[Isolation and purification of antimicrobial polypeptide HMGN2 from human lymph node and analysis of its distribution].

This study was conducted to isolate and purify antimicrobial polypeptides HMGN2 (high mobility group nucleosomal-binding domain2) from human lymph node, to detect the antimicrobial activity of HMGN2, and to determine the subcellular location of HMGN2 in human lymph node. The antimicrobial polypeptides were purified by the Reverse Phase HPLC and identified by Tricine-SDS-PAGE. The antimicrobial activity was detected by agar diffusion test. Mass spectrum and Western-blot analysis indicated the individual character of protein. HMGN2 was isolated and purified from human lymph node, and it showed antimicrobial potency against the pathogenic strain E. coli 54,080. The immunocytochemistry staining indicated that HMGN2 was present both in human lymph node cells' nucleus and cytoplasm. In conclusion, HMGN2 protein is of antimicrobial activity and it is probably involved in the defence of innate immunity in vivo.

Inhibitory effect of HMGN2 protein on human hepatitis B virus expression and replication in the HepG2.2.15 cell line.

Natural killer (NK) cells and cytolytic T lymphocytes (CTL) have been implicated as important effectors of antiviral defense. We previously isolated a novel antibacterial polypeptide, which was identified as high mobility group nucleosomal-binding domain 2 (HMGN2), from human mononuclear leukocytes. This study examined the antiviral activity of HMGN2 against human hepatitis virus B. HMGN2 was isolated and purified from the acid soluble proteins of the human THP-1 cell line, and identified by mass spectrum, Western blot and antibacterial assay. The hepatitis B virus (HBV)-transfected HepG2.2.15 cell line was used in the in vitro assay system. In the range of 1-100 microg/ml HMGN2, no cytotoxicity for HepG2.2.15 cells was detected by MTT assay. When incubated with HMGN2 at 1-100 microg/ml for 72 or 144 h, there was a significant reduction in hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) expression, which were detected by ELISA, and a significant reduction in HBV DNA copies, which was determined by the real time quantitative PCR, in the supernatant of HepG2.2.15 cells. Northern and Southern blot analysis also showed that the levels of the HBV 3.5 kb and the 2.4/2.1 kb mRNA species and HBV replicative intermediate DNA were significantly reduced in the HMGN2-treated HepG2.2.15 cells. These results indicated that HMGN2 protein could markedly inhibit HBV protein expression and replication in vitro.

HMGN2: a novel antimicrobial effector molecule of human mononuclear leukocytes?

Leukocytes are a central cellular element of innate-immune defense in mammals. In addition to the generation of toxic oxygen radicals and nitric oxide, leukocytes express and secrete a broad array of antimicrobial proteins and peptides. In the study, an antimicrobial polypeptide was isolated and purified from human peripheral blood mononuclear leukocytes in the presence of interleukin (IL)-2. Microsequencing provided that its N-terminal amino sequence was PKRKAEGDAK, which was identical to high mobility group nucleosomal-binding domain 2 (HMGN2). Mass spectrometric value and Western blot also indicated its individual character of HMGN2. The antimicrobial assays showed that the Escherichia coli-based production of HMGN2 had a potent antimicrobial activity against E. coli ML-35p, Pseudomonas aeruginosa ATCC 27853, and to some extent, against Candida albicans ATCC 10231. The HMGN2 alpha-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.

[Study of antmicrobial mechanisms of human cervical mucus: isolation and characterization of antibacterial polypeptides].

OBJECTIVE: To isolate and purify antibacterial polypeptides from human cervical mucus. METHODS: Human cervical mucus was collected from human healthy subjects and acid-soluble extract was obtained by solving the mucus with 5% acetic acid in the presence of protease inhibitors. The antibacterial components were identified by Agar radial diffusion assay and gel overlay technique. For further purification, Preparative acid-urea gel electrophoresis and Reverse Phase HPLC were performed. The N-terminal sequencing and degenerate PCR-directered cDNA cloning were performed. The E. coli-based recombinant product was prepared and its antibacterial property was determined by minimal inhibitory concentration and minimal bactericidal concentration. RESULTS: A purified antibacterial polypeptide was obtained. Agar radial diffusion assay showed that the purified polypeptide had antibacterial activities against E. coli ML-35P and Pseudomanas aeruginosa ATCC 27853. The N-terminal amino acid sequence and its full length of cDNA were identical to High Mobility Group Chromosal protein N2 (HMGN2). The purified recombinant HMGN2 was obtained. Its MIC against E. coli ML-35p and P. aeruginosa ATCC27853 were 10.42 microg/ml +/- 3.13 microg/ml and 27.78 microg/ml +/- 8.33 microg/ml respectively, which were equal to human neutrophil defensins HNP, and the MBC were 20.83 microg/ml +/- 6.25 microg/ml and 55.56 microg/ml +/- 16.67 microg/ml respectively. CONCLUSION: HMGN2 may be another antibacterial effecter in the defense mechanisms of human cervical mucus.