title: Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue.
abstract: Human leukocyte antigen (HLA) class I molecules bind peptides derived from the intracellular degradation of endogenous proteins and present them to cytotoxic T lymphocytes, allowing the immune system to detect transformed or virally infected cells. It is known that HLA class I-associated peptides may harbor posttranslational modifications. In particular, phosphorylated ligands have raised much interest as potential targets for cancer immunotherapy. By combining affinity purification with high-resolution mass spectrometry, we identified more than 2000 unique ligands bound to HLA-B40. Sequence analysis revealed two major anchor motifs: aspartic or glutamic acid at peptide position 2 (P2) and methionine, phenylalanine, or aliphatic residues at the C terminus. The use of immobilized metal ion and TiO2 affinity chromatography allowed the characterization of 85 phosphorylated ligands. We further confirmed every sequence belonging to this subset by comparing its experimental MS2 spectrum with that obtained upon fragmentation of the corresponding synthetic peptide. Remarkably, three phospholigands lacked a canonical anchor residue at P2, containing phosphoserine instead. Binding assays showed that these peptides bound to HLA-B40 with high affinity. Together, our data demonstrate that the peptidome of a given HLA allotype can be broadened by the presentation of peptides with posttranslational modifications at major anchor positions. We suggest that ligands with phosphorylated residues at P2 might be optimal targets for T-cell-based cancer immunotherapy.
authors.affiliation: Proteomics Unit, Centro Nacional de Biotecnología (CSIC), 28049 Madrid, Spain;
authors.forename: Miguel
authors.initials: M
authors.lastname: Marcilla
authors.forename: Adán
authors.initials: A
authors.lastname: Alpízar
authors.forename: Manuel
authors.initials: M
authors.lastname: Lombardía
authors.forename: Antonio
authors.initials: A
authors.lastname: Ramos-Fernandez
authors.forename: Manuel
authors.initials: M
authors.lastname: Ramos
authors.forename: Juan Pablo
authors.initials: JP
authors.lastname: Albar
mesh_terms: D000595:Amino Acid Sequence; D017951:Antigen Presentation; D000940:Antigenic Variation; D001665:Binding Sites; D018604:Epitope Mapping; D059947:HLA-B40 Antigen / Q000276:immunology / Q000378:metabolism*; D006801:Humans; D008024:Ligands; D010446:Peptide Fragments / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D020543:Proteome / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D013602:T-Lymphocytes, Cytotoxic / Q000276:immunology / Q000378:metabolism
chemical_list: D059947:HLA-B40 Antigen; D008024:Ligands; D010446:Peptide Fragments; D010750:Phosphoproteins; D020543:Proteome