Understanding the presentation of phosphopeptides by HLA-B antigens through immunopeptidomics and structural biology

Miguel Marcilla (mmarcilla@cnb.csic.es)

 

HLA-I antigens (encoded in the HLA-A, -B and –C loci) play a key role in the eradication of intracellular pathogens and tumor cells by presenting peptides derived from endogenous proteins to cytotoxic T lymphocytes. The peptidome displayed by these molecules includes phosphorylated ligands which have been proposed as optimal targets for cancer immunotherapy. In this work, we set out to study the phosphopeptidomes displayed by several HLA-B allotypes to gain insight into the structural determinants that govern the presentation of phosphorylated ligands.

HLA-I-bound peptidomes were purified, subjected to phosphopeptide enrichment and analyzed by LC-MS/MS using CID or EThcD for peptide fragmentation. With this approach, we identified more than 9500 ligands associated to HLA-B*40 with a common anchor motif: acidic residues (mainly Glu) at P2 and hydrophobic ones at PΩ. We also characterized about 150 phosphopeptides displayed by this allotype. Analysis of this set of sequences revealed two striking facts: 1) some ligands had phosphoserine (pSer) instead of Glu at P2 and 2) phosphorylation was frequently found at P4 and was usually accompanied by a basic residue at P1.

By performing peptide binding assays to HLA-B*40 and solving the crystal structures of 4 B*40-peptide complexes, we determined that the presentation of ligands phosphorylated P2 was due to the structural similarities between Glu and pSer. In addition, we demonstrated that the preference for peptides phosphorylated at P4 was linked to the presence of Arg62 in HLA-B*40. Finally, the analysis of the phosphopeptidomes displayed by other allotypes (B*39, B*27, B*07) indicates that preferent phosphorylation at P4 is a common feature for most HLA-B molecules while the bias towards basic residues at P1 is allotype-dependent. Overall, our results can contribute to the prediction and identification of phosphorylated neo-antigens as putative targets for cancer immunotherapy.

 

REFERENCES

Marcilla M, Alpízar A, Lombardía M, Ramos-Fernandez A, Ramos M, Albar JP. Increased diversity of the HLA-B40 ligandome by the presentation of peptides phosphorylated at their main anchor residue. Mol Cell Proteomics. 2014 Feb;13(2):462-74.

Alpízar A, Marino F, Ramos-Fernández A, Lombardía M, Jeko A, Pazos F, Paradela A, Santiago C, Heck AJ, Marcilla M. A Molecular Basis for the Presentation of Phosphorylated Peptides by HLA-B Antigens. Mol Cell Proteomics. 2017 Feb;16(2):181-193.