An antigenic peptide produced by reverse splicing and double asparagine deamidation

Abstract

A variety of unconventional translational and posttranslational mechanisms contribute to the production of antigenic peptides, thereby increasing the diversity of the peptide repertoire presented by MHC class I molecules. Here, we describe a class I-restricted peptide that combines several posttranslational modifications. It is derived from tyrosinase and recognized by tumor-infiltrating lymphocytes isolated from a melanoma patient. This unusual antigenic peptide is made of two noncontiguous tyrosinase fragments that are spliced together in the reverse order. In addition, it contains two aspartate residues that replace the asparagines encoded in the tyrosinase sequence. We confirmed that this peptide is naturally presented at the surface of melanoma cells, and we showed that its processing sequentially requires translation of tyrosinase into the endoplasmic reticulum and its retrotranslocation into the cytosol, where deglycosylation of the two asparagines by peptide-N-glycanase turns them into aspartates by deamidation. This process is followed by cleavage and splicing of the appropriate fragments by the standard proteasome and additional transport of the resulting peptide into the endoplasmic reticulum through the transporter associated with antigen processing (TAP).antigen processing | peptide splicing | tumor antigen C D8 + cytotoxic T lymphocytes (CTLs) are the principal effectors recognizing, through their specific T cell receptor, peptide fragments bound to MHC class I molecules present on the surface of malignant cells. Most of the genes encoding class I-restricted tumor antigenic peptides were discovered by isolating T lymphocytes from melanoma patients. After in vitro stimulation by autologous melanoma cells, antitumor T lymphocytes were obtained and used to clone the genes encoding the tumor antigens. These genes included cancer germ-line genes, such as those of the melanoma antigen (MAGE) family, which encode tumor-specific antigens expressed in various tumors (1-4), but also differentiation genes encoding antigens expressed in both melanomas and normal melanocytes, such as Melan-A (MART-1), gp100, and tyrosinase (5-8). Precise identification of the antigenic peptides present at the surface of tumor cells is crucial to develop efficient immunotherapy strategies. These peptides can be used, for example, to generate melanoma-specific T cells for adoptive immunotherapy or in strategies using epitope-based vaccination. Understanding the processing and presentation pathways of such antigenic peptides may also help in designing appropriate protocols of immunotherapy. Class I-restricted antigenic peptides are usually made up of fragments of 8-11 aa directly derived from the degradation of the parental protein by the proteasome (9). Proteasomal substrates mostly comprise nuclear and cytosolic proteins but also include endoplasmic reticulum (ER) proteins returned to the cytosol during the process of ER-associated degradation (ERAD) (10, 11). In most cases, the sequence of the antigenic peptides can be easily p...

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

“…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).…”

Driving Forces of Proteasome-catalyzed Peptide Splicing in Yeast and Humans

“…Thus, PCPS would only depends on the proteasomal site-specific cleavage strength (SCS), which determines how frequently proteasomes cleaves specific peptide bond (5,6). Interestingly, Dalet et al identified a single PSP to be generated in absence of proteolysis, albeit with an extremely low efficiency, in a reaction that they defined as "condensation" (8) and that will be reported here as hydrolysis ϩ transpeptidation to discriminate it from the direct transpeptidation reaction.…”

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

“…A second peptide, encoded by the melanoma differentiation Ag gp100 PMEL17 is made by the association of fragments RTK and QLYPEW, which are distant of 4 aa in the parent protein (25). The two other spliced peptides described were produced by re-*Ludwig Institute for Cancer Research, B-1200 Brussels, Belgium; † Waloon Excellence in Life Sciences and Biotechnology, B-1200 Brussels, Belgium; ‡ de Duve Institute, Université Catholique de Louvain, B-1200 Brussels, Belgium; verse splicing, that is, splicing of peptide fragments in the reverse order to that in which they appear in the parental protein (26,27). One of them is a minor histocompatibility Ag created by a polymorphism in the SP110 nuclear phosphoprotein gene and made after splicing of peptide segments STPK and SLPRGT to create the antigenic peptide SLPRGTSTPK (27).…”