PROCESSING OF HUMAN CATHEPSIN-G AFTER TRANSFECTION TO THE RAT BASOPHILIC MAST-CELL TUMOR LINE RBL

The azurophil granules of neutrophil granulocytes contain neutral prot eases such as leukocyte elastase and cathepsin G. These are synthesize d as inactive precursors, but following proteolytic processing, they a re stored in granules as active enzymes. We describe the establishment of a transgenic cellular model for expression of the human myeloid se rine protease cathepsin G. The cDNA for preprocathepsin G was stably e xpressed in the rat basophilic/mast cell line RBL-1 and the translatio n product was characterized by use of biosynthetic labeling followed b y immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and flu orography. Conversion into complex form of an asparagine-linked carboh ydrate unit of approximately 3.5 kDa was shown, as judged by the produ cts obtained upon treatment with endoglycosidase H and N-glycanase. Pr oteolytic processing of 32.5 kDa procathepsin G into a 31-kDa form, wi thin 1-2 h after synthesis, was demonstrated by pulse-chase experiment s. Further processing into a 30-kDa form also occurred to a minor exte nt. The processed forms were enzymatically active, as judged by affini ty for the serine protease inhibitors diisopropylfluorophosphate phosp hate and aprotinin. Translocation of processed forms of cathepsin G to high density fractions, indicating targeting of the protease to granu les, was demonstrated by subcellular fractionation. The weak base NH4C l was shown to delay the processing and enzymatic activation of cathep sin G, whereas the monovalent ionophore monensin completely inhibited both events. Our data demonstrate that human cathepsin G transfected t o rat RBL-1 cells, is proteolytically processed into enzymatically act ive forms and that subcellular transfer to granular organelles occurs. As the processing of transgenic human cathepsin G corresponds to that of endogenous protease of myeloid cells, the model should provide new unique possibilities to further characterize the activation and granu lar targeting of myeloid serine proteases.