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

Abstract

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.

Citrulline Not a Major Determinant in the Recognition of Peptidylarginine Deiminase 2 and 4 by Autoantibodies in Rheumatoid Arthritis

“…Our study also supports the growing appreciation that, in addition to the emerging list of modified antigens, unmodified proteins are also prominent targets of the autoimmune response in RA (13,19, 20). While anti‐PAD antibodies have historically been detected in their native form, this study confirms that these antibodies bind irrespective of citrullination in most patients.…”

Definition of naturally processed peptides reveals convergent presentation of autoantigenic topoisomerase-I epitopes in scleroderma

“…The 10 regions from which TOP1 peptides were presented are from diverse domains in the TOP1 protein and largely reside within structural elements, most dominantly alpha helices flanked by unstructured loops on the surface of the TOP1 protein ( Fig.3A and B). The localization of putative TOP1 epitopes within structural elements adjacent to flexible loops is similar to what has been observed for CD4+ T cell epitopes derived from other infectious and self-antigens (14,15). Using NAPA, we successfully identified a common set of TOP1 peptides derived from the whole TOP1 protein, which were naturally processed and presented by HLA-DR molecules in mo-DCs generated ex vivo from patients with ATA-positive scleroderma.…”

Granzyme B is elevated in autoimmune blistering diseases and cleaves key anchoring proteins of the dermal-epidermal junction

“…Growing evidence suggest that antigenic, GzmB-generated peptide fragments are part of a feed-forward loop that sustains the propagation of several autoimmune diseases (reviewed in 56 ). Studies suggest that GzmB is instrumental in auto-antigen generation in certain autoimmune conditions 57 – 59 . In our study, GzmB cleaves α6/β4 integrin and collagen VII in epitope regions recognized by auto-antibodies present in the sera of patients with certain pemphigoid diseases, and EBA respectively.…”