cathepsin-g and Leukemia--Basophilic--Acute

The serine protease cathepsin G is synthesized during the promyelomonocytic stage of neutrophil and monocyte differentiation. After processing, including removal of an amino-terminal propeptide from the catalytically inactive proform, 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 amino-terminal prodipeptide (CatG/Gly201/triangle upGly19Glu20) 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/Gly201/triangle upGly19Glu20 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/Gly201/triangle upGly19Glu20 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 proform.

Topics: Animals; Anti-Bacterial Agents; Aprotinin; Binding Sites; Biological Transport; Brefeldin A; Cathepsin G; Cathepsins; COS Cells; Cyclopentanes; Cytoplasmic Granules; DNA, Complementary; Enzyme Precursors; Glycosylation; Golgi Apparatus; Hematopoietic Stem Cells; Hexosaminidases; Humans; Leukemia, Basophilic, Acute; Macrolides; Mice; Mutagenesis, Site-Directed; Protein Conformation; Protein Folding; Protein Processing, Post-Translational; Rats; Recombinant Fusion Proteins; Sequence Deletion; Serine Endopeptidases; Substrate Specificity; Transfection; Tumor Cells, Cultured

The neutral protease cathepsin G belongs to a family of hematopoietic serine proteases stored in the azurophil granules of the neutrophil granulocyte. To investigate the function of asparagine-linked carbohydrates in neutrophil serine proteases, we constructed a mutant cDNA, coding for human cathepsin G deficient of a functional glycosylation site, for use in a transgenic cellular model. Wild type and mutant cDNA were stably expressed in the rat basophilic/mast cell line RBL and in the murine myeloblast-like cell line 32D. Biosynthetic labeling, followed by immunoprecipitation, SDS-polyacrylamide gel electrophoresis, and fluorography, showed that carbohydrate-deficient cathepsin G was synthesized as a 29-kDa proform in both cell lines. The proform was proteolytically processed into a stable form with an apparent molecular mass of 27.5 kDa, indicating removal of the carboxyl-terminal prodomain. The mutant cathepsin G was enzymatically activated as determined by acquisition of affinity to aprotinin, a serine protease inhibitor. As for wild type cathepsin G, small amounts of the unprocessed form of the mutated enzyme were released from the cells, while the major part was transferred to a granular compartment as demonstrated by subcellular fractionation. Thus, neither processing leading to enzymatic activation nor granular sorting was obviously affected by the lack of oligosaccharides on the mutant cathepsin G. Our results therefore indicate that glycosylation is not essential for these processes. In addition to the previously utilized cell line RBL, we propose the 32D cell line as a suitable cellular model for transgenic expression of human neutrophil serine proteases.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cathepsin G; Cathepsins; Cell Line; Chromatography, Affinity; Cytoplasmic Granules; DNA Primers; Glutamine; Glycosylation; Humans; Kinetics; Leukemia, Basophilic, Acute; Mast Cells; Mice; Molecular Sequence Data; Mutagenesis, Site-Directed; Point Mutation; Polymerase Chain Reaction; Protein Processing, Post-Translational; Rats; Recombinant Proteins; Sequence Deletion; Serine Endopeptidases; Transfection; Tumor Cells, Cultured