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

Abstract

Proteinase 3 (PR3) is a neutrophil serine protease targeted by anti-neutrophil cytoplasmic antibodies (ANCAs) in the autoimmune disease granulomatosis with polyangiitis (GPA)1–5. PR3 mutants were developed to investigate how PR3 interacts with the ANCAs and whether the interactions can be intervened by therapeutics. One mutant with a Ser195Ala mutation (iPR3-Val103) recognized as many ANCAs as wild-type PR3 (PR3-Val103)6–9, indicating that PR3-Val103 and iPR3-Val103 have equivalent ANCA-binding capabilities. A triple mutant of the latter (iHm5-Val103) bound a monoclonal antibody (moANCA518) from a patient with GPA on an epitope remote from the mutation sites. Unexpectedly, the corresponding epitope of iPR3-Val103 was inaccessible to moANCA518 under the same experimental conditions10,11. These observations demonstrate that a latent epitope of PR3 can be activated surprisingly by remote mutations in PR311. Here we report a comparative analysis of simulated B-factors (i.e., measurements of local mobility) of PR3-Val103, iPR3-Val103, and iHm5-Val103, demonstrating that the binding of moANCA518 to iHm5-Val103 is enabled by an increase in main-chain flexibility in the latent epitope caused by the remote muta-tions in iHm5-Val103. This epitope activation—achieved in vitro by remote mutations as we demonstrated or in vivo conceivably by remote protein»protein interactions12 or remote po-lymorphisms—may be a fundamental feature of antibody-mediated autoimmune diseases. Rigidifying B-cell epitopes on an autoantigen with therapeutics designed using the B-factor analysis disclosed here may lead to effective treatments for these autoimmune diseases by making the autoantigen inaccessible to existing autoantibodies. This analysis may also be used to predict and characterize epitopes and remote mutation effects.