Invited lecture: Peptide splicing by the proteasome: a new mechanism to produce antigenic peptides presented by MHC class I molecules

Peptides presented to CTL by MHC class I molecules usually consist of 9 amino-acid fragments of intracellular proteins which are produced through protein degradation by the proteasome. By studying the antigen recognized by a CTL clone isolated from a melanoma patient, we identified an antigenic peptide composed of two non-contiguous fragments of the same protein, namely the melanocytic protein gp100. The production of this peptide requires the excision of an intervening fragment of 4 amino acids and the splicing of a fragment of 3 residues with a fragment of 6 residues. We have shown that this splicing is exerted by the proteasome and can be reproduced in vitro when incubating a precursor peptide with purified proteasomes. Splicing is coupled directly to peptide bond cleavage by the proteasome and appears to occur by transpeptidation involving an acyl-enzyme intermediate (Vigneron et al, Science 2004). We have now identified a second antigenic peptide produced by peptide splicing in the proteasome. This peptide is recognized by CTL directed against a minor histocompatibility antigen. The CTL was isolated from a multiple myeloma patient treated with HLA-identical bone-marrow transplantation. The peptide is encoded by the polymorphic region of a gene ubiquitously expressed. Again it is composed by the joining of two fragments which are initially non-contiguous is the parental protein. In addition, the two fragments are inverted in the spliced peptide, i.e. the fragment that was more N-terminal in the parental protein ends up at the C-terminal side of the spliced peptide, and vice-versa. We showed that splicing and inversion could be reproduced in vitro with purified proteasomes. The splicing mechanism based on transpeptidation immediately after peptide bond cleavage is compatible with a re-ordering of the fragments prior to splicing. Together with the previous description of a peptide produced by protein splicing of FGF-5 (Hanada et al, Nature 2004), this is the third example of antigenic peptide produced by splicing. These results indicate that spliced peptides are not uncommon and may represent a significant part of the peptide repertoire presented by MHC class I molecules