Proteolysis by Granzyme B Enhances Presentation of Autoantigenic Peptidylarginine Deiminase 4 Epitopes in Rheumatoid Arthritis

J Proteome Res. 2017 Jan 6;16(1):355-365. doi: 10.1021/acs.jproteome.6b00617. Epub 2016 Oct 20.


Proteolysis of autoantigens can alter normal MHC class II antigen processing and has been implicated in the induction of autoimmune diseases. Many autoantigens are substrates for the protease granzyme B (GrB), but the mechanistic significance of this association is unknown. Peptidylarginine deiminase 4 (PAD4) is a frequent target of autoantibodies in patients with rheumatoid arthritis (RA) and a substrate for GrB. RA is strongly associated with specific MHC class II alleles, and elevated levels of GrB and PAD4 are found in the joints of RA patients, suggesting that GrB may alter the presentation of PAD4 by RA-associated class II alleles. In this study, complementary proteomic and immunologic approaches were utilized to define the effects of GrB cleavage on the structure, processing, and immunogenicity of PAD4. Hydrogen-deuterium exchange and a cell-free MHC class II antigen processing system revealed that proteolysis of PAD4 by GrB induced discrete structural changes in PAD4 that promoted enhanced presentation of several immunogenic peptides capable of stimulating PAD4-specific CD4+ T cells from patients with RA. This work demonstrates the existence of PAD4-specific T cells in patients with RA and supports a mechanistic role for GrB in enhancing the presentation of autoantigenic CD4+ T cell epitopes.

Keywords: antigen presentation; antigen processing; autoantigen; autoimmunity; epitope; hydrogen−deuterium exchange; mass spectrometry; peptidylarginine deiminase; rheumatoid arthritis; shared epitope.

MeSH terms

  • Aged
  • Amino Acid Sequence
  • Antigen Presentation
  • Arthritis, Rheumatoid / genetics
  • Arthritis, Rheumatoid / immunology*
  • Arthritis, Rheumatoid / pathology
  • Autoantibodies / biosynthesis
  • Autoantigens / chemistry
  • Autoantigens / genetics
  • Autoantigens / immunology*
  • Binding Sites
  • CD4-Positive T-Lymphocytes / immunology*
  • CD4-Positive T-Lymphocytes / pathology
  • Case-Control Studies
  • Deuterium Exchange Measurement
  • Epitopes, T-Lymphocyte / chemistry
  • Epitopes, T-Lymphocyte / genetics
  • Epitopes, T-Lymphocyte / immunology*
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Female
  • Gene Expression
  • Granzymes / chemistry
  • Granzymes / genetics
  • Granzymes / immunology*
  • Humans
  • Hydrolases / chemistry
  • Hydrolases / genetics
  • Hydrolases / immunology*
  • Male
  • Middle Aged
  • Models, Molecular
  • Protein Binding
  • Protein Conformation, alpha-Helical
  • Protein Conformation, beta-Strand
  • Protein Interaction Domains and Motifs
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases
  • Proteolysis
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / immunology
  • Substrate Specificity


  • Autoantibodies
  • Autoantigens
  • Epitopes, T-Lymphocyte
  • Recombinant Proteins
  • Hydrolases
  • GZMB protein, human
  • Granzymes
  • PADI4 protein, human
  • Protein-Arginine Deiminase Type 4
  • Protein-Arginine Deiminases