Abstract
HLA-B27 is strongly associated with ankylosing spondylitis (AS). We analyzed the relationship between structure, peptide specificity, folding, and stability of the seven major HLA-B27 subtypes to determine the role of their constitutive peptidomes in the pathogenicity of this molecule. Identification of large numbers of ligands allowed us to define the differences among subtype-bound peptidomes and to elucidate the peptide features associated with AS and molecular stability. The peptides identified only in AS-associated or high thermostability subtypes with identical A and B pockets were longer and had bulkier and more diverse C-terminal residues than those found only among non-AS-associated/lower-thermostability subtypes. Peptides sequenced from all AS-associated subtypes and not from non-AS-associated ones, thus strictly correlating with disease, were very rare. Residue 116 was critical in determining peptide binding, thermodynamic properties, and folding, thus emerging as a key feature that unified HLA-B27 biology. HLA-B27 ligands were better suited to TAP transport than their N-terminal precursors, and AS-associated subtype ligands were better than those from non-AS-associated subtypes, suggesting a particular capacity of AS-associated subtypes to bind epitopes directly produced in the cytosol. Peptides identified only from AS-associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-AS-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group. Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes.
Highlights
Associated/high-thermostability subtypes showed a higher frequency of ERAP1-resistant N-terminal residues than ligands found only in non-ankylosing spondylitis (AS)-associated/low-thermostability subtypes, reflecting a more pronounced effect of ERAP1 on the former group
Our results reveal the basis for the relationship between peptide specificity and other features of HLA-B27, provide a unified view of HLA-B27 biology and pathogenicity, and suggest a larger influence of ERAP1 polymorphism on AS-associated than non-AS-associated subtypes
The fact that CD8ϩ T cells are not required for the HLA-B27-associated disease in transgenic rats [7, 8], and the failure to identify specific arthritogenic peptides, point out to a pathogenetic role of HLA-B27 based on its folding and/or non-canonical forms, rather than to an autoimmune mechanism based on molecular mimicry between foreign and self-derived peptides
Summary
Cell Lines and Antibodies—HMy2.C1R (C1R) is a human lymphoid cell line expressing low levels of its endogenous HLA-I antigens [26]. The peptides were identified using multiple search engines: Pep-Miner, Sequest (Thermo-Fisher) [37] and Mascot (server 2.2, Matrix Science Inc. Boston, USA) [38], and searched against the human part of the Uniprot database (http://www.uniprot.org, May 2011) including 20,381 proteins. Search parameters were set as follows: No enzyme, peptide tolerance: 10 ppm, MS/MS tolerance: 25 mDa, instrument: ESI-QUAD-TOF, and variable modifications: dioxidation of C, oxidation of M and pyroglutamic acid formation from N-terminal Q. TAP-binding Affinity—This was estimated for HLA-B27 ligands and their N-terminally extended precursors using the TAPREG tool [43], which makes use of support vector machines trained with large peptide sets to predict the TAP binding affinity of peptides of eight to 16 residues. The method assigned a score, ranging from 0 to 100, to each amino acid, based on its susceptibility to ERAP1, as established in experimental trimming assays [44]
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