n-methoxysuccinyl-alanyl-alanyl-prolyl-valine-4-nitroanilide and Granulomatosis-with-Polyangiitis

Anti-neutrophil cytoplasmic antibodies (ANCA) against proteinase 3 (PR3) are strongly associated with Wegener's granulomatosis (WG) and are thought to be involved in its pathogenesis. Levels of PR3-ANCA do not always correspond to clinical disease activity. To investigate the relationship between functional effects of PR3-ANCA and disease activity, we tested the effect of IgG samples from sera of 43 WG patients, taken during active disease or remission, for their capacity to interfere with the proteolytic activity of PR3. Furthermore, longitudinal sera of seven WG patients were included. The enzymatic activity of PR3 was determined (1) with casein or with a small synthetic substrate and (2) by complexation of PR3 with alpha1-antitrypsin (alpha1-AT). With a fixed concentration (100 microg/ml) of IgG, PR3-ANCA from patients during an active phase of WG had a higher inhibitory capacity towards the proteolytic activity of PR3 and complexation of PR3 with alpha1-AT than did PR3-ANCA from WG patients during remission. However, the number of PR3-ANCA units that gave 50% inhibition of the PR3 enzymatic activity and its complexation with alpha1-AT was lower for patients during remission than for patients during an active phase of WG, indicating a stronger inhibitory capacity at a molar base. In conclusion, PR3-ANCA from patients during remission had a relatively higher inhibitory capacity towards the enzymatic activity of PR3 than PR3-ANCA from patients during an active phase. This may indicate that during active disease the ANCA titre is increased, but the number of active ANCA molecules that recognize the enzyme-inhibiting epitopes is not increased.

Topics: Adult; Aged; alpha 1-Antitrypsin; Antibodies, Antineutrophil Cytoplasmic; Caseins; Female; Granulomatosis with Polyangiitis; Humans; Longitudinal Studies; Male; Middle Aged; Myeloblastin; Oligopeptides; Recurrence; Remission, Spontaneous; Serine Endopeptidases; Serine Proteinase Inhibitors

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.

Topics: Antibodies, Antineutrophil Cytoplasmic; Autoantibodies; Base Sequence; Cell Line; DNA Primers; Enzyme-Linked Immunosorbent Assay; Gene Expression; Granulomatosis with Polyangiitis; Humans; Hydrolysis; Isoflurophate; Mast Cells; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Molecular Sequence Data; Myeloblastin; Oligopeptides; Phenotype; Protein Processing, Post-Translational; Recombinant Proteins; Serine Endopeptidases; Transfection