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 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.
| Original language | English (US) |
|---|---|
| Pages (from-to) | 265-270 |
| Number of pages | 6 |
| Journal | FEBS Letters |
| Volume | 390 |
| Issue number | 3 |
| DOIs | |
| State | Published - Jul 29 1996 |
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Keywords
- Anti-neutrophil cytoplasmic antibody
- Expression system
- Human
- Mast cell
- Neutral serine protease
- Proteinase 3
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Molecular Biology
Cite this
Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA. / Specks, Ulrich; Fass, David N.; Fautsch, Michael P; Hummel, Amber M.; Viss, Margaret A.
In: FEBS Letters, Vol. 390, No. 3, 29.07.1996, p. 265-270.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Recombinant human proteinase 3, the Wegener's autoantigen, expressed in HMC-1 cells is enzymatically active and recognized by c-ANCA
AU - Specks, Ulrich
AU - Fass, David N.
AU - Fautsch, Michael P
AU - Hummel, Amber M.
AU - Viss, Margaret A.
PY - 1996/7/29
Y1 - 1996/7/29
N2 - 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 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.
AB - 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 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.
KW - Anti-neutrophil cytoplasmic antibody
KW - Expression system
KW - Human
KW - Mast cell
KW - Neutral serine protease
KW - Proteinase 3
UR - http://www.scopus.com/inward/record.url?scp=0030605891&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=0030605891&partnerID=8YFLogxK
U2 - 10.1016/0014-5793(96)00669-2
DO - 10.1016/0014-5793(96)00669-2
M3 - Article
C2 - 8706874
AN - SCOPUS:0030605891
VL - 390
SP - 265
EP - 270
JO - FEBS Letters
JF - FEBS Letters
SN - 0014-5793
IS - 3
ER -