Analysis Of Antibacterial-associated Functional Sites In Human HMGB1 Acidic Tail And Anti-inflammation Strategies Against HMGB1

Part I Analysis of antibacterial-associated functional sites in human HMGB1 acidic tailObjective: High-mobility group box 1(HMGB1) belongs to high-mobility group superfamily named for its characteristic rapid mobility in polyacrylamide gel electrophoresis (PAGE). Structurally, HMGB1 has 215 amino acid residues, including three main functional domains: A box, B box and C-terminal acidic tail (C tail). HMGB1 has been shown multiple important functions in nucleus, cytoplasm even extracellular enviroment. In physiological situation, as a potent antibacterial protein, HMGB1 is an important part of innate immunity defensive barrier of the human body that can resist bacterial infection in vivo. But the functional domain for this new effect is presently unknown. In our previous reseaches, we identified that the full-length recombination human HMGB1 presented antibacterial activity, nevertheless, the A box and the B box domains of the molecule and the truncated HMGB1 lacking its C tail failed to inhibit bacterial multiplication, which demonstrated that the C tail was the key domain for the antibacterial activity of HMGB1. But there are still remained some futhur reseaches such as which region in C tail is mainly responsible for the antibacterial activity of HMGB1, and whether the acidic tail alone peptide has antibacterial function.Methods and Results: The encoding fragements of eleven different deleted mutants in C tail of human HMGB1 were constructed by PCR or one-step opposite-direction PCR, which were laking its amino acid residues 211-215, 206-215, 201-215, 196-215, 191-215, 186-200, 196-210, 196-205, 198-207, 201-210, 201-205 respectively. They were consistent with the sequence reported in GenBank. These mutants were successfully expressed in the prokaryotic expression systerm. The relative molecular masses of them were about from 30kDa to 25kDa. The expressed proteins were purified by Ni2+-NTA chromatography. SDS-PAGE analysis showed that the target proteins were highly purified. The acidic tail peptide alone (C peptide) was synthesized. The antibacterial activities of these mutants and C peptide to Staphylococcus aureus (SA), E.coli (JM109, ATCC25922, DH5α), P. aeruginosa (PA) were compared by bacteria dilution in the test tubes and dispersion method. The results showed that the truncated mutants which lacking its amino acid residues 211-215, 206-215, 186-200 respectively and C peptide could inhibit the proliferation of SA, E.coli.JM109, ATCC25922 and DH5αefficiently in vitro. However, other mutants had no antibacterial function. Moreover, all of the mutants and C peptide couldn't inhibit PA growth.Conclusion: We concluded that the amino acid residues 201-205 in C-terminal acidic tail region are the core functional site for the antibacterial activity of the molecule. And C peptide could exert antibacterial function. Part II Analysis of anti-inflammation strategies against HMGB1Objective: HMGB1 has been identified as a mediator of endotoxin lethality and become a new therapeutic target in recent years, which plays a critical role in the processes of many kinds of acute and chronic inflammation, including autoimmune diseases such as rheumatoid arthritis (RA), systemic lupus erythematosus (SLE), Sj?gren Syndrome (SS) and so on. B box of HMGB1 is the inflammation functional domain. Interestingly, HMGB1 A box can antagonize HMGB1-induced inflammation. Moreover, the C tail contributed to the spatial structure of both A box and B box and regulated HMGB1 DNA binding specificity. However, it is unknown whether the C tail can enhance the anti-inflammatory activity of the A box, and whether the fusion proteins based on the A box and C tail have antibacterial and anti-inflammation activity. Then in order to investigate whether the novel fusion molecules have antibacterial activity and anti-inflammation activity, which consist of the human HMGB1 antibacterial acidic tail and A box possessing anti-inflammation action, we prepared two fusion molecules consisting of the human HMGB1 A box and acidic tail linked directly and by a flexible linker sequence (Gly4Ser)3 respectively. The effects of these fusion molecules were compared by antibacterial and anti-inflammation assays in vitro and in vivo.Sinominine, a purified alkaloid extracted from sinomenium acutum, has a variety of pharmacological effects including anti-inflammation, immunosuppression, and anti-angiogenesis, which is treated to RA, SLE. It is not sure whether sinominine can influence the expression of HMGB1. Finally, we tested whether sinomenine inhibits the expression of HMGB1.Methods and Results: The fusion molecules consisting of the human HMGB1 antibacterial acidic tail and A box possessing anti-inflammation action linked directly and by a flexible linker sequence (Gly4Ser)3 were successfully prepared. The relative molecular masses of the two fusion molecules were about 16kDa and 18kDa. Antibacterial assays showed that these two molecules had equal abilities to inhibit the proliferation of SA, E.coli.JM109, ATCC25922 and DH5αin vitro. They could inhibit TNF-a and IL-6 release in vitro and in vivo and increase the survival rate of endotoxemia mouse. And the flexible linker fusion molecule had more powerful anti-inflammation activity. We also detected that sinomenine could inhibit both basal and LPS stimulated HMGB1 expression in human endothelial cell EA.Hy926 by Real-time PCR and Western blotting. Morover, we cloned the promoter of HMGB1. Luciferase assay showed that sinomenine could suppress the transcription activity of HMGB1 promoter and tightly related with -1198～-798 of the promoter region.Conclusion: In summary, the novel fusion molecules have antibacterial and anti-inflammation activity. The fused C tail can enhance the anti-inflammatory effect of the A box. And sinomenine could inhibit HMGB1 expression by suppressing the transcription activity of -1198～-798 in HMGB1 promoter region.