Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC‐1 cells is enzymatically active and recognized by c‐ANCA

FEBS Letters

Hummel, Amber M.²  Fass, David N.¹  Viss, Margaret A.²  Fautsch, Michael P.²  Specks, Ulrich² 

[1]Hematology Research Unit, Mayo Clinic and Foundation, Rochester, MN, USA

[2]Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Guggenheim Bldg, 642A, 200 First Street SW, Rochester, MN 55905, USA

关键词: Proteinase 3;    Human;    Mast cell;    Neutral serine protease;    Anti-neutrophil cytoplasmic antibody;    Expression system;    αl-PI;    α1-protease inhibitor;    ANCA;    anti-neutrophil cytoplasmic antibodies;    c-ANCA;    cytoplasmic fluorescence pattern ANCA;    p-ANCA;    perinuclear fluorescence pattern ANCA;    BCS;    bovine calf serum;    DFP;    diisopropylfluorophosphate;    DPPI;    dipeptidylpeptidase I;    II17;    indirect immunofluorescence;    moAB;    monoclanal antibody;    PBS;    phosphate-buffered saline;    PCR;    polymerase chain reaction;    pNA;    p-nitroanilide;    PR3;    proteinase 3;    rPR3;    recombinant PR3;    WG;    Wegener's granulomatosis;   

DOI  :  10.1016/0014-5793(96)00669-2

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

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-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.

【 授权许可】

Unknown