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

Sary Ben-Shahar, Arthur Komlosh, Eran Nadav, Isabella Shaked, Tamar Ziv, Arie Admon, George N. DeMartino, Yuval Reiss

Research output: Contribution to journalArticle

48 Scopus citations

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-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.

Original languageEnglish (US)
Pages (from-to)21963-21972
Number of pages10
JournalJournal of Biological Chemistry
Volume274
Issue number31
DOIs
StatePublished - Jul 30 1999

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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