A study of conformational restraints on reactivity of human PR3-specific autoantibodies (ANCA) facilitated through protein folding manipulations of a new recombinant proteinase 3 protein

Abstract

Proteinase 3 (PR3)-specific antineutrophil cytoplasmic autoantibodies (PR3-ANCA) recognize conformational epitopes on PR3. This study evaluates PR3-ANCA target epitopes utilizing a novel recombinant PR3 (rPR3) produced to accommodate manipulations of the N-terminal domain. The rPR3 molecule contains an N-terminus six histidine tag, which can be removed by enterokinase (EK) cleavage of an adjacent EK cleavage site. Once cleaved the remaining amino acids correspond to the mature N-terminus of PR3. This rPR3 can be manipulated to produce three variant forms: tagged rPR3^+his, EK-cleaved (his-tag removed) rPR3^{− his}, and EK-cleaved, denatured/refolded rPR3^{− his/dr} (the proteolytically active form). Patients with clinically positive PR3-ANCA titers (n = 40) were confirmed for reactivity against purchased native PR3 in our system. Controls included 29 healthy volunteers and 34 MPO-ANCA patients. All PR3-ANCA sera samples tested were reactive with one or more forms of the recombinant protein (greater than mean ELISA OD 405 + 2 SDs of controls). Of significance, three sera were reactive with non-active forms only and three others were more reactive with rPR3^{− his/dr} than with native PR3. The results of our evaluation of PR3-ANCA sera for reactivity against the three forms of our rPR3 protein uniquely exemplify the diverse array of epitopes within the PR3-ANCA population. This new recombinant form of PR3 should provide a suitable approach to mapping ANCA epitopes using site-directed mutagenesis.

• Recombinant proteinase 3
• anti-neutrophil cytoplasmic autoantibodies
• ANCA
• vasculitis

Acknowledgements

The authors wish to especially thank Thomas Hellmark, PhD of Lund University, Lund, Sweden for his generous guidance and discussion in preparation for production of the rPR3 molecules. These studies were supported by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK, grant # NOT-DK-01-006). Research performed by W.F.P. was also supported in part by the Scott Neil Schwirck Research Fellowship and the Holderness Research Fellowship at UNC-Chapel Hill.