title: 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.
authors.affiliation: Division of Rheumatic Diseases, Hospital for Special Surgery, Cornell University Medical Center, New York, NY 10021.
authors.forename: P
authors.initials: P
authors.lastname: Bini
authors.forename: J E
authors.initials: JE
authors.lastname: Gabay
authors.forename: A
authors.initials: A
authors.lastname: Teitel
authors.forename: M
authors.initials: M
authors.lastname: Melchior
authors.forename: J L
authors.initials: JL
authors.lastname: Zhou
authors.forename: K B
authors.initials: KB
authors.lastname: Elkon
mesh_terms: D000918:Antibody Specificity; D001323:Autoantibodies / Q000276:immunology*; D001324:Autoantigens / Q000737:chemistry*; D015153:Blotting, Western; D003593:Cytoplasm / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D014890:Granulomatosis with Polyangiitis / Q000276:immunology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008623:Mercaptoethanol / Q000494:pharmacology; D053146:Myeloblastin; D009504:Neutrophils / Q000201:enzymology / Q000276:immunology*; D009195:Peroxidase / Q000276:immunology; D011233:Precipitin Tests; D011487:Protein Conformation / Q000187:drug effects; D011489:Protein Denaturation / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000276:immunology; D012697:Serine Endopeptidases / Q000276:immunology* / Q000648:ultrastructure; D012967:Sodium Dodecyl Sulfate / Q000494:pharmacology
chemical_list: D001323:Autoantibodies; D001324:Autoantigens; D000939:Epitopes; D011993:Recombinant Fusion Proteins; D012967:Sodium Dodecyl Sulfate; D008623:Mercaptoethanol; D009195:Peroxidase; D012697:Serine Endopeptidases; D053146:Myeloblastin