A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide.
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.
Sun
J
J
Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Fass
D N
DN
Viss
M A
MA
Hummel
A M
AM
Tang
H
H
Homburger
H A
HA
Specks
U
U
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*
D019268:Antibodies, Antineutrophil Cytoplasmic; D004151:Dipeptides; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D053146:Myeloblastin