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...

Peptide epitopes presented through class I major histocompatability complex (MHC class I) on the cell surface, are generated by proteolytic processing of proteinantigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the peptide binding groove of class I heavy chain molecules and subsequently be presented to the immune system. The mode of action of the processing pathway is therefore of great interest. To study the processing mechanism of MHC class I-restricted intracellular antigens, we reconstituted the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resulted in partial degradation of the antigen. Degradation of the antigen depended on the presence of ATP. Addition of methylated ubiquitin abolished the reaction which was then restored by addition of an excess of native ubiquitin, indicating that the breakdown of the antigen in lymphocyte lysate is mediated by the ubiquitin proteolytic system. Upon incubation of modified OVA in lymphocyte lysate, a specific antigenic peptide was generated. The peptide was recognized by cytotoxic T lymphocytes directed against OVA-derived, H-2K b -restricted peptide (SIINFEKL), and by a monoclonal antibody that recognizes cell-bound K b -SIINFEKL complexes. Formation of the peptide epitope depended on the presence of ATP and ubiquitin. These results indicate that proteolytic processing of modified OVA is carried out by the ubiquitin-mediated degradation system. The experimental system described provides a tool to analyze the molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.Cells display foreign and altered intracellular protein-antigens to cytotoxic T-lymphocytes through MHC 1 class I molecules. Intracellular antigens are not presented directly. The intracellular antigen is first proteolyzed in the cytoplasm of the antigen presenting cell to yield short peptides. Suitable peptides are then translocated through specialized peptide transporters (termed TAP) into the lumen of the endoplasmic reticulum where they bind to newly synthesized MHC class I molecules. The entire complex is then stabilized and transported to the plasma membrane where it is presented to the immune system (1).Proteolysis and generation of antigenic peptides is carried out by the multicatalytic protease (26 S proteasome). Direct evidence for the involvement of the proteasome in the processing of MHC class-I restricted intracellular antigens came from experiments in which membrane-permeable inhibitors of proteasomes were added to cells and the capacity of these cells to present antigens was monitored. These inhibitors, which block proteasome activity in vitro, inhibited the cellular turnover of short-lived and long-lived proteins, assembly of class I molecules as well as presentation of OVA introduced into the cytoplasm (2).The proteasome constitutes the major proteolytic activity in the cytosol and nucleus of all eukaryotes (3)...