CARBOXYL-TERMINAL PRODOMAIN-DELETED HUMAN-LEUKOCYTE ELASTASE AND CATHEPSIN-G ARE EFFICIENTLY TARGETED TO GRANULES AND ENZYMATICALLY ACTIVATED IN THE RAT BASOPHILIC MAST-CELL LINE RBL

The hematopoietic neutral serine proteases leukocyte elastase and cath epsin G are synthesized as inactive precursors, but become activated b y removal of an amino-terminal dipeptide and are stored in granules, M oreover, the pro forms of elastase and cathepsin G show carboxyl-termi nal prodomains of 20 and 11 amino acids, respectively, which are not p resent in the mature enzymes. To investigate mechanisms for processing , activation, and granular targeting, we have utilized transgenic expr ession of myeloid serine proteases in the rat basophilic/mast cell lin e RBL-1 (Gullberg, U., Lindmark, A., Nilsson, E., Persson, A.-M.,, and Olsson, I. (1994) J. Biol. Chem. 269, 25219-25225). Leukocyte elastas e was stably expressed in RBL-1 cells, and the translation products we re characterized by biosynthetic labeling followed by immunoprecipitat ion, SDS-polyacrylamide gel electrophoresis, and fluorography. Process ing of a main pro form of 34 kDa into mature 31- and 29-kDa forms was demonstrated. Translocation of mature forms to granule-containing frac tions was shown by subcellular fractionation experiments. The processe d forms were enzymatically active, judging by the occurrence of amino- terminal processing demonstrated by radiosequence analysis, the acquis ition of affinity for the protease inhibitor aprotinin, and the appear ance of elastase activity in transfected RBL cells. To investigate the function of the carboxyl-terminal prodomains, deletion mutants of leu kocyte elastase and cathepsin G lacking the carboxyl-terminal extensio n were constructed and transfected into RBL cells. Our results show th at as full-length proteins, the deletion mutants were converted to act ive enzymes and transferred to granules with kinetics similar to that of wild-type enzymes. me conclude that human leukocyte elastase and ca thepsin G are converted into enzymatically active forms when expressed in RBL cells and targeted for storage in granules; the carboxyl-termi nal prodomains are necessary neither for enzymatic activation nor for targeting to granules in RBL cells.