A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

Abstract

SUMMARYANCA directed against PR3 are highly specific for Wegener's granulomatosis and microscopic polyangiitis, and have been implicated in the pathogenesis of small vessel vasculitis. Most PR3-ANCA are directed against conformational epitopes on PR3. This study was designed to determine whether the cleavage of the N-terminal activation dipeptide of PR3 is required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variants were expressed in the epithelial cell line, 293. As confirmed by radiosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants were expressed in 293 cells: rPR3-S176A and D-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, D-rPR3-S176A does not. Culture supernatants of rPR3-S176A and D-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA þ sera were tested. With D-rPR3-S176A as antigen all 40 were recognized, compared with only 34 of 40 when rPR3-S176A served as target antigen. The majority of the serum samples contained a mixture of antibodies reacting with epitopes accessible on the mature and on the proform of PR3. In conclusion, the cleavage of the Nterminal activation dipeptide of PR3 is not an absolute requirement for recognition by all PR3-ANCA. However, a substantial proportion of PR3-ANCA recognize (a) target antigen(s) exposed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred exclusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mammalian cells without regulated secretory pathway can be used for their expression.

A Monoclonal Antibody (MCPR3-7) Interfering with the Activity of Proteinase 3 by an Allosteric Mechanism

“…Similarly, the preparation and characterization of the active site mutant carrying a carboxyl-terminal c-myc-His tag (PR3-S195A-cmyc) has been described elsewhere (27). When expressed in HEK 293 cells and secreted into the cell culture supernatant, pro-PR3ctp and PR3ctp-S195A-cmyc have the conformation of pro-PR3, whereas ⌬PR3ctp-S195A has the conformation of the mature enzyme (20,27).…”

“…Stably transfected proP-PR3ctp-expressing HEK 293 cells were selected and cultured as described previously (20,27), and proP-PR3ctp was purified from HEK 293 culture supernatant by immunoaffinity chromatography using MCPR3-2 coupled to a cyanogen bromide-activated Sepharose 4B column and 3 M KSCN in 1% NaHCO 3 as elution buffer. ProP-PR3ctp in the eluted fractions was quantified by capture ELISA as described (25), pooled, and concentrated after dialysis.…”

“…At this stage, it carries a short amino-terminal extension, the dipeptide Ala-Glu. This dipeptide prevents the molecule from assuming its active enzyme conformation prematurely during biosynthesis but is cleaved off by the dipeptidyl aminopeptidase I (cathepsin C) just before storage in primary granules (17)(18)(19)(20). After the removal of the amino-terminal dipeptide, the free positively charged amino terminus of Ile 16 (chymotrypsinogen numbering) forms an internal salt bridge with the side chain carboxylate of Asp 194 .…”

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

“…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).…”

Neutrophils: Molecules, Functions and Pathophysiological Aspects

“…Anti-PR3 recognize conformational epitopes (Bini et al, 1992). Of note, both anti-MPO (Short et al, 1995) and anti- PR3 (Sun et al, 1998) seem to recognize a pro-form that is not completely processed. The relevance of such findings in the pathophysiological mechanisms of vasculitis are still unknown.…”