On the Biosynthesis and Processing of Cathepsin G, Leukocyte Elastase, and Azurocidin ­ neutrophil granule members of a hematopoietic serine protease superfamily

Abstract: The serine proteases cathepsin G, leukocyte elastase, proteinase 3, and the inactive protease homologue azurocidin, stored in the azurophil granules of neutrophils, belong to a hematopoietic serine protease superfamily with members in mature effector cells of bone marrow origin. Promonocytic U-937 cells were used for studying the biosynthesis of cathepsin G and leukocyte elastase by biosynthetic radiolabelling. The proteases were found to be synthesized as precursor forms that were subjected to processing, including acquisition of N-linked complex oligosaccharides. Targeting to a granule compartment was also demonstrated. Using brefeldin A, monensin, and ammonium chloride, the subcellular localization of distinct processing events was characterized. To make possible an evaluation of the importance of distinct protein motifs, a transfectable cellular model for studying the processing was established. cDNA encoding human cathepsin G was transfected to the rat RBL cell line, after which the model was proven valid by characterizing the activation, processing, and sorting of cathepsin G. Mutant cDNAs encoding cathepsin G and leukocyte elastase lacking carboxyl-terminal peptide extensions, normally removed during processing, were transfected to RBL cells. The mutant proteins were found to be activated and sorted to granules, approximately as efficiently as the wild type proteins, hence ruling out a critical role of the propeptides for folding, stability or molecular targeting. The granule thiol protease dipeptidyl peptidase I (DPP I) has been suggested as responsible for the activation of all hematopoietic serine proteases. It was, however, not possible to activate procathepsin G, expressed in COS-7 cells, neither by co-transfection of rat DPP I cDNA nor by in vitro incubation with purified bovine DPP I. After transfection to RBL cells, a novel amino-terminal processing pattern distinct from that of other granule serine proteases was demonstrated for the synthesis and processing of azurocidin.