26 S proteasome-mediated production of an authentic major histocompatibility class I-restricted epitope from an intact protein substrate

Peptides displayed on the cell surface by major histocompatibility class I molecules (MHC class I) are generated by proteolytic processing of protein- antigens in the cytoplasm. Initially, antigens are degraded by the 26 S pro teasome, most probably following ubiquitination, However, it is unclear whe ther this proteolysis results in the generation of MHC class I ligands or i f further processing is required. To investigate the role of the 26 S prote asome in antigen presentation, we analyzed the processing of an intact anti gen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2K(b)-restricted peptide epitope, derived from ova lbumin SIINFEKL (termed ODC-ova), Utilizing recombinant antizyme to target the antigen to the 26 S proteasome, we found that proteolysis of ODC-ova by the 26 S proteasome resulted in the generation of the K-b-ligand. Mass spe ctrometry analysis indicated that in addition to SIINFEKL, the N-terminally extended ligand, HSIINFEKL, was also generated. Production of SIINFEKL was linear with time and directly proportional to the rate of ODC-ova degradat ion. The overall yield of SIINFEKL was approximately 5% of the amount of OD C-ova degraded. The addition of PA28, the 20 S, or the 20 S-PA28 complex to the 26 S proteasome did not significantly affect the yield of the antigeni c peptide. These findings demonstrate that the 26 S proteasome can efficien tly digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.