Identification of destabilizing residues in HLA class II-selected bacteriophage display libraries edited by HLA-DM

HLA-DM (DM) functions as a peptide editor by catalyzing the release of clas s Ii-associated invariant chain peptides (CLIP) and other unstable peptides , thus supporting the formation of stable class If-peptide complexes for pr esentation. To investigate the general features that determine the DM susce ptibility of HLA-DR1/peptide complexes, we generated a large DM-sensitive p eptide repertoire from an M13 bacteriophage display library using a novel d ouble selection protocol: we selected bacteriophage capable of binding to D R1 molecules and, subsequently we enriched DR1-bound bacteriophage suscepti ble to elution by purified DM molecules. Sequence and mutational analyses o f the DR1/DM double-selected peptides revealed that the amino acids Gly and Pro play a destabilizing role in the dissociation kinetics of DR1 ligands. This observation was confirmed also in natural peptide sequences such as C LIP 89-101, HA 307-319 and bovine collagen II (CII) 261-273. Our results de monstrate that DM susceptibility does not only depend on the number and nat ure of anchor residues, or the peptide length. Instead, less obvious sequen ce characteristics play a major role in the DM editing process and ultimate ly in the composition of peptide repertoires presented to T cells.