title: Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage.
abstract: The proteasome is able to create spliced Ags, in which two distant parts of a protein are excised and ligated together to form a novel peptide, for presentation by MHC class I molecules. These noncontiguous epitopes are generated via a transpeptidation reaction catalyzed by the proteasomal active sites. Transpeptidation reactions in the proteasome follow explicit rules and occur particularly efficiently when the C-terminal ligation partner contains a lysine or arginine residue at the site of ligation. Lysine contains two amino groups that theoretically may both participate in ligation reactions, implying that potentially not only peptide but also isopeptide linkages could be formed. Using nuclear magnetic resonance spectroscopy, we demonstrate in the present study that the proteasome can use the ε-amino group of an N-terminal lysine residue in transpeptidation reactions to create a novel type of posttranslationally modified epitopes. We show that the overall efficiency of ε ligation is only 10-fold lower as compared with α ligation, suggesting that the proteasome can produce sufficient isopeptide Ag to evoke a T cell response. Additionally, we show that isopeptides are more stable toward further proteasomal processing than are normal peptides, and we demonstrate that isopeptides can bind to HLA-A2.1 and HLA-A3 with high affinity. These properties likely increase the fraction of ε-ligated peptides presented on the cell surface for CD8(+) T cell surveillance. Finally, we show that isopeptide Ags are immunogenic in vivo. We postulate that ε ligation is a genuine posttranslational modification, suggesting that the proteasome can create a novel type of Ag that is likely to play a role in immunity.
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;
authors.forename: Celia R
authors.initials: CR
authors.lastname: Berkers
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;
authors.forename: Annemieke
authors.initials: A
authors.lastname: de Jong
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands;
authors.forename: Karianne G
authors.initials: KG
authors.lastname: Schuurman
authors.affiliation: Division of Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands; and.
authors.forename: Carsten
authors.initials: C
authors.lastname: Linnemann
authors.affiliation: Van 't Hoff Institute for Molecular Sciences, University of Amsterdam, 1098 XH Amsterdam, the Netherlands.
authors.forename: Jan A J
authors.initials: JA
authors.lastname: Geenevasen
authors.affiliation: Division of Immunology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands; and.
authors.forename: Ton N M
authors.initials: TN
authors.lastname: Schumacher
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands; h.ovaa@nki.nl boris.rodenko@glasgow.ac.uk.
authors.forename: Boris
authors.initials: B
authors.lastname: Rodenko
authors.affiliation: Division of Cell Biology, Netherlands Cancer Institute, 1066 CX Amsterdam, the Netherlands; h.ovaa@nki.nl boris.rodenko@glasgow.ac.uk.
authors.forename: Huib
authors.initials: H
authors.lastname: Ovaa
mesh_terms: D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D010455:Peptides / Q000737:chemistry* / Q000276:immunology; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000276:immunology; D020033:Protein Isoforms / Q000737:chemistry / Q000276:immunology; D011499:Protein Processing, Post-Translational; D019154:Protein Splicing; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D010455:Peptides; D020033:Protein Isoforms; D046988:Proteasome Endopeptidase Complex