The 20S Proteasome Splicing Activity Discovered by SpliceMet

The identification of proteasome-generated spliced peptides (PSP) revealed a new unpredicted activity of the major cellular protease. However, so far characterization of PSP was entirely dependent on the availability of patient-derived cytotoxic CD8+ T lymphocytes (CTL) thus preventing a systematic investigation of proteasome-catalyzed peptide splicing (PCPS). For an unrestricted PSP identification we here developed SpliceMet, combining the computer-based algorithm ProteaJ with in vitro proteasomal degradation assays and mass spectrometry. By applying SpliceMet for the analysis of proteasomal processing products of four different substrate polypeptides, derived from human tumor as well as viral antigens, we identified fifteen new spliced peptides generated by PCPS either by cis or from two separate substrate molecules, i.e., by trans splicing. Our data suggest that 20S proteasomes represent a molecular machine that, due to its catalytic and structural properties, facilitates the generation of spliced peptides, thereby providing a pool of qualitatively new peptides from which functionally relevant products may be selected.Author Summary: MHC class I molecules present antigenic peptides derived from endogenously expressed foreign or aberrant protein molecules to the outside world so that they can be specifically recognised by cytotoxic T lymphocytes (CTLs) at the cell surface. Responsible for the generation of these peptides is the 20S proteasome, which is the major proteolytic enzyme of the cell. These peptides were so far believed to exhibit a linear sequence identical to that found in the unprocessed parental protein. Using patient derived CTL it was previously shown that by proteasome catalyzed peptide splicing, i.e., by fusion of two proteasome generated peptide fragments in a reversed proteolysis reaction, novel spliced antigenic peptides can be generated. To resolve the CTL dependence of spliced-peptide identification we here performed experiments, which combined mass spectrometric analysis of proteasome generated peptides with a computer based algorithm that predicts the masses of all theoretically possible spliced peptides from a given substrate molecule (SpliceMet). Using this unrestricted approach we here identified several new spliced peptides of which some were derived from two distinct substrate molecules. Our data reveal that peptide splicing is an intrinsic additional catalytic property of the proteasome, which may provide a qualitatively new peptide pool for immune selection.