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Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines

Garwicz, Daniel LU ; Lindmark, Anders LU ; Hellmark, Thomas LU ; Gladh, Mathias; Jögi, Jonas LU and Gullberg, Urban LU (1997) In Journal of Leukocyte Biology 61(1). p.113-123
Abstract
Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D c13. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and... (More)
Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D c13. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA. (Less)
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author
organization
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type
Contribution to journal
publication status
published
subject
keywords
serine protease, neutrophil, biosynthesis, transgenic, anti-neutrophil cytoplasmic autoantibodies
in
Journal of Leukocyte Biology
volume
61
issue
1
pages
113 - 123
publisher
Society for Leukocyte Biology
external identifiers
  • pmid:9000544
  • scopus:0031056526
ISSN
1938-3673
language
English
LU publication?
yes
id
f34182ca-a9e5-4bc3-8440-860ff68fbae6 (old id 1111432)
alternative location
http://jleuk.highwire.org/cgi/reprint/61/1/113
date added to LUP
2008-07-17 13:30:29
date last changed
2017-01-01 05:13:14
@article{f34182ca-a9e5-4bc3-8440-860ff68fbae6,
  abstract     = {Proteinase 3 (PR3) is a neutrophil serine protease stored in the azurophil granules of the promyelocyte and its successors. The protease has been identified as an autoantigen for anti-neutrophil cytoplasmic autoantibodies (ANCA) occurring in patients with Wegener's granulomatosis. To characterize the biosynthesis and processing of human PR3 in a transgenic cellular model, cDNA encoding human pre-proproteinase 3 cloned from U-937 cells was transfected to the rat basophilic/mast cell line RBL-1 and the murine myeloblast-like cell line 32D c13. The stable expression of transgenic proteinase 3 was characterized by biosynthetic labeling, followed by immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. After pulse labeling for 30 min two proforms of PR3 (32 and 35 kDa), differing in carbohydrate content but with protein cores of identical size, were demonstrated. Chase of the label resulted in a processed 32-kDa form clearly visible in RBL, but only faintly in 32D cells, probably indicating delayed intracellular transfer in the latter cell line. Partial digestion with N-glycosidase F showed that both potential N-glycosylation sites on PR3 were occupied and conversion of the oligosaccharide side chains into complex forms was demonstrated by acquisition of resistance to endoglycosidase H. Translocation of PR3 to granules was shown by subcellular fractionation and immunocytochemistry. Enzymatic activation of PR3 was suggested by affinity to diisopropylfluorophosphate and removal of an amino-terminal propeptide. Cells transfected with PR3 showed positive immunofluorescence for ANCA-containing sera from patients with Wegener's granulomatosis. Our results show that human PR3 transfected to RBL or 32D cells is synthesized as a 29-kDa protein core glycosylated on two distinct sites. Oligosaccharide trimming and proteolytic processing occur and the protein is targeted for granular storage in a form antigenic for ANCA.},
  author       = {Garwicz, Daniel and Lindmark, Anders and Hellmark, Thomas and Gladh, Mathias and Jögi, Jonas and Gullberg, Urban},
  issn         = {1938-3673},
  keyword      = {serine protease,neutrophil,biosynthesis,transgenic,anti-neutrophil cytoplasmic autoantibodies},
  language     = {eng},
  number       = {1},
  pages        = {113--123},
  publisher    = {Society for Leukocyte Biology},
  series       = {Journal of Leukocyte Biology},
  title        = {Characterization of the processing and granular targeting of human proteinase 3 after transfection to the rat RBL or the murine 32D leukemic cell lines},
  volume       = {61},
  year         = {1997},
}