4.1R interacts with BLNK kinase in the BCR signaling pathway to inhibit B cell activation and down-regulate B cell proliferation by inhibiting CD19mediated phosphorylation of AKT.(3)Protein 4.1R regulates B cell activation and immune response by inhibiting the phosphorylation of downstream signals P38 and AKT mediated by CD40 molecules.In summary,this study investigates the role of protein 4.1R in B cell activation mediated by TLR4,BCR and CD40 to clarify the significant regulatory functions and mechanisms of protein 4.1R on B cell activation and immune response,which provide new ideas and scientific basis for deeply exploring the mechanism of B cell activation and the pathogenesis of diseases related to abnormal B cell activation.