Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/azurocidin

Azurocidin is a multifunctional endotoxin binding serine protease homolog s ynthesized du-ibg the promyelocytic stage of neutrophil development, To cha racterize the biosynthesis and processing of azurocidin, cDNA encoding huma n preproazurocidin was stably transfected to the rat basophilic leukemia ce ll line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines preciously utilized to study the related proteins cathepsin G and proteinas e 3, After 30 min of pulse radiolabeling, two forms of newly synthesized pr oazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with p rotein cores of identical sizes, were recognized, With time, the 34.5-kDa f orm disappeared, while the 37-kDa form was further processed proteolyticall y, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete re sistance to endoglycosidase H, Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids w as followed by cleavage of a dipeptide, Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the clea vage of the dipeptide, thus indicating the involvement of at least two amin o-terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynt hesis, processing, and targeting to granules in both cell lines, were obtai ned with a mutant form of human preproazurocidin lacking the amino-terminal heptapropeptide, In conclusion, this investigation is an important additio n to our precious studies on related azurophil granule proteins, and provid es novel information concerning the biosynthesis and distinctive amino-term inal processing of human azurocidin.