Identification And Characterization Of Novel Antimicrobial Peptides From Female Productive Duct

[Background] Antimicrobial peptides are endogenous, gene-encoded cationic peptides found in virtually every life form. It is a key mediator of innate immune, and exhibits broad-spectrum activity against bacteria, fungi and virus infection. Female reproductive duct is also important site of innate immunity and several antimicrobial peptides or proteins such as defensins, lysozyme, lactoferrin, and secretory leukocyte protease inhibitor (SLPI) are identified from it. Now, it is concerned whether existing unknown antimicrobial peptides or proteins in female reproductive duct by researchers. On the other hand, the antimicrobial peptides or proteins, which have been identified from female reproductive duct, are originating from epithelial cells and/or leucocytes. So it is interesting whether antimicrobial peptides molecules have new cellular sources. Therefore, in this study the novel antimicrobial molecule in the acid-soluble extracts of human uterine mucus and cervical mucus were isolated and purified. Their cellular source,histological distribution were further assayed. In addition, antimicrobial peptides usually are constitutive or induced expression in mucosal tissues. So we investigated the expression manner of the new antimicrobial peptide molecule-HMGN2 in cervical tissue. These results would help for our understanding innate immune mechanisms of female reproductive duct, and might provide a new source of antibiotics for preventing and curing infections of female reproductive duct.[Methods and Results] The antimicrobial activity of the acid-soluble extracts of human uterine mucus and cervical mucus were assayed by gel overlay technique, the corresponding antimicrobial protein bands were cut out from the gel of prepared acid-urea polyacrylamide gel electrophoresis (AU-PAGE), and the proteins in the gel was eluted by electrophoresis. The elution proteins were dialyzed with distill water, lyophilized, and dissolved in 0.01% acetic acid, then were stored in -20"C.The elution sample of the uterine antimicrobial protein was purified by RP-HPLC. A molecule at 39 minute of retention time was named HUP-39 (human uterine protein 39). The HUP-39 was identified by N-terminal amino acid sequencing with Edman degradation response. The result showed that the sequence was FLSFPTTKTY. It was 100% identical to amino acid residues 33-42 of human hemoglobin a-chain (hHEM-a) by GenBank BLAST searching. The molecular mass of HUP-39 was 6776.8-6777.1 Da determined by Mass Spectrometry. According to the results of the N-terminal sequencing and Mass Spectrometry analysis, HUP-39 should be hHEM-a.fragment 33-95. The fragment further analyzed by ExPASy and OMIGA softs. Its pi was 8.38, and contained three intact a-helical transmembrane domains. Agarose radialdiffusion assay showed that HUP-39 was mainly against E.coli ML-35p, E.coli ATCC 25922 and clinical isolated strain E.coli 54080, no effective on S. aureus ATCC 25923 and C. albicans ATCC 90028.HCP-21 was purified by RP-HPLC at 21 minute of retention time from the elution sample of cervical mucus (human cervix protein 21, HCP-21). It could effectively kill E.coli ML-35p determined by agarose radial diffusion assay. The N-terminal amino acid sequence of HCP-21 was PKRKAEGDAK and its molecular mass was 9263.62Da identified by amino acid sequencing and mass spectrometry analysis. HCP-21 showed 100% identity to HMGN2 (high mobility group protein N2, HMGN2) fragment 2-11 by GenBank BLAST searching, and had the same molecular mass as HMGN2. So it was certain that HCP-21 was HMGN2. Total RNA was extracted from primary culture cervical epithelial cells, and the specific primer was designed based on HMGN2 cDNA sequence. A fragment about 270bp was amplificated by RT-PCR, which was same to HMGN2 cDNA. It was suggested that cervical epithelial cells could express HMGN2 mRNA in physiological condition. The fusion protein GST- HMGN2 was purified by low-pressure chromatography. The antiserum of HMGN2 was prepared from the immune rabbit with the fusion protein. Cervical tissue paraffin section and cervical mucus smear were detected by immunhistochemistry. The staining results showed that HMGN2 existed in cervical mucus, and mainly distributed in mucosa surface of cervix. There was no statistical significance of HMGN2 contents between the inflammatory group and normal group samples by immunodot blot detection. It was suggested that bacteria or cytokines could not induce HMGN2 expression.[ Conclusion ] HUP-39 was the fragment of hHEM-a. It could effectively kill Gram-negative bacteria. We believed that antimicrobial peptides of uterine mucus were not only produced by epithelial cells or leucocytes, but also by erythrocytes. HCP-21 was HMGN2 and we first identified it in cervical mucus. It constitutively expressed in epithelial tissues of cervical mucosa. HMGN2 was also one of the members of innate defensive molecule of cervical mucus.