Bactenecins, defense polypeptides of bovine neutrophils, are generated from precursor molecules stored in the large granules.
Bactenecins are highly cationic polypeptides of bovine neutrophil granules and exert in vitro a potent antimicrobial activity. We have previously purified two bactenecins, designated in an abbreviated form Bac7 and Bac5 from their approximate molecular masses of 7 and 5 kD (Gennaro, R., B. Skerlavaj, and D. Romeo. 1989. Infect. Immun. 57:3142-3146). Here we have studied the biosynthesis, processing, and localization of precursors of Bac7 and Bac5 in bovine bone marrow cells of the myeloid lineage. In vitro translation directed by mRNA isolated from these cells has shown that the primary translation products are preprobactenecins of 23.5 and 21 kD, and are processed to polypeptides of 20 and 15.8 kD, respectively. The 20-kD polypeptide is the granule storage form of Bac7, or proBac7, as also demonstrated by Western blot analysis of lysates of peripheral neutrophils. Between 15 and 50 min from the beginning of its biosynthesis the 15.8-kD polypeptide is converted into the 15-kD granule storage form of Bac5, or proBac5. As shown by immunogold EM, proBac7 and proBac5 are sorted and targeted to the matrix of the so called large granules, which are the predominant organelles in the cytoplasm of bovine neutrophils and are the exclusive store of the nonoxidative antimicrobial system of these cells. Solubilization of granules with Triton X-100 with concomitant unmasking of proteases leads to cleavage of the proforms to Bac7 and Bac5. Experiments performed with protease inhibitors suggest that the proteolytic cleavage is catalyzed in detergent-solubilized neutrophils by neutral serine protease(s), very likely derived from the azurophil granules.
Dipartimento di Biochimca, Università di Trieste, Italy.
M
M
Zanetti
L
L
Litteri
R
R
Gennaro
H
H
Horstmann
D
D
Romeo
D000818:Animals; D000906:Antibodies; D001853:Bone Marrow / Q000276:immunology / Q000378:metabolism; D001854:Bone Marrow Cells; D002417:Cattle; D002474:Cell-Free System; D003594:Cytoplasmic Granules / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D009504:Neutrophils / Q000276:immunology / Q000378:metabolism* / Q000648:ultrastructure; D010456:Peptides, Cyclic / Q000096:biosynthesis*; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000096:biosynthesis; D011499:Protein Processing, Post-Translational; D012697:Serine Endopeptidases
D000906:Antibodies; D010456:Peptides, Cyclic; D011498:Protein Precursors; C056454:bactenecin; D012697:Serine Endopeptidases