Tissue Localization And Immunological Function Analysis Of HMGB1 Protein From Lampetra Japonica

High mobility group box-1 protein(HMGB1),as a nuclear protein bound to chromatin,a highly conserved protein.HMGB1 plays a critical role at the activation of innate immune responses.The release of HMGB1 into the extracellular space can trigger inflammation.The lamprey is the representative of the oldest jawless vertebrates,Presently,the immune function of Lampetra japonica HMGB1 is still unknown.In this study,fluorescence-activated cell sorting(FACS)analysis reveals that the Lj-HMGB1 protein is expressed in approximately 39.36% of leukocytes and 46.58% of supraneural body cells,laser confocal microscopy result shows that HMGB1 is distributed in the cytoplasm of the cell,but is not in the nucleus.The immunohistochemistry result demonstrates that HMGB1 is distributed in the epithelial cells of the supraneural body,gills,intestinals and kidneys.Western blotting analysis reveals that the Lj-HMGB1 protein is expressed highly in supraneural body,Lj-HMGB1 is a secrected protein and secretes to the medium directly.HMGB1 gene expression levels is significant higher in the E.coli stimulated group than the control group by quantitative real-time PCR.The recombinant Lampetra japonica HMGB1(rLj-HMGB1)protein is induced and obtained,Super-Resolution Microscopy result showes that the Lj-HMGB1 protein and LIP protein existe in a kind of supraneural body cell,with Co-immunoprecipitation analysis of native HMGB1 protein interacts with native LIP protein,and ITC result analysis of rLj-HMGB1 protein interacts with recombinant Lamprey immune protein(LIP)protein,so Co-immunoprecipitation and ITC results show that Lampetra japonica HMGB1 protein interacts with LIP protein.Semiquantitative and Quantitative Real-time PCR(qRT-PCR)analysis reveal that the gene of MIF、IL-8 and IL-17 expression levels are lower in the rLj-HMGB1-stimulated group than the control group,and IL-8 and IL-17 gene expression levels are significant lower in the rLj-HMGB1-stimulated group than the control group in vivo and in vitro.HMGB1 in the lamprey don’t play a role in the inflammatory response.Here,we study the immune function of Lj-HMGB1.These results provide a foundation for studying the origin and evolution of the innate immune system in lampreys.