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 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.
Garwicz
D
D
Department of Hematology, Lund University, Lund, Sweden. Daniel.Garwicz@hematologi.lu.se
Lindmark
A
A
Persson
A M
AM
Gullberg
U
U
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology; D007611:Aprotinin / Q000378:metabolism; D001665:Binding Sites; D001692:Biological Transport; D020126:Brefeldin A; D019556:COS Cells; D056649:Cathepsin G; D002403:Cathepsins / Q000737:chemistry / Q000378:metabolism*; D003517:Cyclopentanes / Q000494:pharmacology; D003594:Cytoplasmic Granules / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004792:Enzyme Precursors / Q000737:chemistry / Q000378:metabolism*; D006031:Glycosylation; D006056:Golgi Apparatus / Q000187:drug effects; D006412:Hematopoietic Stem Cells / Q000378:metabolism; D006596:Hexosaminidases / Q000494:pharmacology; D006801:Humans; D015471:Leukemia, Basophilic, Acute / Q000473:pathology; D018942:Macrolides; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017510:Protein Folding; D011499:Protein Processing, Post-Translational; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017384:Sequence Deletion; D012697:Serine Endopeptidases; D013379:Substrate Specificity; D014162:Transfection; D014407:Tumor Cells, Cultured
D000900:Anti-Bacterial Agents; D003517:Cyclopentanes; D018076:DNA, Complementary; D004792:Enzyme Precursors; D018942:Macrolides; D011993:Recombinant Fusion Proteins; D020126:Brefeldin A; D007611:Aprotinin; D006596:Hexosaminidases; D002403:Cathepsins; D012697:Serine Endopeptidases; C534094:CTSG protein, human; D056649:Cathepsin G; C534096:Ctsg protein, mouse; C534097:Ctsg protein, rat