On the role of the proform-conformation for processing and intracellular sorting of human cathepsin G

The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an aminoterminal propeptide from the catalytically inactive preform, the active protease acquires a mature conformation and is stored in azurophil granules. To investigate the importance of the proform-conformation for targeting to granules, a cDNA encoding a double-mutant form of human preprocathepsin G lacking functional catalytic site and aminoterminal prodipeptide (CatG/Gly(201)/Delta Gly(19)Glu(20)) was constructed, because we were not able to stably express a mutant lacking only the propeptide. Transfection of the cDNA to the rat basophilic leukemia RBL-1 and the murine myeloblast-like 32D cl3 cell lines resulted in stable, protein-expressing clones. In contrast to wild-type proenzyme, CatG/Gly(201)/Delta Gly(19)Glu(20) adopted a mature conformation cotranslationally, as judged by the early acquisition of affinity to the serine protease inhibitor aprotinin, appearing before the carboxyl-terminal processing and also in the presence of the Golgi-disrupting agent brefeldin A. The presence of a mature amino-terminus was confirmed by amino-terminal radiosequencing. As with wild-type proenzyme, CatG/Gly(201)/Delta Gly(19)Glu(20) was proteolytically processed carboxyl-terminally and glycosylated with asparagine-linked carbohydrates that were converted into complex forms. Furthermore, it was targeted to granules, as determined by subcellular fractionation, Our results show that the initial proform-conformation is not critical for intracellular sorting of human cathepsin G, Moreover, we demonstrate that double-mutant cathepsin G can achieve a mature conformation before carboxyl-terminal processing of the preform. (C) 1998 by The American Society of Hematology.