Purification And Characterization Of A Novel Antibacterial Peptide From Larvae Of Musca Domestica

Musca domestica, which belongs to insecta, diptera, cyclorrhapha, muscidae, is the most common muscae and the richest resource. It is very significant and valuable to isolate antibacterial peptides from Musca domestica and to develop these peptides into antibacterial medicine. Due to purify a pure peptide from the natural materials (animal, plant and microorganism tissue) is very difficult and complex, few research is going on. For the first time, we have purified a novel antibacterial peptide and designed as Muscatoxin based on its bioactivity and resource from larvae of Musca domestica. We have also chemically and biologically characterized the peptide Muscatoxin.1,A group of antibacterial peptides had been isolated from larvae of Musca domestica by a number of batch-wise biochemistry separation followed by several chromatography steps. The antibacterial peptides had characters of broad antibacterial spectrum,low minimum bactericidal concentration (MBC),high thermostable and freeze-thaw stable. The bactericidal ability order was Bacillus subtilis> Bacillus thuringiensis> Staphylococcus aureus> Pseudomonas aeruginosa> Escherichia coli. and the MBC is 0.039,0.078,1.25,5.0 and 20μg/μl respectively. It is significant to develop antibacterial medicine for that the peptides are not agglutinin and have stronger activity to G⁺ than G^-. Furthermore, a plenty of weakly acid protein in the peptides also had bactericidal activity.2,Using reverse-phase HPLC,polyacrylamide gel electrophoresis and electroelution, the novel antibacterial peptide Muscatoxin had been purified in high degree of purity. It was determined pI 8.88 with IEF-PAGE, Mr 7095 D with MALDI-TOF-MS spectrometer. PMF showed Muscatoxin was a novel peptide and the N-terminal amino acid sequence was SQLGD LGSGA GKGGG GGGSI REAGG AFGKL EAARE EEYFY. Muscatoxin was belong to glysine-rich peptides with (Gly)₆,(Gly)₂ and (Glu)₃ domains and maybe had been post-translation modified.3,Searching in Genebank database, Muscatoxin was a novel peptide which had not been reported. Muscatoxin was 97.5% homologenous with pseudoprotein of Drosophila melanogaster (NM₁66597 and AY071315, 24～63aa) from fruit fly gene bank except the third residue Leu vs. Val.4,Obseved by scanning electric mirror (SEM), antibacterial peptide Muscatoxin could destroy the cell membrane of G⁺ (Bacillus subtilis,Bacillus thuringiensis,Staphylococcus aureus) and G^- (Pseudomonas aeruginosa,Escherichia coli. ) bacterium. The mechanism was to perforate the cell membrane and lead to bacterium lysis and die;5,Muscatoxin could kill mononuclear leukaemia cancer cells (THP-1). After treated by Muscatoxin ( 2mg/ml ) , most leukaemia cells suspended for the destroyed anchorage-dependent characterization and died. Obseved by SEM, the intercellular substance decreased obviously and cell morphology had changed. The mechanism was also to perforate the cell membrane and lead to cancer cells lysis and die;6,Muscatoxin showed certain cytotoxicity to mammalian cells. In high concentration (2mg/ml), it could totally lysis all blood cells membrane; but in low concentration (0.08 mg/ml), it just killed leucocyte and had few effect on rat akaryocyte except more round morphology;7,Using patch clamp technique, it was found that Muscatoxin in mM could form cationic channels in pancreas 3-cells but not in human embryo-kidney cells (HEK293) which had few innate receptors and ion channels. It was suggested that Muscatoxin could interact with certain receptors inβ-cells and then perforated the cells membrane to form ion channels. The perforating effect did not depend on extracellular Ca²⁺;8,Using intracellular Ca²⁺ fluorimetry technique, it was found that Muscatoxin in mM could perforateβ-cells and make the extracellular Ca²⁺ flow inwardly to increase the intracellular Ca²⁺ concentration. The effect is cumulative and not related to Muscatoxin dose. It was also apparently effective even the Muscatoxin concentration decreased to fM.9,Another novel antibacterial peptide named SK84 was purified from larvae ofDrosophila virilis and characterized total protein sequence. SK84 had no haemolytic activity from 1 to 100μM. SK84 could inhibite THP-1,HepG2 and MCF-7 cancer cells, but not affect HEK293 and CHO cells.