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)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1111432
- author
- Garwicz, Daniel
LU
; Lindmark, Anders
LU
; Hellmark, Thomas
LU
; Gladh, Mathias
; Jögi, Jonas
LU
and Gullberg, Urban
LU
- organization
- publishing date
- 1997
- 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
- John Wiley & Sons Inc.
- 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
- 2016-04-01 12:31:31
- date last changed
- 2022-01-27 06:16:24
@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}},
keywords = {{serine protease; neutrophil; biosynthesis; transgenic; anti-neutrophil cytoplasmic autoantibodies}},
language = {{eng}},
number = {{1}},
pages = {{113--123}},
publisher = {{John Wiley & Sons Inc.}},
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}},
url = {{http://jleuk.highwire.org/cgi/reprint/61/1/113}},
volume = {{61}},
year = {{1997}},
}