Granzyme B as an indicator and generator of altered antigen structures in autoimmunity

Systemic autoimmune disease is characterized by specific autoantibody responses that associate with distinct clinical phenotypes. While the mechanism for generating this disease-specific autoreactivity is unclear, unique antigen structures within the target tissue may drive autoimmune responses. It has been observed that a majority of autoantigens, but not nonautoantigens, are cleaved by the cytotoxic lymphocyte granule protease granzyme B (GrB). In a search for GrB substrates from amongst tumor antigens, we identified a GrB cleavage site (ICTD249) within the melanoma tumor antigen and vitiligo autoantigen, tyrosinase. Surprisingly, this site is required for proper tyrosinase folding and trafficking. Two distinct conformations of tyrosinase were observed in cells: a GrB-susceptible and a GrB-resistant form, of which the cleavable form is preferentially expressed in human melanomas. We also describe here, the GrB-susceptibility of two rheumatoid arthritis (RA) antigens: (1) the chaperone protein, BiP, and (2) the intermediate filament protein, vimentin. Interestingly, BiP is cleaved only in the absence of nucleotide, and calcium alters the GrB-cleavage profile of vimentin, again highlighting an influence of protein conformation on GrB-susceptibility. The form of vimentin targeted in RA contains citrulline residues, generated by the post-translational deimination of arginine. In the course of our work, we serendipitously discovered that the proteins that catalyze this amino acid conversion, peptidylarginine deiminases (PADs), are cleavable by GrB, and one of them, PAD4, is itself a highly specific (99%), frequently targeted (32-45%) autoantigen in RA. A prominent antibody-binding epitope (which we designate Type II) occurs within the N-terminal region of PAD4 that includes genetic polymorphisms recently shown to confer susceptibility to RA. Type II autoantibodies are also associated with elevated serum C-reactive protein levels, which mark inflammation. These data suggest that PAD4 is one of the long sought-after propagating autoantigens in RA. All of our observations are consistent with the hypotheses that within disease-specific microenvironments GrB cleavage: (1) can be an indication of novel antigen conformations, and (2) can generate previously nontolerized antigen structures in the form of novel polypeptide fragments. Such altered-antigen structures, arising from conformational changes and/or proteolysis, could drive autoimmune responses.