Antineutrophil cytoplasmic autoantibodies in Wegener's granulomatosis recognize conformational epitope(s) on proteinase 3.

Abstract

Although proteinase 3 (PR3) has been identified as a major autoantigen in Wegener's granulomatosis, the precise antibody specificity(ies) and requirements for epitope recognition have not been characterized. We analyzed 11 sera containing antineutrophil cytoplasmic antibodies (cANCA) for binding to azurophilic granule proteins extracted from neutrophils under various conditions and for binding to native or rPR3. Ten of 11 (91%) of the cANCA sera bound to PR3 extracted by nonionic detergents when tested by immunoprecipitation or by IEF followed by capillary immunoblotting. Antibody binding to PR3 was retained when IEF was performed under dissociating conditions (8 M urea) indicating that PR3 is the major autoantigen in azurophilic granules and that association with other proteins is not required for antigenicity. In contrast, antigenicity was totally destroyed by exposure of PR3 to reducing agents or to low pH (less than 3.0) and was either lost or considerably diminished after boiling in SDS. cANCA sera also showed little or no binding to rPR3 expressed as a fusion protein in Escherichia coli or synthesized by wheat germ ribosomes in vitro. Inasmuch as PR3 enzymatic activity was partially retained after acid extraction, these findings indicate that cANCA bind to a limited number of conformational epitopes on PR3. In addition, IEF followed by capillary immunoblotting appears to be a sensitive and specific method to detect anti-PR3 antibodies in Wegener's granulomatosis.

Activation of a Latent Epitope Causing Differential Binding of Antineutrophil Cytoplasmic Antibodies to Proteinase 3

“…The oligoclonal PR3-ANCAs from patients with GPA are known to bind to different epitopes of the folded PR3 antigen, whereas denatured PR3 or improperly folded recombinant PR3 generated in nonmammalian expression systems do not reliably bind PR3-ANCAs (12)(13)(14). Consistently, studies of continuous epitope mapping of PR3-ANCAs using oligopeptides have generated inconclusive results (15)(16)(17)(18).…”

Defensin Interactions in Relation to Monoclonal and Disease-Related Proteinase 3 Antibodies Binding at the Catalytic Site

“…Its granules harbor multiple antimicrobial agents, including proteases and antimicrobial peptides [ 1 , 2 ]. Among the proteases, PR3 has attracted much interest due to the presence and diagnostic value of PR3 AuAbs in GPA [ 3 , 4 , 5 , 6 , 7 , 8 , 9 ]. Additionally, the human neutrophil peptides (HNPs (α-defensins)) have been the subject of much research to elucidate their antimicrobial properties [ 44 , 45 , 46 ].…”

“…PR3 is also an autoantigen found in various forms of vasculitis [ 3 , 4 , 5 , 6 ]. Molecular interaction studies, e.g., using epitope mapping, may shed some light on autoimmune vasculitis and, therefore, the major focus has been on elucidating the immunogenicity and antigenicity of PR3 in relation to diagnostics and pathophysiology of vasculitis [ 6 , 7 , 8 , 9 ]. PR3 is encoded by a single gene located together with two other elastase-like genes at a single genetic locus, and it is synthesized as a pre-pro-protease, which is processed to mature PR3 by proteolytic removal of a signal peptide, an N-terminal pro-dipeptide and a C-terminal pro-peptide [ 2 , 10 , 11 , 12 , 13 ].…”

“…The location and nature of antibody (Ab) epitopes on PR3 have been studied extensively using sera from patients with ANCA (anti-neutrophil cytoplasm antibody) vasculitis and monoclonal antibodies (MAbs), in combination with purified native mature PR3, recombinant PR3 constructs and synthetic peptides. Most Abs react with conformational epitopes and depend on intact disulfide bridges of PR3 [ 9 , 10 , 18 ]. This may partly explain why many studies have yielded different results.…”

“…In general, PR3 expressed in E. coli has shown no or only weak binding of patients’ autoantibodies (AuAbs), presumably due to incorrect folding. However, PR3 expressed in eukaryotic/mammalian expression systems has shown binding of Abs from some but not all patients and binding depended for a subset on removal of the pro-dipeptide and correct glycosylation [ 9 , 19 , 20 , 21 , 22 , 23 , 24 , 25 , 26 , 27 , 28 , 29 ]. Studies with synthetic peptides and peptides from proteolytic digestion of PR3 have yielded some detailed molecular information on epitopes for patient PR3 Abs, pointing to the involvement of regions around the catalytic site [ 18 , 30 , 31 , 32 , 33 ].…”