title: Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA.
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.
authors.affiliation: Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA. specks.ulrich@mayo.edu
authors.forename: U
authors.initials: U
authors.lastname: Specks
authors.forename: D N
authors.initials: DN
authors.lastname: Fass
authors.forename: M P
authors.initials: MP
authors.lastname: Fautsch
authors.forename: A M
authors.initials: AM
authors.lastname: Hummel
authors.forename: M A
authors.initials: MA
authors.lastname: Viss
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies / Q000276:immunology*; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D015870:Gene Expression; D014890:Granulomatosis with Polyangiitis; D006801:Humans; D006868:Hydrolysis; D007531:Isoflurophate / Q000378:metabolism; D008407:Mast Cells / Q000201:enzymology* / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008969:Molecular Sequence Data; D053146:Myeloblastin; D009842:Oligopeptides / Q000378:metabolism; D010641:Phenotype; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D014162:Transfection
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D001323:Autoantibodies; D017931:DNA Primers; D009842:Oligopeptides; D011994:Recombinant Proteins; D007531:Isoflurophate; C067253:N-methoxysuccinyl-alanyl-alanyl-prolyl-valine-4-nitroanilide; D012697:Serine Endopeptidases; D053146:Myeloblastin