Increased Diversity of the HLA-B40 Ligandome by the Presentation of Peptides Phosphorylated at Their Main Anchor Residue

Manuel Ramos,Juan Pablo Albar,Adán Alpízar,Manuel Lombardía,Antonio Ramos-Fernandez,Miguel Marcilla

doi:10.1074/mcp.m113.034314

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.

Highlights

From the ‡Proteomics Unit, Centro Nacional de Biotecnología (CSIC), 28049 Madrid, Spain; ¶Viral Immunology Unit, Centro Nacional de Microbiología (ISCIII), Madrid, 28220 Spain
N-terminal acetylation [10], phosphorylation [11,12,13], methylation [14], and glycosylation [15] have been described in Major histocompatibility complex (MHC) class I– bound peptidomes. In this context, phosphorylated ligands have raised much interest owing to their potential as targets in T-cellbased cancer immunotherapy [12, 13], given that aberrant phosphorylation is a hallmark of malignant transformation [16, 17] and phosphorylated epitopes can be recognized by CTLs [11]
The characterization of the phosphopeptidome associated with MHC class I molecules and the identification of tumor-derived phosphopeptides are necessary in order for such immunotherapeutic approaches to be implemented

Summary

Introduction

From the ‡Proteomics Unit, Centro Nacional de Biotecnología (CSIC), 28049 Madrid, Spain; ¶Viral Immunology Unit, Centro Nacional de Microbiología (ISCIII), Madrid, 28220 Spain. The IMGT/HLA database [7] currently contains about 7000 allele sequences that encode more than 5000 different human class I antigens Most of these polymorphisms are located within the ␣1 and ␣2 domains of the heavy chain and modulate the peptide binding preferences of each allotype [8]. It is thought that the great diversity of HLA class I allotypes, and of their associated ligandomes, is an adaptation to guarantee immunity against intracellular pathogens [6] In this regard, the existence of a large number of different class I molecules capable of presenting diverse peptidomes hampers immune evasion by means of viral genetic mutation. The characterization of the phosphopeptidome associated with MHC class I molecules and the identification of tumor-derived phosphopeptides are necessary in order for such immunotherapeutic approaches to be implemented

Objectives

Results

Conclusion