The association of ERAP1 with ankylosing spondylitis (AS)1 among HLA-B27-positive individuals suggests that ERAP1 polymorphism may affect pathogenesis by altering peptide-dependent features of the HLA-B27 molecule. Comparisons of HLA-B*27:04-bound peptidomes from cells expressing different natural variants of ERAP1 revealed significant differences in the size, length, and amount of many ligands, as well as in HLA-B27 stability. Peptide analyses suggested that the mechanism of ERAP1/HLA-B27 interaction is a variant-dependent alteration in the balance between epitope generation and destruction determined by the susceptibility of N-terminal flanking and P1 residues to trimming. ERAP1 polymorphism associated with AS susceptibility ensured efficient peptide trimming and high HLA-B27 stability. Protective polymorphism resulted in diminished ERAP1 activity, less efficient trimming, suboptimal HLA-B27 peptidomes, and decreased molecular stability. This study demonstrates that natural ERAP1 polymorphism affects HLA-B27 antigen presentation and stability in vivo and proposes a mechanism for the interaction between these molecules in AS.

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 ASassociated 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 ASassociated/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. Molecular & Cellular

The transporter associated with antigen processing (TAP) translocates the cytosol-derived proteolytic peptides to the endoplasmic reticulum lumen where they complex with nascent human leukocyte antigen (HLA) class I molecules. Non-functional TAP complexes and viral or tumoral blocking of these transporters leads to reduced HLA class I surface expression and a drastic change in the available peptide repertoire. Using mass spectrometry to analyze complex human leukocyte antigen HLA-bound peptide pools isolated from large numbers of TAP-deficient cells, we identified 334 TAP-independent ligands naturally presented by four different HLA-A, -B, and -C class I molecules with very different TAP dependency from the same cell line. The repertoire of TAP-independent peptides examined favored increased peptide lengths and a lack of strict binding motifs for all four HLA class I molecules studied. The TAP-independent peptidome arose from 182 parental proteins, the majority of which yielded one HLA ligand. In contrast, TAP-independent antigen processing of very few cellular proteins generated multiple HLA ligands. Comparison between TAP-independent peptidome and proteome of several subcellular locations suggests that the secretory vesicle-like organelles could be a relevant source of parental proteins for TAP-independent HLA ligands. Finally, a predominant endoproteolytic peptidase specificity for Arg/Lys or Leu/Phe residues in the P1 position of the scissile bond was found for the TAP-independent ligands. These data draw a new and intricate picture of TAP-independent pathways.

Chlamydia trachomatis triggers reactive arthritis, a spondyloarthropathy linked to the human major histocompatibility complex molecule HLA-B27, through an unknown mechanism that might involve molecular mimicry between chlamydial and self-derived HLA-B27 ligands. Chlamydia-specific CD8؉ T-cells are found in reactive arthritis patients, but the immunogenic epitopes are unknown. A previous screening of the chlamydial genome for putative HLA-B27 ligands predicted multiple peptides that were recognized in vitro by CD8 ؉ T-lymphocytes from patients. Here stable transfectants expressing bacterial fusion proteins in human cells were generated to investigate the endogenous processing and presentation by HLA-B27 of two such epitopes through comparative immunoproteomics of HLA-B27-bound peptide repertoires. A predicted T-cell epitope, from the CT610 gene product, was presented by HLA-B27. This is, to our knowledge, the first endogenously processed epitope involved in HLA-B27-restricted responses against C. trachomatis in reactive arthritis. A second predicted epitope, from the CT634 gene product, was not detected. Instead a nonpredicted nonamer from the same protein was identified. Both bacterial peptides showed very high homology with human sequences containing the HLA-B27 binding motif.

Objective To evaluate similarity of the peptide repertoires bound to HLA–DR molecules that are differentially associated with rheumatoid arthritis (RA), and to define structural features of the shared peptides. Methods Peptide pools bound to HLA–DRB1*01:01, HLA–DRB1*04:01, and HLA–DRB1*10:01 (RA associated) and those bound to HLA–DRB1*15:01 (non–RA–associated) were purified and analyzed by liquid chromatography (LC) matrix‐assisted laser desorption ionization–time‐of‐flight mass spectrometry (MS) and LC–ion‐trap MS. Peptide pools from each allotype were compared in terms of size, protein origin, composition, and affinity (both theoretical and experimental with some peptides). Finally, 1 peptide sequenced from DR1, DR4, and DR10, but not from DR15, was modeled in complex with all 4 HLA–DRB1 molecules and HLA–DRB5*01:01. Results A total of 6,309 masses and 962 unique peptide sequences were compared. DR10 shared 29 peptides with DR1, 9 with DR4, and 1 with DR15; DR1 shared 6 peptides with DR4 and 9 with DR15; and DR4 and DR15 shared 4 peptides. The direct identification of peptide ligands indicated that DR1 and DR10 were the most similar molecules regarding the peptides that they could share. The peptides common to these molecules contained a high proportion of Leu at P4 and basic residues at P8 binding core positions. Conclusion The degree of overlap between peptide repertoires associated with different HLA–DR molecules is low. The repertoires associated with DR1 and DR10 have the highest similarity among the molecules analyzed (∼10% overlap). Among the peptides shared between DR1 and DR10, a high proportion contained Leu4 and basic residues at the P8 position of the binding core.

Some HLA class I molecules bind a significant fraction of their constitutive peptidomes in the presence of proteasome inhibitors. In this study, A*68:01-bound peptides, and their parental proteins, were characterized through massive mass spectrometry sequencing to refine its binding motif, including the nearly exclusive preference for C-terminal basic residues. Stable isotope tagging was used to distinguish proteasome-inhibitor sensitive and resistant ligands. The latter accounted for less than 20% of the peptidome and, like in HLA-B27, arose predominantly from small and basic proteins. Under the conditions used for proteasome inhibition in vivo, epoxomicin and MG-132 incompletely inhibited the hydrolysis of fluorogenic substrates specific for the tryptic or for both the tryptic and chymotryptic subspecificities, respectively. This incomplete inhibition was also reflected in the cleavage of synthetic peptide precursors of A*68:01 ligands. For these substrates, the inhibition of the proteasome resulted in altered cleavage patterns. However these alterations did not upset the balance between cleavage at peptide bonds resulting in epitope destruction and those leading to their generation. The results indicate that inhibitor-resistant HLA class I ligands are not necessarily produced by non-proteasomal pathways. However, their generation is not simply explained by decreased epitope destruction upon incomplete proteasomal inhibition and may require additional proteolytic steps acting on incompletely processed proteasomal products. Molecular &

HLA-B27 binds peptides with R at position 2. Additionally, a substantial fraction of the HLA-B27-bound peptide repertoire has basic residues at position 1. It is unclear whether this is determined by structural complementarity with the A pocket of the peptide-binding site, by the increased availability of peptides with dibasic N-terminal sequences resulting from their cytosolic stability, or both. To distinguish between these possibilities two B*2705 mutants were generated in which one or two A pocket surface residues stabilizing the peptidic R1 side chain were changed: E163T and E163T-W167S. Both mutants bound a large fraction of the constitutive peptide repertoire of B*2705. Moreover, 90 B*2705 ligands of known sequence were examined for their endogenous presentation by the mutants. The E163T mutation alone had a limited effect on binding of peptides with R1 or K1 and on the relative frequencies of N-terminal residues. However, it decreased the overall stability of the molecule. The E163T-W167S mutant also bound many of the B*2705 ligands with N-terminal basic residues, but its preference for G1 was significantly decreased. The results indicate that the capacity of HLA-B27 to bind peptides with N-terminal basic residues is largely independent of the canonic interactions that stabilize at least the R1 side chain. Thus, the prevalence of HLA-B27 ligands with dibasic N-terminal sequences may be significantly influenced by the increased availability of these peptides resulting from their cytosolic stability. This confers to HLA-B27 a unique capacity to present Ags generated in low amounts, but resistant to intracellular degradation.

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