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Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA
Author(s) -
Specks Ulrich,
Fass David N.,
Fautsch Michael P.,
Hummel Amber M.,
Viss Margaret A.
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00669-2
Subject(s) - recombinant dna , chemistry , intracellular , microbiology and biotechnology , biochemistry , mutant , oligosaccharide , cell culture , enzyme , biology , gene , genetics
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 [ 3 H]DFP and hydrolyzes the substrate N ‐methoxysuccinyl‐Ala‐Ala‐Ala‐Pro‐Val‐pNA. The enzymatic activity is inhibited by greater than 95% by α1‐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.