Characterization of a recombinant proteinase 3, the autoantigen in Wegener's granulomatosis and its reactivity with anti‐neutrophil cytoplasmic autoantibodies

Abstract

Using the bnculovlrus/Insect cells system, we have expmmed n recombinant protelnase 3 (PR3) --the neutrophil.derived sedne protease autonntlgen In Wegener's gntnulomatosis --as a illyemyiated Intrncellular and membrane-associated protein, OIIgosnochnrldes accounted for the difference in molecular weights between recombinant (34 kDa) and neutrophiI-PR3 (29 kDn), Whereas rabbit-anti-PR3 igG recognized both recombinant and nentrophll-derlved PR3, autoantibodies from Wegener patient sent reeognl~l only neutrophil-derived PR3, Although ollgosaeeharides were not involved in PR3 epitope recognition, autoantibodles did not recognize the amino acid primary structure of recombinant PR3, Improper disulfide bond formation and/or lack of post-translational events in insect cells, may affect the conformation of PR3, precluding its reactivity with sera from WG patients.

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

“…The most recent report of rPR3 expression in a baculovirus system has confirmed that that the majority of c-ANCA recognize conformational epitopes on PR3, and that insect cells are not able to process rPR3 into a conformationally intact form [13]. To determine whether rPR3 expressed by HMC-1/ PR3 cells is recognized by c-ANCA, 40 consecutive serum samples that had previously been determined to be c-ANCA positive by standard IIF on neutrophil cytospin preparations [21] and control sera (5 normal, 5 anti-myeloperoxidase ANCA positive, 10 containing various patterns of ANA and 5 containing anti-mitochondrial antibodies) were obtained from the clinical laboratory.…”

“…The expression of recombinant PR3 (rPR3) as a fusion protein in E. coli and in baculovirus systems failed to generate an enzymatically active recombinant product that is recognized by the majority of c-ANCA sera [11][12][13]. Thereby, these reports have confirmed that most c-ANCA recognize conformational epitopes of PR3.…”

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

“…Recently, different expression systems have been used to express recombinant PR3 (rPR3) variants [8][9][10][11][12]. Full length cDNA constructs encoding the PR3 zymogen cloned into baculovirus vectors have been expressed in Sf9 insect cells [8][9][10].…”

“…Full length cDNA constructs encoding the PR3 zymogen cloned into baculovirus vectors have been expressed in Sf9 insect cells [8][9][10]. It has been reported that the N-terminal activation dipeptide needs to be removed in vitro from the expressed protein in order to obtain the active enzyme with the N-terminal amino acid sequence Ile-ValGly-Gly [10].…”

“…It has been reported that the N-terminal activation dipeptide needs to be removed in vitro from the expressed protein in order to obtain the active enzyme with the N-terminal amino acid sequence Ile-ValGly-Gly [10]. The N-terminally unprocessed rPR3 zymogen from Sf9 cells is recognized only by a small number of PR3-ANCAcontaining sera [8,9]. In contrast, enzymatically active, N-terminally processed rPR3 expressed in the human mast cell line, HMC-1, appears to be recognized by all PR3-ANCA [13,14].…”