Amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial activity of HMGB1
© Gong et al; licensee BioMed Central Ltd. 2009
Received: 21 April 2009
Accepted: 14 September 2009
Published: 14 September 2009
Antibacterial activity is a novel function of high-mobility group box 1 (HMGB1). However, the functional site for this new effect is presently unknown.
Methods and Results
In this study, recombinant human HMGB1 A box and B box (rHMGB1 A box, rHMGB1 B box), recombinant human HMGB1 (rHMGB1) and the truncated C-terminal acidic tail mutant (tHMGB1) were prepared by the prokaryotic expression system. The C-terminal acidic tail (C peptide) was synthesized, which was composed of 30 amino acid residues. Antibacterial assays showed that both the full length rHMGB1 and the synthetic C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. These results suggest that C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. Furthermore, we prepared eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1. Antibacterial assays of these mutants demonstrate that the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of the molecule.
In sum, these results define the key region and the crucial site in HMGB1 for its antibacterial function, which is helpful to illustrating the antibacterial mechanisms of HMGB1.
High-mobility group box 1(HMGB1) belongs to high-mobility group superfamily named for its characteristic rapid mobility in polyacrylamide gel electrophoresis (PAGE) . It is highly conserved across species and wildly distribute in eukaryotic cells from yeast to human [2–4]. Structurally, human HMGB1 has 215 amino acid residues, including three main functional domains (A box, B box and a highly conservation, repetitive C terminus). The C terminus is also called C-terminal acidic tail because it is full of acidic amino acid residues such as aspartate and glutamic acid [5–8].
HMGB1 is an endogenous nuclear protein that can be translocated to the cytoplasm and then released extracellularly. In the nucleus, HMGB1 binds to DNA which functions as a co-factor critical for proper transcriptional regulation and DNA repair [9–14]. Cytoplasmal and extracellular HMGB1 has been extensively related to the growth of neurite, malignant tumor metastasis, artherosclerosis and vascellum restenosis [15–23]. HMGB1 is also detected in serum of patients with sepsis. Thus, it has been identified as a late mediator of endotoxin lethality, which plays a critical role in the processes of many kinds of acute and chronic inflammation[24, 25]. Recently it is reported that the cytoplasmal and extracellular HMGB1 can act as an antibacterial factor, which is a novel function of the molecule[26, 27]. 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. However, the functional site for this new effect is presently unknown.
The goal of this study was to determine which region is mainly responsible for the antibacterial activity of HMGB1. We showed that the recombinant human HMGB1 (rHMGB1) and the synthetic C-terminal acidic tail (C peptide) presented antibacterial activity, nevertheless, the A box, B box domains of the molecule and the truncated HMGB1 lacking its C-terminal acidic tail failed to inhibit bacterial multiplication, which demonstrated that the C-terminal acidic tail is the key region for the antibacterial activity of HMGB1. We further showed that the amino acid residues 201-205 in C-terminal acidic tail region play a crucial role in antibacterial function of HMGB1. Therefore, our study defines the key region and the crucial site in HMGB1 for its antibacterial activity, which may provide important insights to antibacterial mechanisms of the molecule.
Materials and methods
Preparation of recombinant proteins and C peptide
Primers used for PCR.
Antibacterial assay with bacterial dilution in the test tubes
Briefly, the bacteria were grown overnight at 37°C in Luria-Bertani (LB) agar plate, and the fresh single clone of the bacteria was picked up and inoculated into LB liquid medium, shaking cultivated at 37°C until the optical density at 600 nm (OD600) reaching 0.4. Five microliter aliquot of the bacteria was 1:1000 diluted in each tube containing LB liquid medium, and then the tested recombinant protein or C peptide was added into different tubes at a final concentration of 0, 2, 4, 6, 8 and 10 μmol/L. After shaking cultivated at 37°C for 8 hours, the OD600 of the bacteria was measured using a spectrophotometer. Each experiment was performed in triplicate and repeated three times. DHFR protein purified by the same system was used as a negative control. The bacteria used in this experiment included Staphylococcus aureus (SA), E. coli JM109 (JM109), E. coli ATCC 25922 (ATCC 25922), E. coli DH5α (DH5α) and Pseudomonas aeruginosa (PA).
Antibacterial assay with dispersion method
Briefly, the double-layer LB agar plates were prepared (the upper layer containing 50 μl bacteria with OD600 = 0.4 and 7 g/L agar, the nether layer with agar at 15 g/L). The holes in the upper layer of the plate were made by a sterile hoop with a diameter of 5 mm. Then tested recombinant protein or C peptide was loaded into the holes (300 nmol/hole). After incubation at 37°C for 8 hours, the diameters of the zones of growth inhibition were measured as reflections of the ability of the corresponding protein or C peptide to inhibit the bacteria multiplication. DHFR protein purified by the same system was used as a negative control. The bacteria used in this experiment were same as in "Antibacterial assay with bacterial dilution in the test tubes".
Data were plotted as mean ± standard deviation and normalized relative to the negative control. Comparison between data sets obtained for different recombinant proteins and peptide were performed with one-way ANOVA using software SPSS13.0.
Preparation of recombinant proteins
C-terminal acidic tail is the key region for the antibacterial activity of HMGB1
Amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for the antibacterial activity of HMGB1
We have primarily demonstrated in this study that the C-terminal acidic tail is the key region for the antibacterial activity of HMGB1, and the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for this activity of the molecule. Moreover, the sensitivity of different bacteria to HMGB1 varies greatly.
The antibacterial activity is a novel function of HMGB1. Because of the new effect the cytoplasmal and extracellular HMGB1 have been considered an important part of innate immunity defensive barrier of the human body in physiological situation. The recent study shows that the purified testicular HMGB1 has antibacterial activity, which means this protein may function as a paracrine host defense factor in the testis[26, 27]. In addition, it has been reported that the recombinant rat HMGB1 expressed by prokaryotic system has similar antibacterial activity as native human HMGB1. However, no information is available at present as to which region in HMGB1 is mainly responsible for its antibacterial activity. In this study we identified that the C-terminal acidic tail is the key region for the antibacterial activity of HMGB1, because both the full length rHMGB1 and C peptide alone could efficiently inhibit bacteria proliferation, but rHMGB1 A box and B box, and tHMGB1 lacking the C-terminal acidic tail had no antibacterial function. Furthermore, we identified that the amino acid residues 201-205 in C-terminal acidic tail region may play a crucial role in the antibacterial activity of HMGB1. Our finding would be of helpful to define the antibacterial mechanisms of HMGB1.
Because of the antibacterial activity it is difficult to express high level HMGB1 with prokaryotic system. Previous studies have shown the difficulties in expressing the molecule in E. coli, indicating that the choice of expressing vector is crucial for the yield of recombinant HMGB1[24, 29]. It is reported that pQE-80L is suitable for expressing toxic protein because there is no leaky expression before IPTG inducing. Therefore, this vector was used in this study. Although we can get enough rHMGB1 for the in vitro experiment, the expression level is still low no matter how the expressional conditions were optimized. However the proteins without antibacterial activity could be highly expressed. Our results is consistent with the previously reports[24, 29]. This phenomenon suggests that it is necessary and important to develop novel expression system for preparing abundant rHMGB1 for the in vivo experiment. Moreover, as mentioned below, E. coli DH5α has the weakest sensitivity to HMGB1, which suggest that besides the antibacterial activity of HMGB1 there may be other reasons responsible for the low level expression of the molecule.
Different bacteria may have a varied sensitivity to HMGB1, so we carefully chose experimental bacteria strains in this study. SA is a typical gram-positive cocci genus. E. coli is a large group of gram-negative rode whose natural habitat is the intestinal tract. E. coli JM109 and DH5α are widely used in gene engineering today. E. coli ATCC 25922 is the standard stain of E. coli. PA is a medically important kind of gram-negative genus. However, PA is resistant to all tested proteins. At present we have no idea about the reasons why the antibacterial effect of HMGB1 on different bacteria is varied.
Results from this study may also suggest that it has some potential to develop antibacterial peptide based on C-terminal acidic tail. Antibacterial peptides have been proposed as novel drugs to target different bacteria, so they may have therapeutic implications for bacteria infections[32, 33]. Our results showed that C-terminal acidic tail alone (C peptide) could exert antibacterial function and the amino acid residues 201-205 in C-terminal acidic tail region plays a crucial role in antibacterial function of HMGB1, which suggested that it may be possible to design novel antibacterial peptides derived from C peptide. The C-terminal acidic tail of HMGB1 is highly conservation, which composes of 30 residues entirely of aspartic acid and glutamic acid residues. The sequence of HMGB1 amino acid residues 201-205 is EDEED which are all acidic amino acid residues but no data and references for the reason why these residues, but not other C-terminal residues, are important for HMGB1 antibacterial function so far. More studies are warranted regarding the structure basis and molecular mechanism by which HMGB1 plays antibacterial function.
This study suggests that the C-terminal acidic tail is the key region for the antibacterial activity of HMGB1, and the amino acid residues 201-205 in C-terminal acidic tail region is the core functional site for this activity of HMGB1.
List of Abbreviations
High-mobility group box 1
recombinant human HMGB1
truncated HMGB1 lacking its C-terminal acidic tail
eleven different deleted mutants lacking several amino acid residues in C-terminal acidic tail of HMGB1
- mHMGB1 -211-215:
mHMGB1 - 206-215, mHMGB1 -201-215, mHMGB1 -196-215, mHMGB1 -191-215, mHMGB1 -186-200, mHMGB1 -196-210, mHMGB1 -196-205, mHMGB1 -198-207, mHMGB1 -201-210, mHMGB1 -201-205: recombinant human HMGB1 lacking amino acid residues 211-215, 206-215, 201-215, 196-215, 191-215, 186-200, 196-210, 196-205, 198-207, 201-210 and 201-205 respectively
- C peptide:
the synthetic C-terminal acidic tail
reverse transcription-polymerase chain reaction
peripheral blood mononuclear cell
- E. coli :
This work was supported by the National Natural Science Foundation of China (No.30670411), the Medical Science Research Project of the Eleventh Five-year Plan of PLA 06H027 and the Natural Science Foundation of Chongqing CSCT 2008BB5022.
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