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...