Proteolytic cleavage by neutrophil elastase converts inactive storage proforms to antibacterial bactenecins.
Bac5 and Bac7, antibiotics of the bactenecin (proline/arginine-rich peptide) family, are stored as proforms in the large granules of bovine neutrophils [Zanetti, M., Litteri, L., Gennaro, R., Horstmann, H. and Romeo, D. (1990) J. Cell Biol. 111, 1363-1371]. These proforms have been purified to homogeneity from granule extracts by immunoaffinity and reverse-phase chromatography. While mature bactenecins efficiently kill Escherichia coli, Klebsiella pneumoniae and Salmonella typhimurium with minimal inhibitory concentrations of 6-12 micrograms/ml, proBac5 and proBac7 do not affect the growth of the same microorganisms, even at 500 micrograms/ml. Previous investigations have suggested that the conversion of probactenecins into mature antimicrobial peptides is catalyzed by a neutral serine protease stored in the azurophil granules. Purified proBac5 and proBac7 were thus treated with elastase, cathepsin G or proteinase 3, which constitute the pool of neutral serine proteases of the azurophils, and the reaction products were identified by Western blot analysis, mass spectrometry, and N-terminal sequence analysis. Of the three proteases, only elastase is able to catalyze the stepwise cleavage of probactenecins into the corresponding mature peptides, which have the same mass, N-terminal sequence and antibiotic activity of authentic Bac5 and Bac7. These results point to the importance of cooperation between azurophils and large granules in mounting a defense reaction.
Scocchi
M
M
Department of Biochemistry, Biophysics and Macromolecular Chemistry, University of Trieste, Italy.
Skerlavaj
B
B
Romeo
D
D
Gennaro
R
R
D000818:Animals; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004926:Escherichia coli / Q000187:drug effects; D006801:Humans; D007700:Kinetics; D007711:Klebsiella pneumoniae / Q000187:drug effects; D019272:Leukocyte Elastase; D008826:Microbial Sensitivity Tests; D053146:Myeloblastin; D009504:Neutrophils / Q000502:physiology*; D010196:Pancreatic Elastase / Q000378:metabolism*; D010456:Peptides, Cyclic / Q000302:isolation & purification / Q000378:metabolism* / Q000494:pharmacology; D011498:Protein Precursors / Q000302:isolation & purification / Q000378:metabolism*; D012486:Salmonella typhimurium / Q000187:drug effects; D012697:Serine Endopeptidases / Q000302:isolation & purification / Q000378:metabolism
D000900:Anti-Bacterial Agents; D010456:Peptides, Cyclic; D011498:Protein Precursors; C056454:bactenecin; D012697:Serine Endopeptidases; D010196:Pancreatic Elastase; D019272:Leukocyte Elastase; D053146:Myeloblastin