To characterize the peptidome of the Behçet's disease–associated HLA–B*51:01 allotype as well as the differential features of major peptide subsets and their distinct endoplasmic reticulum aminopeptidase 1 (ERAP‐1)–mediated processing.

The endogenous B*51:01‐bound peptidome was characterized from 721.221 transfectant cells, after affinity chromatography and acid extraction, by tandem mass spectrometry. Recombinant ERAP‐1 variants were used to digest synthetic B*51:01 ligands. HLA and transporter associated with antigen processing (TAP) binding affinities of peptide ligands were calculated with well‐established algorithms. ERAP‐1 and ERAP‐2 from 721.221 cells were characterized by genomic sequencing and Western blotting.

The B*51:01 peptidome consisted of 29.5% octamers, 61.7% nonamers, 4.8% decamers, and 4.0% longer peptides. The major peptide motif consisted of Pro and Ala at position 2, aliphatic/aromatic position 3 residues, and Val and Ile at the C‐terminal position. The ligands with Pro or Ala at position 2 constituted 2 distinct subpeptidomes. Peptides with Pro at position 2 showed higher affinity for B*51:01 and lower affinity for TAP than those with Ala at position 2. Most important, both peptide subsets differed drastically in the susceptibility of their position 1 residues to ERAP‐1, revealing a distinct influence of this enzyme on both subpeptidomes, which may alter their balance, affecting the global affinity of B*51:01–peptide complexes.

ERAP‐1 has a significant influence on the B*51:01 peptidome and its affinity. This influence is based on very distinct effects on the 2 subpeptidomes, whereby only peptides in the subpeptidome with Ala at position 2 are extensively destroyed, except when their position 1 residues are ERAP‐1 resistant. This pattern provides a mechanism for the epistatic association of ERAP‐1 and B*51:01 in Behçet's disease.