TY - JOUR
T1 - Extensive citrullination promotes immunogenicity of hsp90 through protein unfolding and exposure of cryptic epitopes
AU - Travers, Timothy S.
AU - Harlow, Lisa
AU - Rosas, Ivan O.
AU - Gochuico, Bernadette R.
AU - Mikuls, Ted R.
AU - Bhattacharya, Sanjoy K.
AU - Camacho, Carlos J.
AU - Ascherman, Dana P.
N1 - Funding Information:
This work was supported by Veterans Administration Merit Review Award 1I01BX000788 (to D.P.A.), an American College of Rheumatology/Research Education Foundation Within Our Reach Foundation Investigator Award (to D.P.A.), National Institutes of Health Grant R01 GM97082 (to C.J.C.), Commonwealth of Pennsylvania Department of Health Grant SAP 4100062224 (to C.J.C.), and U.S. Department of Defense Grant MS150135 (to S.K.B.). This work was also supported in part by the Intramural Research Program of the National Human Genome Research Institute, National Institutes of Health (to B.R.G.).
PY - 2016/9/1
Y1 - 2016/9/1
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=84983740511&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=84983740511&partnerID=8YFLogxK
U2 - 10.4049/jimmunol.1600162
DO - 10.4049/jimmunol.1600162
M3 - Article
C2 - 27448590
AN - SCOPUS:84983740511
VL - 197
SP - 1926
EP - 1936
JO - Journal of Immunology
JF - Journal of Immunology
SN - 0022-1767
IS - 5
ER -