Extensive citrullination promotes immunogenicity of hsp90 through protein unfolding and exposure of cryptic epitopes

Timothy S. Travers, Lisa Harlow, Ivan O. Rosas, Bernadette R. Gochuico, Ted R. Mikuls, Sanjoy K Bhattacharya, Carlos J. Camacho, Dana Ascherman

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Abstract

Post-translational protein modifications such as citrullination have been linked to the breach of immune tolerance and clinical autoimmunity. Previous studies from our laboratory support this concept, demonstrating that autoantibodies targeting citrullinated isoforms of heat shock protein 90 (HSP90) are associated with rheumatoid arthritis complicated by interstitial lung disease. To further explore the relationship between citrullination and structural determinants of HSP90 immunogenicity, we employed a combination of ELISA-based epitope profiling, computational modeling, and mass-spectrometric sequencing of peptidylarginine deiminase (PAD)-modified protein. Remarkably, ELISAs involving selected citrullinated HSP90b/a peptides identified a key epitope corresponding to an internal Arg residue (R502 [HSP90b]/R510 [HSP90a]) that is normally buried within the crystal structure of native/unmodified HSP90. In vitro time/dose-response experiments reveal an ordered pattern of PAD-mediated deimination events culminating in citrullination of R502/R510. Conventional as well as scaled molecular dynamics simulations further demonstrate that citrullination of selected Arg residues leads to progressive disruption of HSP90 tertiary structure, promoting exposure of R502/R510 to PAD modification and subsequent autoantibody binding. Consistent with this process, ELISAs incorporating variably deiminated HSP90 as substrate Ag indicate a direct relationship between the degree of citrullination and the level of ex vivo Ab recognition. Overall, these data support a novel structural paradigm whereby citrullination-induced shifts in protein structure generate cryptic epitopes capable of bypassing B cell tolerance in the appropriate genetic context.

Original languageEnglish (US)
Pages (from-to)1926-1936
Number of pages11
JournalJournal of Immunology
Volume197
Issue number5
DOIs
StatePublished - Sep 1 2016

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ASJC Scopus subject areas

  • Immunology
  • Medicine(all)

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