Specific proteolytic cleavages limit the diversity of the pool of peptides available to MHC class I molecules in living cells.
MHC class I molecules display peptides selected from a poorly characterized pool of peptides available in the endoplasmic reticulum. We analyzed the diversity of peptides available to MHC class I molecules by monitoring the generation of an OVA-derived octapeptide, OVA257-264 (SL8), and its C-terminally extended analog, SL8-I. The poorly antigenic SL8-I could be detected in cell extracts only after its conversion to the readily detectable SL8 with carboxypeptidase Y. Analysis of extracts from cells expressing the minimal precursor Met-SL8-I by this method revealed the presence of SL8/Kb and the extended SL8-I/Kb complexes, indicating that the peptide pool contained both peptides. In contrast, cells expressing full length OVA generated only the SL8/Kb complex, demonstrating that the peptide pool generated from the full length precursor contained only a subset of potential MHC-binding peptides. Deletion analysis revealed that SL8-I was generated only from precursors lacking additional C-terminal flanking residues, suggesting that the generation of the C terminus of the SL8 peptide involves a specific endopeptidase cleavage. To investigate the protease responsible for this cleavage, we tested the effect of different protease inhibitors on the generation of the SL8 and SL8-I peptides. Only the proteasome inhibitors blocked generation of SL8, but not SL8-I. These findings demonstrate that the specificities of the proteases in the Ag-processing pathway, which include but are not limited to the proteasome, limit the diversity of peptides available for binding by MHC class I molecules in the endoplasmic reticulum.
Serwold
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T
Division of Immunology, Department of Molecular and Cell Biology, University of California, Berkeley 94720, USA.
Shastri
N
N
D000596:Amino Acids / Q000276:immunology / Q000378:metabolism; D000818:Animals; D017951:Antigen Presentation; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004527:Egg Proteins / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D006183:H-2 Antigens / Q000276:immunology / Q000378:metabolism*; D006825:Hybridomas; D006868:Hydrolysis; D046911:Macromolecular Substances; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D010047:Ovalbumin / Q000037:antagonists & inhibitors / Q000276:immunology / Q000378:metabolism; D010446:Peptide Fragments / Q000276:immunology / Q000378:metabolism; D010455:Peptides / Q000276:immunology / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000276:immunology; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology; D013601:T-Lymphocytes / Q000201:enzymology* / Q000276:immunology / Q000378:metabolism
D000596:Amino Acids; D004527:Egg Proteins; D006183:H-2 Antigens; C032876:H-2Kb protein, mouse; D046911:Macromolecular Substances; D009097:Multienzyme Complexes; C110366:OVA-8; D010446:Peptide Fragments; D010455:Peptides; D015842:Serine Proteinase Inhibitors; D010047:Ovalbumin; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex