Characterization of bovine neutrophil antibacterial polypeptides which bind to Escherichia coli.
Bovine neutrophils contain several cationic polypeptides which exert potent microbicidal effects in vitro. To better characterize the repertoire of these polypeptides, we have incubated extracts of bovine neutrophils or neutrophil granules at pH 4 or 7 with either a smooth strain of Escherichia coli or a rough one. Only a few polypeptides interacted with the bacterial surface and were subsequently desorbed with 200 mM MgCl2, as revealed by gel electrophoresis and analysis of Western blots (immunoblots) with appropriate antibodies. Two of the main proteins appearing in Coomassie blue-stained gels have molecular masses of 53 and 15 kDa and correspond to the heavy and light chains of myeloperoxidase. Another prevailing protein band with a molecular mass of 31 kDa was purified and shown to be 87% identical to human azurocidin/CAP37 in its 22-amino-acid N-terminal sequence. Proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and blotted to nitrocellulose did not react with an antiserum to human bactericidal/permeability-increasing protein. Conversely, immunoglobulin G against Bac7 or Bac5, two members of the antimicrobial proline- and arginine-rich polypeptide family, recognized in Western blots both the inactive precursor molecules, proBac7 and proBac5, and the mature polypeptides.
Litteri
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Department of Biochemistry, Biophysics, and Macromolecular Chemistry, University of Trieste, Italy.
Romeo
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D000818:Animals; D023181:Antimicrobial Cationic Peptides; D001770:Blood Bactericidal Activity; D001798:Blood Proteins / Q000737:chemistry*; D002417:Cattle; D003594:Cytoplasmic Granules / Q000737:chemistry; D004926:Escherichia coli / Q000276:immunology*; D008970:Molecular Weight; D009504:Neutrophils / Q000737:chemistry / Q000276:immunology*
D023181:Antimicrobial Cationic Peptides; D001798:Blood Proteins; C053368:cationic antimicrobial protein 57, human