title: A recyclable assay to analyze the NH(2)-terminal trimming of antigenic peptide precursors.
abstract: The proteasome plays an essential role in the production of MHC class I-restricted antigenic peptides. Recent results have indicated that several peptidases, including tripeptidyl peptidase II and puromycin-sensitive aminopeptidase, could act downstream of the proteasome by trimming NH(2)-terminal extensions of antigenic peptide precursors liberated by the proteasome. In this study, we have developed a solid-phase peptidase assay that allowed us to efficiently purify and immobilize proteasome, tripeptidyl peptidase II, and puromycin-sensitive aminopeptidase. Whereas the first peptidase was active against small fluorogenic peptides, the latter two could also digest antigenic peptide precursors and could be used repeatedly with different precursors. Using three distinct antigenic peptide precursors, we found that tripeptidyl peptidase II never cleaved within the antigenic peptide sequence, suggesting that, aside from its proteolytic activities, it may also play a role in protecting antigenic peptides from complete hydrolysis in the cytosol. This method should be valuable for high throughput screenings of substrate specificity and potential inhibitors.
mesh_terms: D000595:Amino Acid Sequence; D000941:Antigens / Q000737:chemistry* / Q000378:metabolism*; D001681:Biological Assay / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D003546:Cysteine Endopeptidases / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism; D010455:Peptides / Q000737:chemistry* / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012680:Sensitivity and Specificity
chemical_list: D000941:Antigens; D009097:Multienzyme Complexes; D010455:Peptides; D011498:Protein Precursors; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex