A Spliced Antigenic Peptide Comprising a Single Spliced Amino Acid Is Produced in the Proteasome by Reverse Splicing of a Longer Peptide Fragment followed by Trimming

Abstract

Peptide splicing is a novel mechanism of production of peptides relying on the proteasome and involving the linkage of fragments originally distant in the parental protein. Peptides produced by splicing can be presented on class I molecules of the MHC and recognized by CTLs. In this study, we describe a new antigenic peptide, which is presented by HLA-A3 and comprises two noncontiguous fragments of the melanoma differentiation Ag gp100PMEL17 spliced together in the reverse order to that in which they appear in the parental protein. Contrary to the previously described spliced peptides, which are produced by the association of fragments of 3–6 aa, the peptide described in this work results from the ultimate association of an 8-aa fragment with a single arginine residue. As described before, peptide splicing takes place in the proteasome by transpeptidation involving an acyl-enzyme intermediate linking one of the peptide fragment to a catalytic subunit of the proteasome. Interestingly, we observe that the peptide causing the nucleophilic attack on the acyl-enzyme intermediate must be at least 3 aa long to give rise to a spliced peptide. The spliced peptide produced from this reaction therefore bears an extended C terminus that needs to be further trimmed to produce the final antigenic peptide. We show that the proteasome is able to perform the final trimming step required to produce the antigenic peptide described in this work.

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

“…Length was not a determining factor because both short and long peptides were equally suited to serve as C-terminal ligation partners. However, C-terminal fragments shorter than 3 aa were not found to participate in ligation reactions, in line with results from previous studies (9).…”

“…Although such combinations are abundant in the human proteome, their prevalence in spliced peptides does confirm that these motifs facilitate the proteasomal splicing reaction and suggests that our splicing rules are an important first step toward predicting the occurrence of spliced epitopes. Two previously described in vivo N-terminal precursors contained a K or H on P 1 (5,9). In our hands, however, peptides with a basic residue on P 1 failed to serve as N-terminal ligation precursors.…”

“…This reversed cleavage has also been shown for other proteases including the proteasome that then would yield new spliced Ags that are not genetically coded yet can be presented to the immune system (5)(6)(7)(8)(9). To date, five spliced Ags that are immunogenic in vivo have been described: [RTK][QLYPEW] (5), [NTYAS][PRFK] (6), [SLPRGT] [STPK] (7), [IYMDGT][ADFSF] (8), and [RSYVPLAH][R] (9), all of which are produced by the proteasome through a transpeptidation mechanism (5,7,10,11). During normal proteasomal peptide hydrolysis, the N-terminal threonine residue of a catalytically active subunit reacts with the scissile peptide bond to form an O-acyl enzyme intermediate, resulting in the release of the C-terminal part of the peptide.…”

“…Because spliced Ags may mediate T cell responses to cancer, transplants, and viral infection, predicting their generation would be important for the development of vaccines and immunotherapies or to pharmacologically suppress T cell responses (12). Although proteasomal peptide ligation can readily be detected in vitro (11,15), identifying spliced Ags in vivo has proven to be difficult with only five immunomodulatory spliced epitopes identified (5)(6)(7)(8)(9). Classically, novel epitopes are identified by cell surface elution and mass spectroscopy (MS) analysis, followed by matching against protein databases (16,17).…”

“…During a proteasomal transpeptidation reaction, in contrast, the amino group of a second peptide competes with water for reaction with the intermediate ester, resulting in the formation of a new peptide bond and thus a spliced peptide. Splicing of ligation partners in the proteasome has been described to occur both in-line [i.e., in the order in which they occurred in the parent protein sequence (5,6)] and in the reverse order [i.e., a posterior sequence is spliced to the anterior end of its neighboring sequence (7)(8)(9)]. In addition, peptide splicing by the proteasome can be combined with other posttranslational processes, such as asparagine deamidation (8), and can be followed by further trimming to produce peptides of the right HLA binding properties (9).…”