A PROPORTION OF PROTEINASE-3 (PR3)-SPECIFIC ANTINEUTROPHIL 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 granulomat osis and microscopic polyangiitis, and have been implicated in the pat hogenesis of small vessel vasculitis. Most PR3-ANCA are directed again st conformational epitopes on PR3. This study was designed to determin e whether the cleavage of the N-terminal activation dipeptide of PR3 i s required for the binding of PR3-ANCA. Recombinant PR3 (rPR3) variant s were expressed in the epithelial cell line, 293. As confirmed by rad iosequencing, the rPR3 secreted into the 293 cell culture supernatant is N-terminally unprocessed. Two enzymatically inactive rPR3 mutants w ere expressed in 293 cells: rPR3-S176A and Delta-rPR3-S176A, rPR3-S176 A contains the N-propetide Ala-2-Glu-1, Delta-rPR3-S176A does not. Cul ture supernatants of rPR3-S176A and Delta-rPR3-S176A expressing 293 ce lls were used as sources of target antigen for PR3-ANCA testing by cap ture ELISA. Forty unselected consecutive PR3-ANCA(+) sera were tested. With Delta-rPR3-S176A as antigen all 40 were recognized, compared wit h 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 preform of PR3. In conclu sion, the cleavage of the N-terminal activation dipeptide of PR3 is no t an absolute requirement for recognition by all PR3-ANCA. However, a. substantial proportion of PR3-ANCA recognize (a) target antigen(s) ex posed only after the conformational change of PR3 associated with the N-terminal processing. In 15% of sera this PR3-ANCA subset occurred ex clusively. PR3-ANCA subtypes can be differentiated using specifically designed rPR3 variants as target antigens, and non-haematopoietic mamm alian cells without regulated secretory pathway can be used for their expression.