Definition of Proteasomal Peptide Splicing Rules for High-Efficiency Spliced Peptide Presentation by MHC Class I Molecules

Abstract

Peptide splicing, in which two distant parts of a protein are excised and then ligated to form a novel peptide, can generate unique MHC class I–restricted responses. Because these peptides are not genetically encoded and the rules behind proteasomal splicing are unknown, it is difficult to predict these spliced Ags. In the current study, small libraries of short peptides were used to identify amino acid sequences that affect the efficiency of this transpeptidation process. We observed that splicing does not occur at random, neither in terms of the amino acid sequences nor through random splicing of peptides from different sources. In contrast, splicing followed distinct rules that we deduced and validated both in vitro and in cells. Peptide ligation was quantified using a model peptide and demonstrated to occur with up to 30% ligation efficiency in vitro, provided that optimal structural requirements for ligation were met by both ligating partners. In addition, many splicing products could be formed from a single protein. Our splicing rules will facilitate prediction and detection of new spliced Ags to expand the peptidome presented by MHC class I Ags.

Peptide Splicing in the Proteasome Creates a Novel Type of Antigen with an Isopeptide Linkage

“…This transpeptidation reaction can apparently compete with normal hydrolysis, in which water reacts with the intermediate ester (12), in such a way that sufficiently new peptide can be formed to invoke an immune response against transpeptidation products. In an accompanying article in this issue by Berkers et al (16), we show that transpeptidation reactions can efficiently compete with hydrolysis when specific structural requirements on the two ligating partners are met. Ligation occurs particularly efficiently when the C-terminal ligation partner has a basic amino acid residue (lysine or arginine) at its N terminus (16).…”

“…In an accompanying article in this issue by Berkers et al (16), we show that transpeptidation reactions can efficiently compete with hydrolysis when specific structural requirements on the two ligating partners are met. Ligation occurs particularly efficiently when the C-terminal ligation partner has a basic amino acid residue (lysine or arginine) at its N terminus (16). As lysine has two amino groups that can theoretically both react with the O-acyl enzyme intermediate, this implies that the proteasome may be able to form isopeptide linkages.…”

“…Indeed, peptide splicing involving a large variety of C-terminal fragments can be readily observed in vitro (14,15). Although the concentration of precursor fragments is one of the driving factors of the splicing reaction (14), we show in an accompanying article (16) that splicing is also governed by explicit rules, as revealed by preferred sequence motifs within both splicing partners (16). For the C-terminal ligation partner, ligation occurs particularly efficiently when a basic amino acid residue (lysine or arginine) is present at the N terminus (16).…”

“…Although the concentration of precursor fragments is one of the driving factors of the splicing reaction (14), we show in an accompanying article (16) that splicing is also governed by explicit rules, as revealed by preferred sequence motifs within both splicing partners (16). For the C-terminal ligation partner, ligation occurs particularly efficiently when a basic amino acid residue (lysine or arginine) is present at the N terminus (16). Lysine has a second free amino group on the C ε position (the ε-amine) in addition to its free N terminus (the a-amine) and, theoretically, both these amino groups may be able to participate in ligation reactions.…”

Current tools for predicting cancer-specific T cell immunity

“…7,10,17 Immunogenic tumors are under pressure to evade immune destruction and some escape mechanisms involve aberrant TA presentation and processing, e.g., altered proteasomal degradation, RNA splicing, and/or post-translational modifications. [18][19][20][21][22] Most cancer cells express low levels of MHC I and no II molecules. However, they are prone to cell death and as a result can be taken up by antigen presenting cells (APC), namely DC and macrophages, which (cross)-present exogenous TA on their MHC molecules and induce TA-specific CD8…”