Mutation Effects on Remote Epitopes of An Autoantigen Decoded with Simulated B-Factors

Abstract

Mutants of a catalytically inactive variant of Proteinase 3 (PR3)-iPR3-Val 103 possessing a Ser195Ala mutation relative to wild-type PR3-Val 103 -offer insights into how autoantigen PR3 interacts with antineutrophil cytoplasmic antibodies (ANCAs) in granulomatosis with polyangiitis (GPA) and whether such interactions can be interrupted. Here we report that iHm5-Val 103 , a triple mutant of iPR3-Val 103 , bound a monoclonal antibody (moANCA518) from a GPA patient on an epitope remote from the mutation sites, whereas the corresponding epitope of iPR3-Val 103 was latent to moANCA518.Simulated B-factor analysis revealed that the binding of moANCA518 to iHm5-Val 103 was due to increased main-chain flexibility of the latent epitope caused by remote mutations, suggesting rigidification of epitopes with therapeutics to alter pathogenic PR3•ANCA interactions as new GPA treatments. MATERIALS AND METHODS MaterialsReagents were obtained from Sigma (St. Louis, MO) unless specified otherwise. The human epithelial kidney cell line 293 used for the expression of recombinant PR3 mutants was obtained from ATCC (Rockville, MD).iPR3-Val 103 and iHm5-Val 103 : The cDNA constructs coding for iPR3-Val 103 and iHm5-Val 103 and their expression in HEK293 cells were described in detail elsewhere (11,12). Both mutants carry a carboxyterminal cmyc-peptide extension and a poly-His peptide extension for purification using nickel columns from GE Healthcare (Chicago, IL) and for anchoring in ELISAs as previously described and specified below (11-15).moANCA518: DNA barcode-enabled sequencing of the antibody repertoire was performed on plasmablasts derived from a PR3-targeting ANCA (PR3-ANCA) positive patient as described elsewhere for rheumatoid arthritis and Sjögren syndrome (16)(17)(18). Phylograms of the antibody repertoires revealed clonal families of affinity matured antibodies with shared heavy and light chain VJ usage.Twenty-five antibodies were selected for recombinant expression (18) and tested for reactivity with recombinant ANCA antigens [including myeloperoxidase (15), human neutrophil elastase (19-21), iPR3-Val 103 , and iHm5-Val 103 ] using the ELISA. As described in Results, one antibody bound iHm5-Val 103 but not iPR3-Val 103 and is termed moANCA518, whereas none of the other 24 antibodies bound either of the two PR3 antigens or other ANCA antigens.Epitope-specific anti-PR3 moAbs: PR3G-2 (22) was a gift from C.G.M. Kallenberg of the University of Groningen. WGM2 (11, 23) was purchased from Hycult Biotech Inc (Wayne, PA). MCPR3-3 was made as previously described (8, 11). Enzyme-linked immunosorbent assaysELISAs used for detection of PR3-ANCA were described in detail elsewhere (12,13,15). In brief, either purified PR3 mutants or culture media supernatants from PR3 mutant expressing 293 cell clones diluted in the IRMA buffer (0.05 mM Tris-HCl, 0.1 M NaCl, pH 7.4, and 0.1% bovine serum albumin) were incubated in Pierce® nickel-coated plates from Thermo Fisher Scientific (Waltham, CA) for 1 hour at room temperature; control wells were...