Abstract
We developed a stable expression system for conformationally intact recombinant human PR3 (rPR3) using the human mast cell line HMC-1. Like in U937 cells, the rPR3 is processed from a 34 kDa precursor to the 29 kDa mature form, primarily as the result of oligosaccharide trimming. The rPR3 binds [3H]DFP and hydrolyzes the substrate N-methoxysuccinyl-Ala-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by alpha 1-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PR3's targeting to granules. This rPR3 is the first to be recognized by most c-ANCA from WG patients and all anti-PR3 ANCA that were detected by standard anti-PR3 specific ELISA. This expression system for rPR3 represents a versatile tool for the analysis of its intracellular processing, structure-function relationships and interaction with autoantibodies.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Antibodies, Antineutrophil Cytoplasmic
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Autoantibodies / immunology*
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Base Sequence
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Cell Line
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DNA Primers
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Enzyme-Linked Immunosorbent Assay
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Gene Expression
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Granulomatosis with Polyangiitis
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Humans
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Hydrolysis
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Isoflurophate / metabolism
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Mast Cells / enzymology*
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Mast Cells / metabolism
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Microscopy, Fluorescence
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Microscopy, Phase-Contrast
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Molecular Sequence Data
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Myeloblastin
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Oligopeptides / metabolism
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Phenotype
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Protein Processing, Post-Translational
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Recombinant Proteins / immunology
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Recombinant Proteins / metabolism
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Serine Endopeptidases / chemistry
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Serine Endopeptidases / genetics
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Serine Endopeptidases / immunology*
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Serine Endopeptidases / metabolism*
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Transfection
Substances
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Antibodies, Antineutrophil Cytoplasmic
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Autoantibodies
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DNA Primers
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Oligopeptides
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Recombinant Proteins
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Isoflurophate
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N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-4-nitroanilide
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Serine Endopeptidases
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Myeloblastin