A large fraction of HLA class I ligands are proteasome-generated spliced peptides

Abstract

catalyzed peptide splicing.Abbreviations: electron-transfer higher-energy collision dissociation (EThcD); higher-energy collision dissociation (HCD); false discovery rate (FDR); HLA class I (HLA-I); lymphoblastoid cell line (LCL); mass spectrometry (MS); molecular weight (MW); proteasome-catalyzed peptide splicing (PCPS). 2The proteasome generates the epitopes presented on HLA class I molecules that elicit CD8 + T cell responses. While reports of proteasome-generated spliced epitopes exist, they have been regarded as rare events. Here, however, we show that the proteasome-generated spliced peptide pool accounts for one third of the entire HLA class I immunopeptidome in terms of diversity and one fourth in terms of abundance. They also represent a unique set of antigens, possessing particular and distinguishing features. We validated this observation using a range of complementary experimental and bioinformatics approaches, and multiple cell types. The widespread appearance and abundance of proteasome-catalyzed peptide splicing events will inform immunobiology and autoimmunity theories, and may provide a previously untapped source of epitopes for uses in vaccines and cancer immunotherapy.3The presentation of epitopes on the cell surface is a key mechanism by which organisms identify the presence of pathogens, metabolic malfunctioning, or tumors. The HLA class I (HLA-I) immunopeptidome, i.e. the epitopes allocated onto the HLA-I molecules, impinges upon the CD8 + T cell repertoire and the cell-mediated immune response (1). HLA-I immunopeptidomes are usually investigated by sequence identification of peptides eluted from HLA-I molecules using LC-tandem mass spectrometry (MS) (Fig. S1). The key step for the transformation of a protein into HLA-Irestricted epitopes is usually processing by the proteasome (1), which cuts proteins into peptides;alternatively, the proteasome can also cut and paste peptide sequences, thereby releasing peptide antigens that do not correspond to the original protein sequence (2) (Fig. S2). This proteasomecatalyzed peptide splicing (PCPS) has long been considered to occur only very rarely; partly this has been because the screening of the HLA-I immunopeptidome for proteasome-generated spliced peptides was impeded by methodological challenges.To overcome these problems we developed an analytical strategy, which accounts for recent discoveries underpinning the PCPS mechanism, and which can handle the vast proteome-wide human spliced peptide database (Fig. S3). With this strategy we initially analyzed the HLA-I-eluted immunopeptidome of the GR lymphoblastoid cell line (GR-LCL) adopting, for a deeper coverage of the immunopeptidome, a 2D peptide pre-fractionation strategy, followed by a hybrid peptide fragmentation method, electron-transfer higher-energy collision dissociation (EThcD) for peptide identification (3, 4) ( Fig. S1), supplemented by an adapted target-decoy approach (Fig. S4). Our analysis led to the identification of 6592 non-spliced and 3417 spliced 9-12mer peptides ( Table S1)....

Peptide splicing by the proteasome

“…This makes it very difficult to compare the splicing of peptides with one or two amino acid substitutions, as these may target the peptide to another catalytic subunit, and the C-terminal reactant itself might not access as efficiently to the primed binding site of each catalytic subunit. Moreover, when Liepe et al (37) compared spliced peptides eluted from distinct cell lines, they observed significant differences in the nature of the amino acids found at key positions (P 1 , P 1 Ј (Fig. 5), N terminus, and C terminus).…”

“…Again, a major limitation in the identification of spliced peptides by peptidomics is the fact that databases used for matching only contain linear protein fragments. Recently, Liepe et al (37) addressed this issue by creating a custom peptide database, which includes any possible short peptides created by the splicing of non-contiguous peptide fragments from the proteins expressed in the cell lines studied. To restrict the size of the database, only peptides produced by the cis-splicing of fragments separated by 25 amino acids or less were taken into account.…”

“…Additionally, when Liepe et al (37), using NetMHC, analyzed the binding affinity of spliced peptides for the HLA-A and -B molecules from which they were eluted, they observed that their IC 50 was quite high when compared with non-spliced peptides. The authors suggested that this low predicted binding affinity might reflect intrinsic differences in the HLA-binding motifs of spliced versus non-spliced peptides, which are not captured by existing algorithms because these have been exclusively trained on non-spliced epitopes.…”

Immune targets and neoantigens for cancer immunotherapy and precision medicine

“…Even though proteasome-generated spliced epitopes have been reported, such protein-splicing derived epitopes are generally rare events. Interestingly, a recent study shows that the proteasome-generated spliced peptide pool could account for one-third of the entire HLA class I immunopeptidome in terms of diversity and one-fourth in terms of abundance [151]. However, it is not clear how many or what percentages of tumor antigens are generated through this mechanism.…”

“…However, it is not clear how many or what percentages of tumor antigens are generated through this mechanism. Nonetheless, proteasome-generated MHC class I-restricted spliced cancer peptides may represent a previously untapped source of epitopes for use in vaccines and cancer immunotherapy [151]. MHC class II-restricted spliced T cell epitopes from cancer cells have not been reported so far.…”