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

Abstract

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-2K b -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 ODCova by the 26 S proteasome resulted in the generation of the K b -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. Cells display foreign and altered intracellular antigens to cytotoxic T lymphocytes (CTL)1 through MHC class I molecules. Antigenic peptides presented through class I molecules are generated in the cytoplasm by proteolytic degradation of endogenously synthesized antigens. Suitable peptides are then translocated through specialized peptide transporters (termed TAP) to the lumen of the endoplasmic reticulum, where they bind and subsequently stabilize newly synthesized MHC class I molecules. Assembled class I molecules then migrate to the cell surface for recognition by T cells (1).There is now substantial evidence implicating the proteasome in antigen processing. When membrane-permeable inhibitors of proteasomes were added to cells, they severely inhibited proteasome activity in vitro, the cellular turnover of short and long lived proteins, and assembly of class I molecules as well as presentation of ovalbumin (OVA) introduced into the cytoplasm (2, 3).Proteasomes are multicatalytic complexes that constitute the major proteolytic activity in the cytosol and nucleus of all eukaryotes. Proteasomes are found in the cytoplasm as 20 and 26 S particles. The 20 S proteasome is a barrel-shaped complex consisting of four stacked rings, each composed of seven related subunits. The outer rings are formed by noncatalytic ␣ subunits, whereas catalytic ␤ subunits occupy the inner two rings. The 20 S proteasome is an ATP-independent protease that in vitro cleaves only peptides. It can also digest several unfolded proteins, but onl...

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