Human leukocyte antigen (HLA) B*51:01 and endoplasmic reticulum aminopeptidase 1 (ERAP1) are strongly genetically associated with Behçet's disease (BD).  Previous studies have defined two subgroups of HLA-B*51 peptidome containing Proline (Pro) or Alanine (Ala) at position 2 (P2).  Little is known about the unconventional non-Pro/Ala2 HLA-B*51-bound peptides.  We aimed to study the features of this novel sub-peptidome, and investigate its regulation by ERAP1.  CRISPR-Cas9 was used to generate an HLA-ABC-triple knockout HeLa cell line (HeLa.ABC-KO), which was subsequently transduced to express HLA-B*51:01 (HeLa.ABC-KO.B51).  ERAP1 was silenced using lentiviral shRNA.  Peptides bound to HLA-B*51:01 were eluted and analyzed by Mass Spectrometry.  The characteristics of non-Pro/Ala2, Pro2 and Ala2 peptides, and their alteration by ERAP1 silencing were investigated.  Effects of ERAP1 silencing on cell surface expression of HLA-B*51:01 were studied using flow cytometry.  More than 20% of peptides eluted from HLA-B*51:01 lacked Proline or Alanine at P2.  This unconventional group of HLA-B*51:01-bound peptides was relatively enriched for 8-mers (with relatively fewer 9-mers) compared to the Pro2 and Ala2 sub-peptidomes, and had similar N-terminal and C-terminal residue usages to Ala2 peptides (with the exception of the less abundant Leucine at position Ω).  Knockdown of ERAP1 increased the percentage of non-Pro/Ala2 from 20% to approximately 40%, increased the percentage of longer (10-mer and 11-mer) peptides eluted from HLA-B*51:01 complexes, and abrogated the predominance of Leucine at P1.  Interestingly knockdown of ERAP1 altered the length and N-terminal residue usage of non-Ala2&Pro2 and Ala2 but not the Pro2 peptides.  Finally, ERAP1 silencing regulated the expression levels of cell surface HLA-B*51 in a cell type-dependent manner. In conclusion, we have used a novel methodology to identify an unconventional but surprisingly abundant non-Pro/Ala2 HLA-B*51:01 sub-peptidome.  It is increased by knockdown of ERAP1, a gene affecting the risk of developing BD. This has implications for theories of disease pathogenesis.

EXPLOR_STEP=$WICRI_ROOT/Sante/explor/MersV1/Data/Main/Exploration

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{{Explor lien
   |wiki=    Sante
   |area=    MersV1
   |flux=    Main
   |étape=   Exploration
   |type=    RBID
   |clé=     pubmed:32161166
   |texte=   Identification of an unconventional sub-peptidome bound to the Behçet's disease - associated HLA-B*51:01 that is regulated by endoplasmic reticulum aminopeptidase 1 (ERAP1).
}}

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