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© by The Society for Leukocyte Biology
Journal of Leukocyte Biology, doi:10.1189/jlb.1104688

Received for publication November 25, 2004.
Revised May 2, 2005.
Accepted for publication May 9, 2005.

Article

Prodefensins are matrix proteins of specific granules in human neutrophils

Mikkel Faurschou *, Søren Kamp *, Jack B. Cowland *, Lene Udby *, Anders H. Johnsen {dagger}, Jero Calafat {ddagger}, Henrik Winther {sect}, and Niels Borregaard *@

Departments of *Hematology, The Granulocyte Research Laboratory, and {dagger}Clinical Biochemistry, Rigshospitalet, Copenhagen, Denmark; {ddagger}Department of Cell Biology, The Netherlands Cancer Institute, Amsterdam; and {sect}Dako-Cytomation, Glostrup, Denmark

@ To whom correspondence should be addressed. E-mail: borregaard{at}rh.dk.


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Abstract

Defensins are potent antimicrobial and proinflammatory peptides. The human neutrophil defensins human neutrophil peptides (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.

Key Words: proHNP • HNP • secondary granules • polymorphonuclear leukocytes




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