Effects Of The Acidic Tail On HMGB1 Antibacterial Activity

High-mobility group box 1 (HMGB1) was originally described as a nuclear non-histone DNA-binding protein which was a structural co-factor critical for proper transcriptional regulation in somatic cells. Cytoplasmic and extracellular HMGB1 had also been identified which had many physiological and pathological functions. HMGB1 is an extremely abundant protein that is widely distributed in all mammalian tissues. Structurally, HMGB1 has organized into two DNA-binding domains (named A box and B box), and a negative charged C terminus called acidic tail. Recent studies showed that antibacterial effect was a novel function of HMGB1. However, the mechanism for this activity is presently unknown. Identification of the basic structure and main domain of HMGB1 as an antibacterial factor will help us understand the mechanism of antibacterial activity of HMGB1. It was reported that native HMGB1 purified by tissue extraction from human adenoid glands without signs of inflammation revealed that HMGB1 may mediate potent, direct antibacterial function. The preliminary experiments demonstrated that recombinant human HMGB1 was expressed by the prokaryotic expression system pQE-80L/DH5αin low level, and couldn't be expressed by other prokaryotic expression system. In the mean time, recombinant human HMGB1 A box and B box (rhHMGB1 A box, B box) had no antibacterial activities. We presume that the acidic tail plays a crucial role in antibacterial activity of HMGB1. In order to prove the presumption, firstly we purified recombinant human HMGB1 A box and B box in our experiments. Secondly, the cDNA fragments encoding the human and mouse HMGB1 (human HMGB1, hHMGB1; mouse HMGB1, mHMGB1) were cloned by RT-PCR and the truncated acidic tail mutants were constructed by PCR respectively. Thirdly, the prokaryotic expression vectors containing the hHMGB1 cDNA, mHMGB1 cDNA and their mutants were transformed respectively into E.coli DH5αand induced by IPTG to express the corresponding proteins, which were purified by Ni2+-NTA chromatography. Finally, the antibacterial activities of these purified proteins were compared by bacterial dilution in the test tubes and dispersion method. The major results and conclusions are summarized as follows:...