Antigen presentation by major histocompatibility complex (MHC) class I molecules is a critical step in adaptive immunity: it enables cytotoxic T lymphocytes to identify infected or abnormal cells. To be efficiently presented, antigenic peptides must reach a precise length of eight to ten residues in order to fit correctly within the groove of the MHC I molecule. In the endoplasmic reticulum, this trimming relies on the action of two human aminopeptidases, ERAP1 and ERAP2, which successively remove amino acids from the ends of precursor peptides. A detailed understanding of this trimming step, and of its regulation, is essential to grasp how the repertoire of peptides displayed at the cell surface is established.
While the notion that ERAPs trim free peptides is widely accepted, data from in vitro and cellular experiments suggest that these enzymes can also act on peptides bound, or partially bound, to MHC I molecules—a configuration likely closer to the physiological situation. To explore this versatility, the authors describe a comprehensive experimental protocol allowing the step-by-step monitoring of residue removal by ERAPs. The method first involves the production of soluble recombinant ERAP1/ERAP2 complexes as heterodimers. These proteins are expressed in insect cells, with Hi5 lines being preferred because they secrete more recombinant protein than Sf9 cells, in a serum-free medium whose lack of proteases promotes the stability of the secreted enzymes. The ERAP complexes are then covalently immobilized on Sepharose beads, which facilitates serial immunoprecipitations and limits antibody contamination during quantification.
The trimming assay itself consists of incubating a precursor peptide, either in free form or bound to MHC I, with the calibrated ERAP1/2 beads, and then analyzing the generated fragments by mass spectrometry. A fifteen-residue control peptide is used to standardize each new batch of beads and to normalize results across experiments. The authors emphasize that free peptides are trimmed considerably faster than MHC I–bound peptides, which requires adapting the sampling times to the objective of each experiment. The preparation of peptide-deficient MHC I molecules, which are intrinsically unstable and stabilized with glycerol, together with the controlled loading of synthetic nine-residue peptides or precursors extended at their N-terminus, complete the setup.
By enabling the monitoring of the trimming of both free and MHC I–bound peptides, this protocol provides a tool to characterize trimming events and to better delineate the role of ERAPs in shaping the peptide repertoire presented to cytotoxic T lymphocytes.