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

Garwicz, Daniel LU ; Lindmark, Anders ; Persson, Ann-Maj LU and Gullberg, Urban LU (1998) In Blood 92(4). p.22-1415
Abstract

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... (More)

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.

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author
; ; and
organization
publishing date
type
Contribution to journal
publication status
published
keywords
Animals, Anti-Bacterial Agents/pharmacology, Aprotinin/metabolism, Binding Sites, Biological Transport, Brefeldin A, COS Cells, Cathepsin G, Cathepsins/chemistry, Cyclopentanes/pharmacology, Cytoplasmic Granules/metabolism, DNA, Complementary/genetics, Enzyme Precursors/chemistry, Glycosylation, Golgi Apparatus/drug effects, Hematopoietic Stem Cells/metabolism, Hexosaminidases/pharmacology, Humans, Leukemia, Basophilic, Acute/pathology, Macrolides, Mice, Mutagenesis, Site-Directed, Protein Conformation, Protein Folding, Protein Processing, Post-Translational, Rats, Recombinant Fusion Proteins/metabolism, Sequence Deletion, Serine Endopeptidases, Substrate Specificity, Transfection, Tumor Cells, Cultured
in
Blood
volume
92
issue
4
pages
22 - 1415
publisher
American Society of Hematology
external identifiers
  • scopus:0032529324
  • pmid:9694731
ISSN
0006-4971
DOI
10.1182/blood.V92.4.1415
language
English
LU publication?
yes
additional info
Copyright 1998 by The American Society of Hematology.
id
03f8cd95-e076-418a-b521-565b1452bba5
date added to LUP
2025-03-03 15:56:57
date last changed
2025-07-08 15:02:10
@article{03f8cd95-e076-418a-b521-565b1452bba5,
  abstract     = {{<p>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.</p>}},
  author       = {{Garwicz, Daniel and Lindmark, Anders and Persson, Ann-Maj and Gullberg, Urban}},
  issn         = {{0006-4971}},
  keywords     = {{Animals; Anti-Bacterial Agents/pharmacology; Aprotinin/metabolism; Binding Sites; Biological Transport; Brefeldin A; COS Cells; Cathepsin G; Cathepsins/chemistry; Cyclopentanes/pharmacology; Cytoplasmic Granules/metabolism; DNA, Complementary/genetics; Enzyme Precursors/chemistry; Glycosylation; Golgi Apparatus/drug effects; Hematopoietic Stem Cells/metabolism; Hexosaminidases/pharmacology; Humans; Leukemia, Basophilic, Acute/pathology; Macrolides; Mice; Mutagenesis, Site-Directed; Protein Conformation; Protein Folding; Protein Processing, Post-Translational; Rats; Recombinant Fusion Proteins/metabolism; Sequence Deletion; Serine Endopeptidases; Substrate Specificity; Transfection; Tumor Cells, Cultured}},
  language     = {{eng}},
  month        = {{08}},
  number       = {{4}},
  pages        = {{22--1415}},
  publisher    = {{American Society of Hematology}},
  series       = {{Blood}},
  title        = {{On the role of the proform-conformation for processing and intracellular sorting of human cathepsin G}},
  url          = {{http://dx.doi.org/10.1182/blood.V92.4.1415}},
  doi          = {{10.1182/blood.V92.4.1415}},
  volume       = {{92}},
  year         = {{1998}},
}