A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

J. Sun, D. N. Fass, M. A. Viss, A. M. Hummel, H. Tang, H. A. Homburger, Ulrich Specks

Research output: Contribution to journalArticle

34 Citations (Scopus)

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 Δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, Δ-rPR3-S176A does not. Culture supernatants of rPR3- S176A and Δ-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 Δ-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.

Original languageEnglish (US)
Pages (from-to)320-326
Number of pages7
JournalClinical and Experimental Immunology
Volume114
Issue number2
DOIs
StatePublished - 1998

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Myeloblastin
Antineutrophil Cytoplasmic Antibodies
Dipeptides
Antigens
Epitopes
Serum
Microscopic Polyangiitis
Granulomatosis with Polyangiitis
Secretory Pathway

Keywords

  • Anti-neutrophil
  • Cytoplasmic antibodies
  • N-terminal propeptide
  • Recombinant proteinase 3
  • Target antigen

ASJC Scopus subject areas

  • Immunology

Cite this

A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide. / Sun, J.; Fass, D. N.; Viss, M. A.; Hummel, A. M.; Tang, H.; Homburger, H. A.; Specks, Ulrich.

In: Clinical and Experimental Immunology, Vol. 114, No. 2, 1998, p. 320-326.

Research output: Contribution to journalArticle

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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 Δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, Δ-rPR3-S176A does not. Culture supernatants of rPR3- S176A and Δ-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 Δ-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.",
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T1 - A proportion of proteinase 3 (PR3)-specific anti-neutrophil cytoplasmic antibodies (ANCA) only react with PR3 after cleavage of its N-terminal activation dipeptide

AU - Sun, J.

AU - Fass, D. N.

AU - Viss, M. A.

AU - Hummel, A. M.

AU - Tang, H.

AU - Homburger, H. A.

AU - Specks, Ulrich

PY - 1998

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N2 - 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 Δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, Δ-rPR3-S176A does not. Culture supernatants of rPR3- S176A and Δ-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 Δ-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.

AB - 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 Δ-rPR3-S176A. rPR3-S176A contains the N-propetide Ala-2-Glu-1, Δ-rPR3-S176A does not. Culture supernatants of rPR3- S176A and Δ-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 Δ-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.

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