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).