Cloning and expression of bovine neutrophil beta-defensins - Biosynthetic profile during neutrophilic maturation and localization of mature peptide to novel cytoplasmic dense granules

beta-Defensins are microbicidal peptides implicated in host defense functio ns of phagocytic leukocytes and certain surface epithelial cells. Here we i nvestigated the genetic structures and cellular expression of BNBD-4, -12, and -13, three prototypic bovine neutrophil beta-defensins. Characterizatio n of the corresponding cDNAs indicated that BNBD-4 (41 residues) derives fr om a 63-amino acid prepropeptide and that BNBD-12 (38 residues) and BNBD-13 (42 residues) derive from a common 60-amino acid precursor (BNBD-12/13). T he peptides were found to be encoded by two-exon genes that are closely rel ated to bovine epithelial beta-defensin genes. BNBD-4 and BNBD-12/13 mRNAs were most abundant in bone marrow, but were expressed differentially in cer tain non-myeloid tissues. In situ hybridization and immunohistochemical stu dies demonstrated that BNBD-4 synthesis is completed early in myelopoiesis. BNBD-12 was localized exclusively to the novel dense granules, organelles that also contain precursors of cathelicidins, antimicrobial peptides that undergo proteolytic processing during phagocytosis. In contrast to cathelic idins, Western blot analyses revealed that mature beta-defensins are the pr edominant organellar form in myeloid cells. Stimulation of neutrophils with phorbol myristate acetate induced secretion of BNBD-12, indicating that it is co-secreted with pro-cathelicidins. The exocytosis of BNBD-12 by activa ted neutrophils reveals different mobilization pathways for myeloid alpha- and beta-defensins.