Faridi et al. developed an approach using high quality MS/MS de novo sequencing of HLA-bound peptides (p-HLA) to search for evidence demonstrating that some p-HLA arise by a trans-peptide splicing (linkage of segments from two different proteins into one presented peptide). Approximately 5 to 30% of p-HLA across 17 HLA-A and -B alleles were attributable to trans-splicing. Comparison of binding motifs, peptide:HLA binding strength, and generation of canonical crystal structures validated the trans-spliced peptides as bona fide HLA binders. Trans-splicing (and previously identified cis-splicing) may dramatically expand the repertoire of presented peptides.
The diversity of peptides displayed by class I human leukocyte antigen (HLA) plays an essential role in T cell immunity. The peptide repertoire is extended by various posttranslational modifications, including proteasomal splicing of peptide fragments from distinct regions of an antigen to form nongenomically templated cis-spliced sequences. Previously, it has been suggested that a fraction of the immunopeptidome constitutes such cis-spliced peptides; however, because of computational limitations, it has not been possible to assess whether trans-spliced peptides (i.e., the fusion of peptide segments from distinct antigens) are also bound and presented by HLA molecules, and if so, in what proportion. Here, we have developed and applied a bioinformatic workflow and demonstrated that trans-spliced peptides are presented by HLA-I, and their abundance challenges current models of proteasomal splicing that predict cis-splicing as the most probable outcome. These trans-spliced peptides display canonical HLA-binding sequence features and are as frequently identified as cis-spliced peptides found bound to a number of different HLA-A and HLA-B allotypes. Structural analysis reveals that the junction between spliced peptides is highly solvent exposed and likely to participate in T cell receptor interactions. These results highlight the unanticipated diversity of the immunopeptidome and have important implications for autoimmunity, vaccine design, and immunotherapy.
Author Info: (1) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (2)
Author Info: (1) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (2) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. Department of Biology, Institute of Molecular Systems Biology,ETH Zurich, Zurich 8093, Switzerland. (3) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (4) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia. (5) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (6) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (7) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. (8) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. Monash Centre for Data Science, Faculty of Information Technology, Monash University, Melbourne, Victoria 3800, Australia. (9) Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research and Department of Molecular and Translational Science, School of Clinical Science, Monash University, Clayton, Victoria 3168, Australia. (10) Centre for Innate Immunity and Infectious Diseases, Hudson Institute of Medical Research and Department of Molecular and Translational Science, School of Clinical Science, Monash University, Clayton, Victoria 3168, Australia. (11) Jenner Institute, University of Oxford, Oxford OX3 7FZ, UK. (12) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. Australian Research Council Centre of Excellence in Advanced Molecular Imaging, Monash University, Clayton, Victoria 3800, Australia. Institute of Infection and Immunity, Cardiff University School of Medicine,Heath Park, Cardiff CF14 4XN, UK. (13) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. anthony.purcell@monash.edu nathan.croft@monash.edu. (14) Infection and Immunity Program and Department of Biochemistry and Molecular Biology, Biomedicine Discovery Institute, Monash University, Clayton, Victoria 3800, Australia. anthony.purcell@monash.edu nathan.croft@monash.edu.
