Invited lecture: Antigenic peptides produced by peptide splicing in the proteasome

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 by 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 (1). The splicing reaction appears not to involve a particular motif, but rather to result from a low-efficiency reversal of the proteolysis reaction. Its occurrence is depending only on the occurrence of peptide cleavage. 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 made by the joining of two fragments that are initially non-contiguous in 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 transposition could be reproduced in vitro with purified proteasomes. The splicing mechanism based on transpeptidation immediately after peptide bond cleavage is compatible with a transposition of the fragments prior to splicing. Together with the previous description of a peptide produced by protein splicing of FGF-5 (2), 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. 1. Vigneron, N., V. Stroobant, J. Chapiro, A. Ooms, G. Degiovanni, S. Morel, P. van der Bruggen, T. Boon, and B. Van den Eynde. 2004. An antigenic peptide produced by peptide splicing in the proteasome. Science 304:587-590. 2. Hanada, K., J.W. Yewdell, and J.C. Yang. 2004. Immune recognition of a human renal cancer antigen through post-translational protein splicing. Nature 427:252-256