Longitudinal studies of patients with ANCA vasculitis demonstrate concurrent reactivity to complementary PR3 protein segments cPR3m and cPR3C and with no reactivity to cPR3N

Abstract

Antibodies recognizing the complement of the middle of PR3 (cPR3m) occur in ∼30% of PR3-anti-neutrophil cytoplasmic autoantibodies (ANCA)-vasculitis patients and immunization of animals with a peptide complementary to the middle of PR3 (cPR3m) induces not only anti-complementary PR3 antibodies, but also anti-PR3 antibodies derived through an anti-idiotypic response. PR3 epitopes recognized by patient ANCA, however, are not restricted to the middle of PR3. This prompted us to test for antibodies that react with proteins complementary to the terminal regions of PR3 (cPR3C and cPR3N) in PR3-ANCA patients. Anti-cPR3C reactivity was detected in 28% of patients but anti-cPR3N reactivity in only 15%. Ranked anti-cPR3C and anti-cPR3m reactivity correlated in the cohort, whereas there was no significant relationship between cPR3C and cPR3N reactivity. Serial samples from 3 patients' revealed that anti-cPR3C and anti-cPR3m reactivity followed a similar pattern over time. Serial samples from a fourth patient demonstrated an anti-cPR3N response without concurrent cPR3m or cPR3C reactivity. Epitope determination by mass spectrometry identified a 13-amino acid sequence on cPR3C that contained a common binding site recognized by antibodies from three patients. This peptide sequence contains a “PHQ” motif which was reported to be the basis for cross-reactivity of anti-cPR3m antibodies with plasminogen. Why these antibodies are detected in only ∼30% of the patients remains unclear. The data reveal that it is not due to lack of inclusion of flanking regions of complementary PR3 during screening. Instead, quite unexpectedly, the data demonstrate that patients' antibodies react with a restricted epitope that exists in both cPR3m and cPR3C.

Keywords::

• Autoantigen complementarity
• ANCA vasculitis
• anti-idiotypic antibodies
• PR3
• complementary protein pairs

Acknowledgments

This work was supported by the Wellcome Trust UK and by NIDDK/NIH grant PO1DK058335. We wish to thank Dr Susan Hogan for assistance in statistical analysis of the data. We thank Michael Brown and Libby Berg for locating patients' sample and compiling clinical information. We are indebted to Drs Carol Parker and Viorel Mocanu of the UNC-Duke Michael Hooker Proteomics Center for protein identification and epitope mapping.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.