Identification of the primary antimicrobial domains in human neutrophil cathepsin G.
Lysosomal cathepsin G from human neutrophils is a chymotrypsin-like protease which also possesses antimicrobial activity. The antimicrobial activity, however, is independent of protease activity, because treatment of this enzyme with the irreversible serine protease inhibitor diisopropylfluorophosphate has no effect on its antimicrobial action. In this study, we found that digestion of cathepsin G with clostripain caused a loss of proteolytic activity in this neutrophil proteinase. However, bactericidal activity in in vitro assays against Staphylococcus aureus and Neisseria gonorrhoeae was retained. Fractionation of the clostripain-digested cathepsin G mixture yielded two distinct antimicrobial peptides. The sequences of these peptides were IIGGR and HPQYNQR (residues 1-5 and 77-83 in cathepsin G, respectively). Synthetic peptides corresponding to these sequences were also prepared and found to exert broad-spectrum antimicrobial activity in vitro, displaying conditions of temperature- and pH-dependent optima for antimicrobial action resembling that of the full-length enzyme. Depending on the target bacterial strain, these peptides exhibited antimicrobial activity between 5.0 x 10(-5) and 4.0 x 10(-4) M. Significantly, replacement of certain residues within these peptides with either alanine or valine significantly reduced their antibacterial capacities. Our studies suggest that cathepsin G has two antimicrobial sequences, either or both of which may contribute to its bactericidal activity.
Bangalore
N
N
Department of Biochemistry, University of Georgia, Athens 30602.
Travis
J
J
Onunka
V C
VC
Pohl
J
J
Shafer
W M
WM
D000595:Amino Acid Sequence; D000900:Anti-Bacterial Agents; D056649:Cathepsin G; D002403:Cathepsins / Q000097:blood* / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008247:Lysosomes / Q000201:enzymology; D008969:Molecular Sequence Data; D009344:Neisseria gonorrhoeae / Q000187:drug effects; D009504:Neutrophils / Q000201:enzymology*; D009842:Oligopeptides / Q000138:chemical synthesis / Q000494:pharmacology*; D010196:Pancreatic Elastase / Q000097:blood; D010446:Peptide Fragments / Q000494:pharmacology; D012697:Serine Endopeptidases; D013211:Staphylococcus aureus / Q000187:drug effects
D000900:Anti-Bacterial Agents; D009842:Oligopeptides; D010446:Peptide Fragments; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; D010196:Pancreatic Elastase; D003546:Cysteine Endopeptidases; C019913:clostripain