PRODUCTION OF A SPECIFIC MAJOR HISTOCOMPATIBILITY COMPLEX CLASS I-RESTRICTED EPITOPE BY UBIQUITIN-DEPENDENT DEGRADATION OF MODIFIED OVALBUMIN IN LYMPHOCYTE LYSATE

Peptide epitopes presented through class I major histocompatability co mplex (MHC class I) on the cell! surface, are generated by proteolytic processing of protein-antigens in the cytoplasm. The length and amino acid sequence determine whether a given peptide can fit into the pept ide binding groove of class I heavy chain molecules and subsequently b e presented to the immune system. The mode ai action of the processing pathway Is therefore of great interest. go study the processing mecha nism of MHC class I-restricted intracellular antigens, me reconstitute d the proteolytic processing of a model antigen in a cell-free system. Incubation of oxidized and urea-treated OVA in lymphocyte lysate resu lted in partial degradation of the antigen, Degradation of the antigen depended on the presence of ATP, Addition of methylated ubiquitin abo lished the reaction which was then restored by addition of am excess o f native ubiquitin, indicating that the breakdown of the antigen in ly mphocyte 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 lymph ocytes directed against OVA-derived, H-2K(b)-restricted peptide (SIINF EKL), and by st monoclonal antibody that recognizes cell-bound K-b-SII NFEKL complexes. Formation of the peptide epitope depended on the pres ence of ATP and ubiquitin. These results indicate that proteolytic pro cessing of modified OVA is carried out by the ubiquitin-mediated degra dation system. The experimental system described provides a tool to an alyze else molecular mechanisms underlying the generation of specific, MHC class I-restricted peptide epitopes.