26 S Proteasome-mediated Production of an Authentic Major Histocompatibility Class I-restricted Epitope from an Intact Protein Substrate

• Published in: The Journal of biological chemistry Vol. 274; no. 31; pp. 21963 - 21972
• Main Authors: Ben-Shahar, Sary, Komlosh, Arthur, Nadav, Eran, Shaked, Isabella, Ziv, Tamar, Admon, Arie, DeMartino, George N., Reiss, Yuval
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 30.07.1999; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Animals; Cell-Free System; Chromatography, High Pressure Liquid; Cloning, Molecular; Epitopes - metabolism; H-2 Antigens - metabolism; Histocompatibility Antigens Class I - metabolism; Ligands; Mice; Mutagenesis, Insertional; Ornithine Decarboxylase - genetics; Ornithine Decarboxylase - metabolism; Ovalbumin - metabolism; Peptide Fragments - chemistry; Peptide Fragments - isolation & purification; Peptide Hydrolases - metabolism; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Rabbits; Recombinant Proteins - metabolism; Restriction Mapping
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.274.31.21963

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 proteasome, most probably following ubiquitination. However, it is unclear whether this proteolysis results in the generation of MHC class I ligands or if further processing is required. To investigate the role of the 26 S proteasome in antigen presentation, we analyzed the processing of an intact antigen by purified 26 S proteasome. A recombinant ornithine decarboxylase was produced harboring the H-2Kb-restricted peptide epitope, derived from ovalbumin 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 Kb-ligand. Mass spectrometry 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 degradation. The overall yield of SIINFEKL was approximately 5% of the amount of ODC-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 antigenic peptide. These findings demonstrate that the 26 S proteasome can efficiently digest an intact physiological substrate and generate an authentic MHC class I-restricted epitope.