title: Prodefensins are matrix proteins of specific granules in human neutrophils.
abstract: Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptide (HNP)-1-3 are synthesized as 94 amino acide (aa) preproHNPs, which are converted to 75 aa proHNPs by cotranslational removal of a 19 aa endoplasmic reticulum signal peptide. At the promyelocytic stage of myelopoiesis, proHNPs are further proteolytically modified and accumulate in azurophil granules as 29-30 aa HNPs. In contrast, proHNPs produced by more mature myeloid cells are not subjected to proteolytic cleavage and undergo a high degree of constitutive exocytosis. The proHNPs are devoid of antimicrobial potential, and the significance of their secretion is unknown. To investigate whether mature neutrophils contain proHNPs, we developed antibodies against proHNP-1 by DNA immunization of rabbits. In addition, antibodies against the 45 aa proHNP pro-piece were raised by conventional immunization procedures. These antibodies allowed detection of proHNPs in homogenates of peripheral blood neutrophils. The proHNPs were isolated by affinity chromatography, and their identity was confirmed by mass spectrometry and N-terminal aa sequence analysis. Finally, the neutrophil proHNPs were identified as novel matrix proteins of specific granules by subcellular fractionation experiments, release studies, and immunoelectron microscopy. Thus, human neutrophils not only store large amounts of mature defensins in azurophil granules but also contain a more easily mobilized reservoir of unprocessed prodefensins in specific granules.
authors.affiliation: Department of Hematology, Rigshospitalet 4042, DK-2100 Copenhagen, Denmark.
authors.forename: Mikkel
authors.initials: M
authors.lastname: Faurschou
authors.forename: Søren
authors.initials: S
authors.lastname: Kamp
authors.forename: Jack B
authors.initials: JB
authors.lastname: Cowland
authors.forename: Lene
authors.initials: L
authors.lastname: Udby
authors.forename: Anders H
authors.initials: AH
authors.lastname: Johnsen
authors.forename: Jero
authors.initials: J
authors.lastname: Calafat
authors.forename: Henrik
authors.initials: H
authors.lastname: Winther
authors.forename: Niels
authors.initials: N
authors.lastname: Borregaard
mesh_terms: D000906:Antibodies / Q000494:pharmacology; D003001:Cloning, Molecular; D003594:Cytoplasmic Granules / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D023082:Defensins / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification*; D006801:Humans; D009504:Neutrophils / Q000737:chemistry* / Q000187:drug effects / Q000276:immunology; D011994:Recombinant Proteins / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification; D023084:alpha-Defensins / Q000187:drug effects / Q000276:immunology / Q000302:isolation & purification
chemical_list: D000906:Antibodies; D023082:Defensins; D011994:Recombinant Proteins; D023084:alpha-Defensins; C406413:human neutrophil peptide 1