Published online before print October 4, 2005
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Research Unit of Infection and Immunity, West China Medical Center, Sichuan University
1Correspondence: Research Unit of Infection and Immunity, West China Medical Center, Sichuan University, Chengdu, Sichuan 610041, PR China. E-mail: wangby{at}mail.sc.cninfo.net
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-helical domain had the same antimicrobial activity as HMGN2. The immunocytochemistry staining, enzyme-linked immunosorbent assay, and Western blot revealed that HMGN2 was present in the cytoplasm of mononuclear leukocytes and released to the extracellular environment when stimulated with IL-2. These results suggest that HMGN2 would be a novel antimicrobial effector molecule of human mononuclear leukocyte.
Key Words: antimicrobial activity • -helical domain • subcellular distribution
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Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antiviral defense. In addition, CTL demonstrate activity against intracellular pathogens [2
]. Several APP have been described in human NK and CTL cells. Granulysin is a member of the saponin family of membrane-active peptides with activity against a range of bacteria [3
, 4
]. NK-lysin is an interleukin (IL)-2-inducible, 78 amino acid (aa) porcine homologue of granulysin [5
]. Cationic helical motifs and a disulfide-constrained loop are important determinants of NK-lysin and granulysin’s antimycobacterial activity [4
, 5
]. Agerberth and co-workers [6
] have discovered that freshly isolated lymphocytes grown in vitro in the presence of IL-2, including 
T cells and NK cells, express and secrete -defensins [human neutrophil peptide 1–3 (HNP1–3)], the cathelicidin peptide LL-37, lysozyme, and a fragment of histone H2B. Further, Grimm et al. [7
] found that peripheral blood mononuclear leukocytes activated by IL-2 had strong antitumor activities.
In our study, an antimicrobial polypeptide was isolated and characterized from human peripheral blood mononuclear leukocytes in the presence of IL-2. Its N-terminal amino acid sequence was identical to high mobility group nucleosomal-binding domain 2 (HMGN2). The mass spectrometric value and Western blot also indicated its individual character of HMGN2, which is a highly conserved nucleosomal protein thought to be involved in unfolding a higher-order chromatin structure and facilitating the transcriptional activation of mammalian genes [8
]. However, our present understanding of the physiological role of HMGN2 is in its infancy. We further bioexpressively or chemosynthetically prepared HMGN2 holo-molecule, -helical domain, N-terminal, and C-terminal fragments to determine its antimicrobial spectrum and antimicrobial domain and produced HMGN2 polyclonal antibody to examine its subcellular location.
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Biochemical analysis and purification techniques
Protein concentrations were measured according to the bicinchoninic acid (BCA) protein assay (Pierce, Rockford, IL) using bovine serum albumin (BSA) as a standard. Acid-urea polyacylamide gel electrophoresis (AU-PAGE) [9
] and tricine-sodium dodecyl sulfate (SDS)-PAGE [10
] were performed in mini-gel formats, using the Modular Mini-Protein II electrophoresis system (Bio-Rad, Hercules, CA) and stained with 0.1% Coomassie brilliant blue R-250 or with silver [11
]. All reagents were obtained from Sigma Chemical Co. unless otherwise noted. Molecular weight standards were from Bethesda Research Laboratories (MD).
The purification included preparative AU-PAGE elution and reverse-phase high-performance liquid chromatography (RP-HPLC) on a 4.6 x 250-mm Vydac C18 column, eluted with a 0–60% linear gradient of solvent B [0.1% trifluoracetic acid (TFA), 60% actonitrile, 40% water] in 60 min at a flow rate of 1.0 ml/min. The column effluents collected at every minute were lyophilized.
Sequencing was performed on a 477A Protein Sequencer (Applied Biosystems) according to the Edman degradation procedure.
Mass spectrometric measurement was performed by Shanghai Genecore Biotechnologies Company (China). The method is as follows: Samples were eluted in 1 ml 50% (v/v) acetonitrile/0.1% TFA and mixed 1:1 with a saturated solution of Sinapinic acid in 50% (v/v) acetonitrile/0.3% TFA. Samples (1 µl) were applied to a stainless steel 96 x 2 target matrix-assisted laser desorption-ionization plate and air-dried before analysis in the mass spectrometer, where mass spectrometry was performed using an Applied Biosystems Voyager DE-PRO (ABI, Foster City, CA), equipped with a nitrogen laser (337 nm, 3 ns pulse width, 3.0 Hz REP rate). Mass spectra was acquired in the liner-positive mode with an accelerating voltage of 20 Kv, a grid voltage setting of 95%, and a 400-ns delay using 
150 laser shots.
Escherichia coli-based production of recombinant human (rh)holo-HMGN2 and its -helical domain
Total RNA was isolated with Trizol reagent (Gibco-BRL, Grand Island, NY) from the stimulated mononuclear leukocytes. The full-length HMGN2 cDNA was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) and ligated into a pMD-18T vector (TakaRa, Japan) for DNA sequencing. Generation of cDNA of holo-HMGN2 and the HMGN2 -helical domain was carried out by PCR amplification. Primers containing BamHI and EcoRI restriction sites were designed as follows: P1 (5'-ACGGATCCCCCAAGAGAAAGGCTG-3') and P2 (5'-TAGAATTCCTTGGCATCCTCCAGCAC-3') for amplifying holo-HMGN2 cDNA and P3 (5'-CAGGATCCAAGGACGAACCACAG-3') and P4 (5'-GCGAATTCCTTCTTTGCAGGGGCCT-3') for synthesizing DNA encoding the HMGN2 -helical domain. BamHI and EcoRI restriction sites are underlined. After digestion with BamHI and EcoRI, the PCR products were inserted into the pGEX-1
T vector (Amersham Biosciences, Uppsala, Sweden). DNA sequencing of the recombinant prokaryotic expression vectors pGEX-1T-HMGN2 and pGEX-1T-HMGN2 was carried out to confirm the insert sequences.
The transformed E. coli JM109 carrying pGEX-1T-HMGN2 and pGEX-1T-HMGN2 was cultured in Luria-Bertani medium for 12 h in the presence of isopropylthio-ß-D-galactoside (IPTG; Sigma Chemical Co.) to induce protein expression. The induced cultures were washed with PBS, and cell lysates were obtained by freezing/thawing in the presence of lysozyme. After centrifugation, the glutathione S-transferase (GST)-HMGN2 and GST-HMGN2 -helical domain fusion proteins were purified from the supernatants using a bulk glutathione Sepharose 4B column (Amersham Biosciences). The purified fusion proteins were cleaved by thrombin digestion. The recombinant holo-HMGN2 and its -helical domain were obtained by AU-PAGE elution and HPLC purification.
Synthetic peptide
Synthetic N- and C-terminal fragments and the -helical domain of HMGN2 were prepared by Shanghai Genebase Gen-Tech (China). Their amino acid sequences are as follows: Fragment 1. MPKRKAEGDAKGDKAKV (position 1–17 of the HMGN2 amino acid sequence); Fragment 2. KDEPQRRSARLSAKPAPPKPEPKPKKAPAKK (position 18–48 of the HMGN2 amino acid sequence); Fragment 3. GEKVPKGKKG KADAGKEGNNPAENGDAKTDQAQKAEGAGDAK (position 49–90 of the HMGN2 amino acid sequence).
HPLC and mass spectrometric analysis of these peptides revealed a purity of >95%. The peptides were dissolved in 10 mM potassium phosphate buffer (pH 7.0) to a final concentration of 10 mg/ml.
Antimicrobial testing
The agar radial diffusion assay and gel overlay techniques, developed by Lehrer et al. [12
], were used to identify the antimicrobial proteins. Underlay bacterial agars (10 ml) contained 1% agarose (Sigma A-6013), 0.3 mg/ml trypticase soy broth, 10 mmol phosphate buffer (pH 7.2), and 1 x 105 mid-logarithmic-phase bacteria. Overlay nutrient agars (10 ml) contained 30 g/L tripticase soy broth (Sigma Chemicla Co.) and were poured on the underlay agars after 3 h incubation of the latter. After overnight incubation at 37°C, the clearing zones were observed. In the radial diffusion assay system, antimicrobial activity was determined as a clear zone around the well.
The minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of the peptides were examined using bacteria at 1 x 106 colony-forming units (CFU)/ml in the trypticase soy broth and serial dilutions of the peptides (500, 250, 200, 150, 100, 50, 25, 12.5, and 6.25 mg/L). Inhibition of growth was determined by measuring optical density (OD) at 492 nm on an ultraviolet/visible absorption spectroscopy spectrometer after incubation at 37°C for 12–16 h. Antimicrobial activity was expressed as the MIC, the concentration at which 100% inhibition of growth was observed, and the MBC, the concentration at which no CFU were observed after incubation for 12–16 h on soy broth (for growing bacteria, E. coli ML-35p, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 25923) or sabouraud dextrose broth (for growing, Candida albicans ATCC 10231) solid media. The testing microbes included E. coli ML-35p, P. aeruginosa ATCC 27853, S. aureus ATCC 25923, and C. albicans ATCC 10231.
Rabbit neutrophil petide defensin-1 (NP-1) and HNP1–3 were used as the control antimicrobial peptides and were prepared as described elsewhere [13 , 14 ].
Analysis of HMGN2 subcellular distribution
Preparation of antiserum against the GST-HMGN2 fusion protein [15
]: Antibodies against purified GST-HMGN2 were raised in a New Zealand white rabbit, which was subcutaneously (s.c.) injected with 1 mg antigens mixed with an equal volume of Freund’s complete adjuvant. Every 2 weeks, 0.5 mg antigens mixed with an equal volume of Freund’s incomplete adjuvant were given s.c. for a total of four times. Antigen (0.5 mg) in PBS was given intravenously 1 week before the rabbit was killed. Serum samples collected from the rabbit 1 week after the last injection were assayed. The antiserum titer was determined with an enzyme-linked immunosorbent assay (ELISA) using purified HMGN2, coated onto a microplate (Eurogentec, San Diego, CA).
Immunofluorescence staining of HMGN2: The mononuclear leukocytes were cultured in RPMI-1640 medium in the presence or absence of rIL-2 (100 U/ml) and PHA (100 µg/ml) for 2 days or 8 days and then centrifuged and washed with PBS. The cells were fixed with 4% paraformaldehyde for 20 min and then blocked with 5% normal goat serum for 20 min. The cells were incubated first in HMGN2 polyclonal antibody (1–500 diluted) or preimmunized serum for 60 min, then in biotinylated goat anti-rabbit immunoglobulins (Ig; 1–300 diluted) for 45 min, and finally, in fluorescein isothiocyanate (FITC)-conjugated streptavidin (1–1000 diluted) for 15 min. Glycerin alkaline buffer (50%) was used to seal the slides. Photographs were taken using a laser confocal fluorescent microscope (Bio-Rad).
ELISA: An ELISA procedure [16 ] was used to detect the presence of HMGN2 in the culture supernatant of mononuclear leukocytes in the presence of IL-2. Each well was coated with 100 µl culture supernatant in 0.1 M carbonate buffer (pH 9.6) at 4ºC overnight. Plates were washed three times with PBS/Tween-20 buffer (pH 7.4), and nonspecific binding sites were blocked by incubation with 1% BSA for 1 h at room temperature. After washing, primary rabbit anti-HMGN2 antibody or preimmunized serum (1:500 diluted) was added to each well and incubated at room temperature for 1 h. Biotinylated goat anti-rabbit IgG (100 µl; 1:5000 diluted) in PBS/Tween-20/1% BSA buffer was added to each well after repeating washing and incubated for 1 h at room temperature, and then, H2O2 and o-phenylenediamine (0.4 mg/ml; Sigma Chemical Co.) as a substrate were added and incubated for 20 min. Reaction was stopped by adding 2 N H2SO4, and absorbance was read at 490 nm on a microplate reader (Bio-Rad).
Western blot: Culture supernatants (10 ml) were collected and freeze-dried. The protein concentration was determined by the BCA reagent. The whole soluble proteins were run on a 15% SDS-PAGE at 80 v for 3 h and blotted onto polyvinylidene difluoride membranes in transfer buffer at 30 v overnight at 4ºC. The membranes were blocked in 1x Tris-buffered saline (TBS), 5% (w/v) nonfat dried milk, and 0.1% Tween-20 (v/v) for 2 h at room temperature. The 1:500 dilution of anti-HMGN2 polyclonal antibody in primary antibody buffer (1xTBS, 5% BSA, 0.1% Tween-20) was added and incubated for 1 h at room temperature. The membrane was then incubated with goat anti-rabbit IgG-horseradish peroxidase secondary antibody at 1:5000 blocking buffer for 1 h at room temperature with gentle agitation. Visualization was performed using the 3,3'-diaminobenzidine method.
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Figure 1. AU-PAGE of acid-soluble proteins from IL-2-activated human mononuclear leukocytes. The acid-soluble proteins (30 ng) were subjected to AU-polyacrylamide mini-gel (0.75x100x70 mm) electrophoresis at a constant 150 V for 45 min. The gel was stained with 0.1% Coomassie brilliant blue R-250.
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Figure 2. HPLC of HLP3. AU-PAGE elute of HLP3 was subjected to RP-HPLC on a 4.6 x 250-mm Vydac C18 column and eluted with a 0–60% linear gradient of solvent B (0.1% TFA, 60% actonitrile, 40% water) in 60 min at a flow rate of 1.0 ml/min. Arrow indicates the antimicrobial fraction HLP3–21. (A Inset) Tricine-SDS-PAGE of HLP3–21 with silver stain. (B Inset) Agar radial diffusion assay indicated the antimicrobial activity against E. coli ML-35p (twofold serial dilutions of 500 µg/ml HLP3–21). mAU, .
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Figure 3. Mass spectrum identification of HLP3–21. Mass spectrometric analysis of HLP3–21 revealed the same molecular mass (m/z=9274.04) as HMGN2.
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-helical domain-helical structure, the putative antimicrobial domain, located from position 18 to 48 of the HMGN2 protein sequence (Fig. 4
). To further examine the antimicrobial property of HMGN2, we prepared its recombinant holo-molecule and the -helical domain. The cDNA encoding holo-HMGN2 and its helical domain were obtained by RT-PCR, and their corresponding prokaryotic expression vectors were constructed. Sequence analysis indicated that the insert sequences and their orientation were correct in the recombinant vector. The recombinant vectors pGEX-1T-HMGN2 or pGEX-1T-HMGN2-transformed E. coli produced a bulk amount of HMGN2 and HMGN2 fusion proteins, which were purified by GST affinity chromatography. The purified recombinant holo-HMGN2 and its -helical domain were obtained using AU-PAGE elution from thrombin-digested fusion proteins and RP-HPLC (Fig. 5
).
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Figure 4. OMIGA protein structure software analysis of the transmembrane helices structure of HMGN2
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Figure 5. Tricine-SDS-PAGE of recombinant HMGN2 and its -helical domain. cDNAs encoding HMGN2 and its -helical domain plus BamHI and EcoRI restriction sites were obtained by PCR, and the recombinant prokaryotic expression vectors pGEX-1T-HMGN2 and pGEX-1T-HMGN2 were constructed. After IPTG induction, the fusion proteins were purified by GST affinity chromatography from the transformed E. coli. The purified recombinants were obtained by AU-PAGE elution from thrombin-digested fusion proteins and RP-HPLC and subjected to Tricine-SDS-PAGE. Lanes 1 and 4 represent whole protein extract of E. coli transformed with pGEX-1T-HMGN2 or pGEX-1T-HMGN2. Lanes 2 and 5 represent the purified GST-HMGN2 or GST-HMGN2-helical domain fusion proteins. Lanes 3 and 6 represent the purified recombinant HMGN2 and its -helical domain, respectively.
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-helical domain to further examine the antimicrobial structure. The agar radial diffusion assay indicated that the -helical domain of HMGN2 had a strong antimicrobial activity against E. coli ML-35p. In contrast, no antimicrobial activity was observed for its N-terminal or C-terminal fragments using this assay system (data not shown).
As shown in Table 1
, the MIC and MBC assays indicated that the rhHMGN2 and its transmembrane -helical domain (synthetic and recombinant) had potent antimicrobial activity against E. coli ML-35p, P. aeruginosa ATCC 27853, and to some extent, against C. albicans ATCC 10231. However, HMGN2 was inactive against S. aureus in this system (data not shown).
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Table 1. Antimicrobial Activities of Several Peptides
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The immunofluorescene staining indicated that HMGN2 located mainly in the nucleus of the resting cells. In contrast, a strong HMGN2 signal appeared in the cytoplasm of the stimulating human mononuclear leukocytes (Fig. 6 ). Futher, there were no special signal observed under the laser confocal fluorescent microscope when preimmunized serum was used. The extracellular release of HMGN2 from the stimulated human mononuclear leukocytes was detected by ELISA and Western blot (Fig. 7 ).
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Figure 6. Immunofluorescence staining showing HMGN2 distribution in mononuclear leukocytes in the presence or absence of IL-2. The mononuclear leukocytes were isolated from a healthy person by Hypaque-Ficoll gradient centrifugation and cultured in RPMI-1640 medium in the presence or absence of rIL-2 (100 U/ml) and PHA (100 µg/ml) for 2 days. The cells were fixed with 4% paraformaldehyde for 20 min and then incubated with affinity rabbit anti-HMGN2 polyclonal antibody, then with biotinylated goat anti-rabbit Ig, and finally, with FITC-conjugated streptavidin. (A and B) Light micrographs of hematoxylin and eosin staining of resting human mononuclear leukocytes, and IL-2-activated mononuclear leukocytes (x400). (C and D) Photographs were taken using a laser confocal fluorescent microscope (x600). (C) The immunofluorescence staining of HMGN2 in resting human mononuclear leukocytes (x600); (D) the immunofluorescence staining of HMGN2 in human mononuclear leukocytes in the presence of IL-2 (x600).
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Figure 7. Immunological detection of HMGN2 in the culture supernatant of mononuclear leukocytes in the presence of IL-2. The mononuclear leukocytes were isolated from a healthy person by Hypaque-Ficoll gradient centrifugation and cultured in RPMI-1640 medium in the presence or absence of rIL-2 (100 U/ml) and PHA (100 µg/ml). The supernatant was collected and dialyzed against water at 4ºC for 48 h, lyophilized, and stored at –70ºC for ELISA and Western blot analysis. (Left) ELISA detection of HMGN2 in the supernatant of mononuclear leukocytes cells, n = 3. (Right) Western blot of HMGN2 in the supernatant of mononuclear leukocytes cells. Lane 1, Supernatant of resting mononuclear leukocytes. Lanes 2 and 3, Supernatants of 2-day-cultured and 8-day-cultured mononuclear leukocytes in the presence of IL-2, respectively. Lane 4, Purified HMGN2.
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The nucleosomal-binding domain is the functional motif of the HMGN subfamily [8 ]. The functional gene is located in chromosome 1p36.1 [22 , 23 ], and it contains six exons, with an extremely high GC content and an "HpaII tiny fragment" island, indicative of a housekeeping gene that could be crucial for the regular functioning of cells [24 , 25 ]. However, until now, the biological role of this protein has not been defined fully. A variety of experiments have shown that HMGN2 are preferentially associated with chromatin subunits containing transcribed genes and enhance the transcriptional potential of corresponding genes [26 27 28 29 ]. Furthermore, the abnormal gene or protein expression of HMGN2 is related to some diseases such as neoplasms [30 , 31 ] and autoimmune diseases [32 33 34 ]. The significance of HMGN2 in the host defense against infection is unclear. Frohm et al. [35] attempted to identify antimicrobial polypeptides from human wound and blister fluid. Several known antimicrobial peptides or proteins, e.g., defensins HNP1–3, lysozyme, LL-37, and histone H2B fragments, were found. Although HMGN2 was isolated, its antimicrobial property was not determined. In our study, we found that HMGN2 had an antimicrobial activity, and this made us think that the physiological role of HMGN2 is not so simple.
As early as in 1982, Grimm et al. [7 ] found that peripheral blood mononuclear leukocytes activated by IL-2 had strong antitumor activities. Culture of peripheral blood mononuclear leukocytes in the presence of IL-2 for 2–3 days caused the expression of lymphokine-activated killers (LAK) and lytic activity toward a variety of NK-resistant fresh and cultured tumor targets. Their data present evidence that the LAK system is a phenomenon distinct from NK or CTL systems. In our study, the immunocytochemistry, ELISA, and Western blot analysis revealed that HMGN2 was not only present in the nucleus but also showed up in the cytoplasm of mononuclear leukocytes and released to the extracellular environment when activated with IL-2. Thus, we presume that HMGN2 would be released positively from the activated mononuclear leukocytes to combat invading bacteria or perform other biological functions, which remain to be seen, rather than a tissue damage event.
There has been another interesting finding about HMGN2. Porkka et al. [36
] used phage-displayed cDNA libraries in vivo to search for phage capable of homing to tumors, especially to their vascular endothelium. The cDNA library screening revealed a remarkably potent homing peptide F3, which was a 17- to 48-aa fragment in HMGN2. F3 was just the functional -helical domain of HMGN2, which we had proved to be essential for the antimicrobial activity of HMGN2.
Received May 28, 2005; revised June 20, 2005; accepted July 18, 2005.
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