Modulation of Natural HLA-B*27:05 Ligandome by Ankylosing Spondylitis-associated Endoplasmic Reticulum Aminopeptidase 2 (ERAP2).

The HLA-B*27:05 allele and the endoplasmic reticulum-resident aminopeptidases are strongly associated with AS, a chronic inflammatory spondyloarthropathy. This study examined the effect of ERAP2 in the generation of the natural HLA-B*27:05 ligandome in live cells. Complexes of HLA-B*27:05-bound peptide pools were isolated from human ERAP2-edited cell clones, and the peptides were identified using high-throughput mass spectrometry analyses. The relative abundance of a thousand ligands was established by quantitative tandem mass spectrometry and bioinformatics analysis. The residue frequencies at different peptide position, identified in the presence or absence of ERAP2, determined structural features of ligands and their interactions with specific pockets of the antigen-binding site of the HLA-B*27:05 molecule. Sequence alignment of ligands identified with species of bacteria associated with HLA-B*27-dependent reactive arthritis was performed. In the absence of ERAP2, peptides with N-terminal basic residues and minority canonical P2 residues are enriched in the natural ligandome. Further, alterations of residue frequencies and hydrophobicity profile at P3, P7, and PΩ positions were detected. In addition, several ERAP2-dependent cellular peptides were highly similar to protein sequences of arthritogenic bacteria, including one human HLA-B*27:05 ligand fully conserved in a protein from Campylobacter jejuni These findings highlight the pathogenic role of this aminopeptidase in the triggering of AS autoimmune disease.

Functional interaction of the ankylosing spondylitis-associated endoplasmic reticulum aminopeptidase 1 polymorphism and HLA-B27 in vivo.

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.

Mutational analysis reveals a complex interplay of peptide binding and multiple biological features of HLA-B27.

Molecular polymorphism influences the strong association of HLA-B27 with ankylosing spondylitis through an unknown mechanism. Natural subtypes and site-directed mutants were used to analyze the effect of altering the peptide-binding site of this molecule on its stability, interaction with tapasin, folding, and export. The disease-associated subtypes B*2705, B*2702, and B*2704 showed higher thermostability at 50 °C than all other subtypes and mutants, except some mimicking B*2702 polymorphism. The lowest values were found among pocket B mutants, most of which interacted strongly with tapasin, but otherwise there was no correlation between thermostability and tapasin interaction. Mutants resulting in increased hydrophobicity frequently acquired their maximal thermostability faster than those with increased polarity, suggesting that this process is largely driven by the thermodynamics of peptide binding. Folding, export, and tendency to misfold were influenced by polymorphism all along the peptide-binding site and were not specifically dependent on any particular region or structural feature. Frequent uncoupling of thermostability, folding/misfolding, and export can be explained by the distinct effect of mutations on the acquisition of a folded conformation, the optimization rate of B27-peptide complexes, and their quality control in the endoplasmic reticulum, all of which largely depend on the ways in which mutations alter peptide binding, without excluding additional effects on interactions with tapasin or other proteins involved in folding and export. The similarity of the generally disease-associated B*2707 to nondisease-associated subtypes in all the features analyzed suggests that molecular properties other than antigen presentation may not currently explain the relationship between HLA-B27 polymorphism and ankylosing spondylitis.

Presentation of cytosolically stable peptides by HLA-B27 is not dependent on the canonic interactions of N-terminal basic residues in the A pocket.

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.

Disparate folding and stability of the ankylosing spondylitis-associated HLA-B*1403 and B*2705 proteins.

OBJECTIVE: To investigate the folding, assembly, maturation, and stability of HLA-B*1402 and B*1403, which differ by 1 amino acid change and are differentially associated with ankylosing spondylitis (AS), and to compare these features with those of B*2705. METHODS: Stable transfectants expressing B*1402, B*1403, and B*2705 were used. Folding rates were estimated from the ratio of unfolded heavy chains to folded heavy chains that had been immunoprecipitated with specific antibodies in pulse-chase experiments. Heavy chain misfolding was measured as the half-life of endoglycosidase H (Endo H)-sensitive beta2-microglobulin-free heavy chains. Maturation/export rates were measured by acquisition of Endo H resistance. Association with calnexin or tapasin was analyzed by coprecipitation with chaperone-specific antibodies, and surface expression was estimated by flow cytometry. Thermostability of HLA-peptide complexes was assessed by immunoprecipitation after incubation at various temperatures. Heavy chain expression was quantified by Western blotting. RESULTS: The folding rates of B*1402 and B*1403 were similar, and both were faster and more efficient than B*2705, but some unfolded heavy chains from both B14 subtypes remained in the endoplasmic reticulum (ER) with a long half-life. The export rates of B*1402 and B*1403 were slow, and the heterodimers partially dissociated after exiting the ER, as revealed by significant amounts of Endo H-resistant and surface-expressed free heavy chains. Both interaction with tapasin and thermostability were higher for B*2705 than for B*1402 and higher for B*1402 than for B*1403, suggesting that the repertoires of the B*1402-bound peptide and especially the B*1403-bound peptide were less optimized than that of B*2705. CONCLUSION: Our results indicate that the folding, maturation, and stability of B*1403 differ more from B*2705 than from B*1402. Thus, these features cannot account for the fact that only the 2 former allotypes are associated with AS.

Folding of HLA-B27 subtypes is determined by the global effect of polymorphic residues and shows incomplete correspondence to ankylosing spondylitis.

OBJECTIVE: To investigate the maturation and folding of HLA-B27 subtypes and the relationship of these features to ankylosing spondylitis (AS). METHODS: Stable transfectants expressing B27 subtypes and site-directed mutants were used. Maturation/export rates were measured by acquisition of endoglycosidase H resistance. Folding efficiency was estimated from the ratio of unfolded heavy chain to folded heavy chain, which was immunoprecipitated with specific antibodies, in pulse-chase experiments. Association with calnexin was analyzed in coprecipitation experiments. Cytosolic dislocation was estimated by immunoprecipitation of deglycosylated heavy chain after proteasome inhibition. The level of heavy chain expression on unstimulated or interferon-gamma (IFNgamma)-stimulated cells was quantified by Western blotting. RESULTS: There was no correlation between the export rate and the association of HLA-B27 subtypes with AS. Three of the 4 AS-associated B27 subtypes showed inefficient folding, but B*2707 folded with the same high efficiency as the non-disease-associated subtypes. Some individual mutations that mimicked subtype polymorphism profoundly influenced folding, but in a context-dependent way. The differences in export and folding rates among B27 variants were unrelated to levels of heavy chain expression in the corresponding transfectants, as indicated by the lack of correlation between the two parameters and by heavy chain up-regulation with IFNgamma. Misfolded heavy chain was inefficiently cleared from the endoplasmic reticulum, based on the marginal increase in levels of deglycosylated heavy chain, which resulted from loss of the glycan moiety after cytosolic dislocation, following proteasome inhibition. CONCLUSION: HLA-B27 subtype folding is determined by the overall heavy-chain structure, since the effect of a given polymorphism depends on its structural context. Heavy chain misfolding does not explain the association of B*2707 with AS.

HLA-B*2704, an allotype associated with ankylosing spondylitis, is critically dependent on transporter associated with antigen processing and relatively independent of tapasin and immunoproteasome for maturation, surface expression, and T cell recognition: relationship to B*2705 and B*2706.

B*2704 is strongly associated to ankylosing spondylitis in Asian populations. It differs from the main HLA-B27 allotype, B*2705, in three amino acid changes. We analyzed the influence of tapasin, TAP, and immunoproteasome induction on maturation, surface expression, and T cell allorecognition of B*2704 and compared some of these features with B*2705 and B*2706, allotypes not associated to disease. In the tapasin-deficient .220 cell line, this chaperone significantly influenced the extent of folding of B*2704 and B*2705, but not their egress from the endoplasmic reticulum. In contrast, B*2706 showed faster folding and no accumulation in the endoplasmic reticulum in the absence of tapasin. Surface expression of B*2704 was more tapasin dependent than B*2705. However, expression of free H chain decreased in the presence of this chaperone for B*2705 but not B*2704, suggesting that more suboptimal ligands were loaded on B*2705 in the absence of tapasin. Despite its influence on surface expression, tapasin had little effect on allorecognition of B*2704. Both surface expression and T cell recognition of B*2704 were critically dependent on TAP, as established with TAP-deficient and TAP-proficient T2 cells. Both immunoproteasome and surface levels of B*2704 were induced by IFN-gamma, but this had little effect on allorecognition. Thus, except for the differential effects of tapasin on surface expression, the tapasin, TAP, and immunoproteasome dependency of B*2704 for maturation, surface expression, and T cell recognition are similar to B*2705, indicating that basic immunological features are shared by the two major HLA-B27 allotypes associated to ankylosing spondylitis in human populations.

Qualitative and quantitative differences in peptides bound to HLA-B27 in the presence of mouse versus human tapasin define a role for tapasin as a size-dependent peptide editor.

Tapasin (Tpn) is a chaperone of the endoplasmic reticulum involved in peptide loading to MHC class I proteins. The influence of mouse Tpn (mTpn) on the HLA-B*2705-bound peptide repertoire was analyzed to characterize the species specificity of this chaperone. B*2705 was expressed on Tpn-deficient human 721.220 cells cotransfected with human (hTpn) or mTpn. The heterodimer to beta(2)-microglobulin-free H chain ratio on the cell surface was reduced with mTpn, suggesting lower B*2705 stability. The B*2705-bound peptide repertoires loaded with hTpn or mTpn shared 94-97% identity, although significant differences in peptide amount were observed in 16-17% of the shared ligands. About 3-6% of peptides were bound only with either hTpn or mTpn. Nonamers differentially bound with mTpn had less suitable anchor residues and bound B*2705 less efficiently in vitro than those loaded only with hTpn or shared nonamers. Decamers showed a different pattern: those found only with mTpn had similarly suitable residues as shared decamers and bound B*2705 with high efficiency. Peptides differentially presented by B*2705 on human or mouse cells showed an analogous pattern of residue suitability, suggesting that the effect of mTpn on B*2705 loading is comparable in both cell types. Thus, mTpn has quantitative and qualitative effects on the B*2705-bound peptide repertoire, impairing presentation of some suitable ligands and allowing others with suboptimal anchor residues and lower affinity to be presented. Our results favor a size-dependent peptide editing role of Tpn for HLA-B*2705 that is species-dependent and suboptimally performed, at least for nonamers, by mTpn.

T cell receptor-mediated signal transduction controlled by the beta chain transmembrane domain: apoptosis-deficient cells display unbalanced mitogen-activated protein kinases activities upon T cell receptor engagement.

The bases that support the versatility of the T cell receptor (TCR) to generate distinct T cell responses remain unclear. We have previously shown that mutant cells in the transmembrane domain of TCRbeta chain are impaired in TCR-induced apoptosis but are not affected in other functions. Here we describe the biochemical mechanisms by which this mutant receptor supports some T cell responses but fails to induce apoptosis. Extracellular signal-regulated protein kinase (ERK) is activated at higher and more sustained levels in TCRbeta-mutated than in wild type cells. Conversely, activation of both c-Jun N-terminal kinase and p38 mitogen-activated protein kinase is severely reduced in mutant cells. By attempting to link this unbalanced induction to altered upstream events, we found that ZAP-70 is normally activated. However, although SLP-76 phosphorylation is normally induced, TCR engagement of mutant cells results in lower tyrosine phosphorylation of LAT but in higher tyrosine phosphorylation of Vav than in wild type cells. The results suggest that an altered signaling cascade leading to an imbalance in mitogen-activated protein kinase activities is involved in the selective impairment of apoptosis in these mutant cells. Furthermore, they also provide new insights in the contribution of TCR to decipher the signals that mediate apoptosis distinctly from proliferation.