P190 Structures of proteinase 3 and the CD177 receptor complex reveals major autoantibody epitope

Background/Aims Granulomatosis with polyangiitis is a life-threatening systemic vasculitis, characterised by anti-neutrophil cytoplasmic autoantibodies (ANCA) against proteinase 3 (PR3), a protease expressed intracellularly and on the surface of neutrophils. Most cell surface PR3 is bound to the receptor CD177, however the molecular mechanism of the interactions is not well understood. Previous studies have suggested that the hydrophobic patch on PR3 is involved in binding with CD177. We aimed to solve the structure of the PR3-CD177 complex, and CD177 alone, and determine whether the PR3-CD177 binding site is an important epitope for PR3-ANCA binding. Methods X-ray crystallography was used to solve the structure of PR3-CD177 complex and of an unliganded CD177 construct containing all four LU domains. The study was possible using a site-directed mutagenesis screen to identify stable PR3 variants that are readily expressed and purified from HEK293 cultures. We employed a panel of eight PR3-ANCA positive GPA patient plasma samples to test ANCA binding to our recombinant PR3 variants using ELISAs. Results Crystal structures revealed that the binding interface between PR3 and CD177 is mainly hydrophobic and involves the first two LU domains of CD177. The structure of unliganded CD177 shows that it is composed of two subdomains, each containing two tightly packed LU domains, that are connected by a flexible linker. Our standardised PR3-ANCA ELISAs show that for most GPA plasma samples, there is reduced ANCA binding when a recombinant PR3 variant that has the CD177-binding site masked was used. Conclusion The previously identified hydrophobic patch of PR3 is significantly involved in the binding interface between PR3 and CD177, and this site is a major autoantibody epitope that is targeted in most GPA patients examined in this study. Disclosure E. Seiradake: None. S. Dubey: None. J. Joha: None. C. Zheng-Gerard: None. S. Draper: None. H. Lin: None. A. Salama: None. K. McHugh: None.