Processing of Neutrophil α-Defensins Does Not Rely on Serine Proteases in Vivo

The α-defensins human neutrophil peptides (HNPs) are the predominant antimicrobial peptides of neutrophil granules. They are synthesized in promyelocytes and myelocytes as proHNPs, but only processed and stored as mature HNPs in promyelocytes. Despite decades of search, the mechanisms underlying the posttranslational processing of neutrophil defensins remain unidentified. Thus, neither the enzyme processing proHNPs nor the localization of processing has been identified. It has long been hypothesized that proHNPs are processed by the serine proteases highly expressed in promyelocytes: Neutrophil elastase (NE), cathepsin G (CG), and proteinase 3 (PR3), all of which have shown in vitrocapability to process recombinant proHNP into HNP.

We investigated, whether serine proteases are in fact responsible for proHNP processing in human bone marrow cells and in human and murine myeloid cell lines. Subcellular fractionation of the human promyelocytic cell line PLB-985 demonstrated proHNP processing to commence in fractions containing endoplasmic reticulum. Processing of ³⁵S-proHNP was insensitive to serine protease inhibitors. Simultaneous knockdown of NE, CG, and PR3 did not decrease proHNP processing by primary human neutrophil precursors. Furthermore, introduction of NE, CG, and PR3 into murine promyelocytic cells did not increase processing capability. Finally, two patients suffering from Papillon–Lefèvre syndrome, who lack the ability to activate neutrophil serine proteases, demonstrated normal levels of fully processed HNP in peripheral neutrophils.

Contradicting earlier assumptions, our study found serine proteases dispensable for processing of proHNPs in vivo. This calls for study of other protease classes in the search for the proHNP processing protease(s).