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).

Fig. 1.

Localization of the minimal region of tyrosinase required to produce the antigen. (A–C) COS-7 cells were transiently transfected with a construct encoding HLA-A24 together with the indicated constructs derived from human tyrosinase (hTyr). The next day, these cells were tested for recognition by CTL CL62, a tyrosinase-reactive CTL clone isolated from line TIL888. IFNγ production was measured by ELISA in the culture supernatants after overnight incubation. In B, SP indicates that the plasmid construct encodes the signal peptide of IL-2. In C, where indicated, the Asn residues at position 337 or 371 were replaced by Asp residues by site-directed mutagenesis. (D) HEK293-A24 cells were transiently transfected with the indicated tyrosinase constructs containing an internal deletion. Transfected cells were tested for their recognition by TIL888 1 d later. Where indicated, an additional thymidine (t) was inserted between the codons corresponding to amino acids 342 and 367. The addition of this nucleotide results in a downstream frame shift after translation of the construct. The sequence of the minimal regions of tyrosinase required for recognition by the CTL is indicated below the sequences of the different constructs tested. Data shown are representative of at least two independent experiments. Error bars show SDs of triplicates.

Fig. 2.

Identification of the antigenic peptide presented by melanoma cells and recognized by CTL CL62. (A) CTL recognition of synthetic peptides composed of two noncontiguous fragments of tyrosinase that are joined together in the reverse order to that of the tyrosinase sequence. The indicated peptides were loaded onto HLA-A24⁺ EBV B cells before the CTL assay. IFNγ production was measured after an overnight incubation. (B) Peptides were eluted from HLA class I molecules purified from melanoma cells LB33-MEL and were fractionated on a reversed-phase column by HPLC. The fractions were loaded onto presenting cells and tested for CTL recognition (Upper). IFNγ production was measured after an overnight incubation. Three peaks of CTL activity, indicated by A, B, and C on the graph, were detected. Buffer was run on the column before the eluted peptides to rule out any contamination of the HPLC system, and the fractions were tested similarly. Synthetic peptides IYMDGTADFSF and IYMDGAADFSF were oxidized by treatment with H₂O₂. The nonoxidized (M), methionine sulfoxide (m), and methionine sulfone (m*) forms of the peptides were separated and purified. About 10 ng each peptide form were injected together under the same HPLC conditions as those conditions used for eluates, and the fractions were tested for CTL recognition (Lower). (C) MS/MS fragmentation spectrum of the singly charged ion with m/z 1,282 detected in fraction 32.5 (peak C) from LB33-MEL eluates (Upper) and the fragmentation spectrum of the singly charged ion (m/z 1,282) of the synthetic peptide IYmDGTADFSF (Lower) are shown. The fragments that were detected are indicated above the peptide sequence for N-terminal b ions and their dehydrated forms and indicated below the sequence for C-terminal y ions. The ion with m/z 1,264 is the dehydrated form of ion 1,282, and the ion with m/z 1,218 corresponds to the loss of methanesulfenic acid from the methionine sulfoxide of ion 1,282 (55). Data shown are representative of at least two independent experiments (peaks A, B, and C).

Fig. 3.

Production of the spliced antigenic peptide IYMDGTADFSF by the proteasome. (A) CTL recognition of melanoma cells treated or not treated with proteasome inhibitor. Melanoma cells LB39-MEL naturally express tyrosinase, HLA-A24, and HLA-A2. They were treated for 2 h with epoxomicin (0.5 μM) after acid elution of MHC-bound peptides. Cells were then washed and incubated for 1 more h with a lower dose of epoxomicin (0.01 μM) and where indicated, (white bars), with the corresponding synthetic antigenic peptide as a control. Recognition by three CTL clones was evaluated by flow cytometry in a degranulation assay performed after 5 h of incubation with LB39-MEL cells (SI Results explains the choice of this assay in this context). The percentage of CD8⁺ T cells expressing the degranulation markers CD107a and CD107b is shown. CTL CL62 recognizes tyrosinase/A24 spliced peptide IYMDGTADFSF, CTL IVS-B recognizes tyrosinase/A2 ₃₆₉YMDGTMSQV₃₇₇, and CTL 210/9 recognizes tyrosinase/A2 ₁MLLAVLYCL₉. One of three similar experiments is shown. (B) In vitro production of the tyrosinase spliced peptide by the standard proteasome. The indicated 25-mer precursor peptide was incubated with purified 20S standard proteasomes or immunoproteasomes. The reaction was stopped after 0, 1, 2, 3, and 4 h of incubation. Digests were then loaded onto HLA-A24⁺ EBV B cells. CTL CL62 was added and IFNγ production was measured after an overnight incubation. The degradation of the precursor peptide was determined by MS. One of two similar experiments is shown. (C) Processing of peptide IYMDGTADFSF by HEK293 cells expressing standard proteasomes (293-SP) or immunoproteasomes (293-IP). Both cell lines were transfected with an HLA-A24 plasmid construct and the indicated amounts of plasmid encoding tyrosinase. CTLs TIL888 were added and IFNγ production was measured in supernatants after an overnight incubation. Control cells were transfected with a plasmid coding for HLA-A24 and pulsed with the synthetic peptide IYMDGTADFSF for 1 h before addition of the CTLs. One of two similar experiments is shown. Error bars show SDs of triplicates. (D) Model for the catalytic mechanism producing the tyrosinase spliced peptide in the proteasome. The balls represent catalytically active β-subunits of the proteasome with the hydroxyl group of the side chain of their N-terminal threonine. (E) CTL recognition of digests obtained by incubating purified 20S standard proteasomes with various pairs of synthetic peptides during the indicated periods of time. The different digests were loaded onto presenting cells and tested for CTL stimulation. One of four similar experiments is shown. Ac-ADFSF, N-terminally acetylated peptide with sequence ADFSF.

Fig. 4.

Role of PNGase in the processing of the tyrosinase spliced peptide. (A) Melanoma cells LB39-MEL, expressing both HLA-A24 and HLA-A2, were treated for 5 h with the indicated compounds (50 μM) after acid elution of MHC-bound peptides. Both z-VAD-fmk and Q-VD-OPh are caspase inhibitors. Only z-VAD-fmk blocks PNGase activity. Cells were then tested for recognition by three different CTL clones (Left). CTL CL62 recognizes the tyrosinase/A24 spliced peptide IYMDGTADFSF, CTL IVS-B recognizes tyrosinase/A2 ₃₆₉YMDGTMSQV₃₇₇, and CTL 210/9 recognizes tyrosinase/A2 ₁MLLAVLYCL₉. IFNγ production was measured after an overnight incubation. Cells were pulsed with the corresponding synthetic antigenic peptide before addition of the CTL to check their antigen-presenting capacity (Right). Data shown are representative of at least two independent experiments. (B) COS-7 cells were transiently transfected with plasmids encoding HLA-A24 and a full-length tyrosinase construct encoding either Asn residues at positions 337 and 371 (N337 N371) or Asp residues at the same positions (D337 D371); 5 h after transfection, cells were incubated overnight with 50 μM of the indicated compounds. Supernatants were then removed, and CTL CL62 was added. Where indicated (white bars), cells were pulsed for 1 h with the antigenic peptide before the addition of the CTL. Data shown are representative of four independent experiments. In all panels, error bars show SDs of triplicates.

Fig. P1.

Production of the deamidated reverse-spliced antigenic peptide derived from tyrosinase. Misfolded N-glycosylated tyrosinase proteins are retrotranslocated from the ER into the cytosol (I), where peptide N-glycanase converts two asparagines into aspartates upon removal of the asparagine-associated sugars (II). This process is followed by cleavage and reverse splicing of the deamidated protein by the proteasome (III), leading to production of the final antigenic peptide IYMDGTADFSF, which is then transported back into the ER by the TAP transporter (IV). The peptide/MHC complex then migrates to the cell surface where it can be recognized by the CTL (V). ERAD, ER-associated degradation.