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Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/azurocidin

Lindmark, Anders ; Garwicz, Daniel LU ; Rasmussen, Poul B ; Flodgaard, Hans and Gullberg, U LU (1999) In Journal of Leukocyte Biology 66(4). p.43-634
Abstract

Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed... (More)

Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human azurocidin.

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; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Amino Acids, Animals, Antimicrobial Cationic Peptides, Asparagine/metabolism, Biological Transport, Blood Proteins/biosynthesis, Carbohydrate Metabolism, Carrier Proteins/biosynthesis, Gene Expression, Glycoproteins/biosynthesis, Humans, Isotope Labeling, Mice, Monocyte Chemoattractant Proteins/biosynthesis, Protein Precursors/biosynthesis, Protein Processing, Post-Translational, Rabbits, Rats, Sulfur Radioisotopes, Tumor Cells, Cultured
in
Journal of Leukocyte Biology
volume
66
issue
4
pages
43 - 634
publisher
John Wiley & Sons Inc.
external identifiers
  • pmid:10534120
  • scopus:0032879339
ISSN
0741-5400
DOI
10.1002/jlb.66.4.634
language
English
LU publication?
yes
id
08241db1-0cb4-4e9c-ae2f-83fd32130692
date added to LUP
2025-02-28 16:09:57
date last changed
2025-07-05 15:08:45
@article{08241db1-0cb4-4e9c-ae2f-83fd32130692,
  abstract     = {{<p>Azurocidin is a multifunctional endotoxin-binding serine protease homolog synthesized during the promyelocytic stage of neutrophil development. To characterize the biosynthesis and processing of azurocidin, cDNA encoding human preproazurocidin was stably transfected to the rat basophilic leukemia cell line RBL-1 and the murine myeloblast-like cell line 32D cl3; cell lines previously utilized to study the related proteins cathepsin G and proteinase 3. After 30 min of pulse radiolabeling, two forms of newly synthesized proazurocidin (34.5 and 37 kDa), differing in carbohydrate content but with protein cores of identical sizes, were recognized. With time, the 34.5-kDa form disappeared, while the 37-kDa form was further processed proteolytically, as judged by digestion with N-glycosidase F. Conversion of high-mannose oligosaccharides into complex forms was shown by acquisition of complete resistance to endoglycosidase H. Radiosequence analysis demonstrated that the amino-terminal seven amino acid propeptide of proazurocidin was removed in a stepwise manner during processing; initial removal of five amino acids was followed by cleavage of a dipeptide. Presence of the protease inhibitors Gly-Phe-diazomethyl ketone, bestatin, or leupeptin inhibited only the cleavage of the dipeptide, thus indicating the involvement of at least two amino-terminal processing enzymes. Translocation of azurocidin to granules was shown by subcellular fractionation. Similar results, with efficient biosynthesis, processing, and targeting to granules in both cell lines, were obtained with a mutant form of human preproazurocidin lacking the amino-terminal heptapropeptide. In conclusion, this investigation is an important addition to our previous studies on related azurophil granule proteins, and provides novel information concerning the biosynthesis and distinctive amino-terminal processing of human azurocidin.</p>}},
  author       = {{Lindmark, Anders and Garwicz, Daniel and Rasmussen, Poul B and Flodgaard, Hans and Gullberg, U}},
  issn         = {{0741-5400}},
  keywords     = {{Amino Acids; Animals; Antimicrobial Cationic Peptides; Asparagine/metabolism; Biological Transport; Blood Proteins/biosynthesis; Carbohydrate Metabolism; Carrier Proteins/biosynthesis; Gene Expression; Glycoproteins/biosynthesis; Humans; Isotope Labeling; Mice; Monocyte Chemoattractant Proteins/biosynthesis; Protein Precursors/biosynthesis; Protein Processing, Post-Translational; Rabbits; Rats; Sulfur Radioisotopes; Tumor Cells, Cultured}},
  language     = {{eng}},
  number       = {{4}},
  pages        = {{43--634}},
  publisher    = {{John Wiley & Sons Inc.}},
  series       = {{Journal of Leukocyte Biology}},
  title        = {{Characterization of the biosynthesis, processing, and sorting of human HBP/CAP37/azurocidin}},
  url          = {{http://dx.doi.org/10.1002/jlb.66.4.634}},
  doi          = {{10.1002/jlb.66.4.634}},
  volume       = {{66}},
  year         = {{1999}},
}