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-I. 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 snbstrate N-methoxysuccinylAla-Ala-Pro-Val-pNA. The enzymatic activity is inhibited by greater than 95% by al-PI. The rPR3 and the enzymatically inactive mutant rPR3-S176A are both packaged in granules. Thus, proteolytic autoprocessing is not required for PRYs 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 intraceHular processing, structure-function relationships and interaction with autoantibodies.

Antineutrophil cytoplasmic antibodies reacting with human neutrophil elastase as a diagnostic marker for cocaine‐induced midline destructive lesions but not autoimmune vasculitis

“…As previously reported (21,30), lysates of HMC-1 and HMC-1/VEC cells displayed no endogenous hydrolytic activity against this sensitive substrate for HNE. The hydrolysis of N-MeOSucc-AAPV-pNA by HMC-1/HNE lysates of 10 6 cells was equivalent to that of 40 ng of purified PMN HNE mixed in with cell lysate of 10 6 sham-transfected HMC-1/VEC cells ( Figure 1C).…”

“…The human mast cell line HMC-1, a kind gift from Dr. J. H. Butterfield (Mayo Clinic, Rochester, MN), was cultured in RPMI, 10% bovine calf serum, and 1 g/ml monothioglycerol, as previously described (21). The full-length complementary DNA (cDNA) for wild-type HNE was amplified from total cDNA of human bone marrow cells using the forward primer DJ568 (5Ј-CCGGATCCCCAGCCCCACCAT-3Ј), the backward primer DJ569 (5Ј-GGCAGCCCTTCTCAGTGGGTC-3Ј), and Pfu DNA polymerase.…”

“…The cDNA sequence was verified by comparison with the published sequence (GenBank accession number Y00477). The plasmid was transfected into HMC-1 cells by electroporation (21), and rHNE-expressing HMC-1 cell populations were selected using Zeocin (100 g/ml) and cloning by limiting dilution. Sham-transfected (plasmid pcDNA3 without insert) control cells (HMC-1/VEC) were cultured and selected in parallel under the same conditions.…”

“…The enzymatic activity of rHNE in comparison with that of purified native HNE was determined by measuring the hydrolysis of the elastase substrate N-methoxysuccinyl-Ala-Ala-Pro-Val p-nitroanilide (NMeOSucc-AAPV-pNA), as described in detail elsewhere (21). Inhibitors, when used, were added during the first 30-minute incubation, prior to the addition of substrate, in the following concentrations: for ␣ 1 -plasmin inhibitor (␣ 1 PI), 50 g/ml; for eglin C, 0.1 M; and for aprotinin, 0.1 trypsin-inhibiting units/ml.…”

“…Biosynthetic labeling of HMC-1/HNE and HMC-1/ VEC cells using 35 S-methionine/ 35 S-cysteine (ICN Biomedicals, Costa Mesa, CA), labeling of purified HNE with 3 Hdiisopropylfluorophosphate (DFP), and immunoprecipitation using anti-HNE antibodies and HNE ANCA were performed according to previously published procedures (21,24).…”