title: 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: ANCA 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 delta-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, delta-rPR3-S176A does not. Culture supernatants of rPR3-S176A and delta-rPR3-S176A expressing 293 cells were used as sources of target antigen for PR3-ANCA testing by capture ELISA. Forty unselected consecutive PR3-ANCA+ sera were tested. With delta-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 N-terminal 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.
authors.affiliation: Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: J
authors.initials: J
authors.lastname: Sun
authors.forename: D N
authors.initials: DN
authors.lastname: Fass
authors.forename: M A
authors.initials: MA
authors.lastname: Viss
authors.forename: A M
authors.initials: AM
authors.lastname: Hummel
authors.forename: H
authors.initials: H
authors.lastname: Tang
authors.forename: H A
authors.initials: HA
authors.lastname: Homburger
authors.forename: U
authors.initials: U
authors.lastname: Specks
mesh_terms: D019268:Antibodies, Antineutrophil Cytoplasmic / Q000276:immunology* / Q000378:metabolism; D002460:Cell Line; D002461:Cell Line, Transformed; D004151:Dipeptides / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D053146:Myeloblastin; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology; D012697:Serine Endopeptidases / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*
chemical_list: D019268:Antibodies, Antineutrophil Cytoplasmic; D004151:Dipeptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin