José A. López de Castro

Quantitative and Qualitative Influences of Tapasin on the Class I Peptide Repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and β2-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Differential Association of HLA-B*2705 and B*2709 to Ankylosing Spondylitis Correlates with Limited Peptide Subsets but Not with Altered Cell Surface Stability

In contrast to HLA-B*2705, B*2709 is weakly or not associated to ankylosing spondylitis. Both allotypes differ by a single D116H change. We compared the B*2705- and B*2709-bound peptide repertoires by mass spectrometry to quantify the effect of B*2709 polymorphism on peptide specificity. In addition, shared and differentially bound ligands were sequenced to define the structural features of the various peptide subsets. B*2705 shared 79% of its peptide repertoire with B*2709. Shared ligands accounted for 88% of the B*2709-bound repertoire. All B*2705 ligands not bound to B*2709 had C-terminal basic or Tyr residues. Most B*2709-bound peptides had C-terminal aliphatic and Phe residues, but two showed C-terminal Arg or Tyr. The B*2709-bound repertoire included 12% of peptides not found in B*2705. These had aliphatic C-terminal residues, which are also favored in B*2705. However, these peptides bound weakly B*2705 in vitro, indicating distinct contribution of secondary anchor residues in both subtypes. Differences in peptide binding did not affect the ratio of native to β2-microglobulin-free HLA-B27 heavy chain at the cell surface. Our results suggest that weaker association of B*2709 with ankylosing spondylitis is based on differential binding of a limited subset of natural ligands by this allotype.

Molecular Mimicry of an HLA-B27-derived Ligand of Arthritis-linked Subtypes with Chlamydial Proteins

HLA-B27 is strongly associated with spondyloarthropathies, including ankylosing spondylitis and reactive arthritis. The latter disease is triggered by various Gram-negative bacteria. A dodecamer derived from the intracytoplasmic tail of HLA-B27 was a natural ligand of three disease-associated subtypes (B*2702, B*2704, and B*2705) but not of two (B*2706 and B*2709), weakly or not associated to spondyloarthropathy. This peptide was strikingly homologous to protein sequences from arthritogenic bacteria, particularly to a region of the DNA primase fromChlamydia trachomatis. A synthetic peptide with this bacterial sequence bound in vitro disease-associated subtypes equally as the natural B27-derived ligand. The chlamydial peptide was generated by the 20 S proteasome from a synthetic 28-mer with the sequence of the corresponding region of the bacterial DNA primase. Molecular modeling suggested that the B27-derived and chlamydial peptides adopt very similar conformations in complex with B*2705. The results demonstrate that an HLA-B27-derived peptide mimicking arthritogenic bacterial sequences is a natural ligand of disease-associated HLA-B27 subtypes and suggest that the homologous chlamydial peptide might be presented by HLA-B27 onChlamydia-infected cells.

The Peptide Repertoires of HLA-B27 Subtypes Differentially Associated to Spondyloarthropathy (B*2704 and B*2706) Differ by Specific Changes at Three Anchor Positions

HLA-B*2704 is strongly associated with ankylosing spondylitis. B*2706, which differs from B*2704 by two amino acid changes, is not associated with this disease. A systematic comparison of the B*2704- and B*2706-bound peptide repertoires was carried out to elucidate their overlap and differential features and to correlate them with disease susceptibility. Both subtypes shared about 90% of their peptide repertoires, consisting of peptides with Arg2and C-terminal aliphatic or Phe residues. B*2706 polymorphism influenced specificity at three anchor positions: it favored basic residues at P3 and PΩ-2 and impaired binding of Tyr and Arg at PΩ. Thus, the main structural feature of peptides differentially bound to B*2704 was the presence of C-terminal Tyr or Arg, together with a strong preference for aliphatic/aromatic P3 residues. This is the only known feature of B*2704 and B*2706 that correlates to their differential association with spondyloarthropathy. The concomitant presence of basic P3 and PΩ-2 residues was observed only among peptides differentially bound to B*2706, suggesting that it impairs binding to B*2704. Similarity between peptide overlap and the degree of cross-reaction with alloreactive T lymphocytes suggested that the majority of shared ligands maintain unaltered antigenic features in the context of both subtypes.

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.

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.

ERAP1 structure, function and pathogenetic role in ankylosing spondylitis and other MHC-associated diseases ☆

The endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in the final processing of Major Histocompatibility Complex class I (MHC-I) ligands and with a significant influence in the stability and immunological properties of MHC-I proteins. ERAP1 polymorphism is associated with ankylosing spondylitis among HLA-B27-positive individuals and the altered enzymatic activity of natural variants has significant effects on the HLA-B27 peptidome, suggesting a critical pathogenetic role of peptides in this disease. Likewise, the association of ERAP1 with other MHC-I associated disorders and its epistasis with their susceptibility MHC alleles point out to a general role of the MHC-I peptidome in these diseases. The functional interaction between ERAP1 and HLA-B27 or other MHC-I molecules may be related to the processing of specific epitopes, or to a more general peptide-dependent influence on other biological features of the MHC-I proteins. In addition, from a consideration of the reported functions of ERAP1, including its involvement in angiogenesis and macrophage activation, a more complex and multi-level influence in the inflammatory and immune pathways operating in these diseases cannot be ruled out.

Modulation at multiple anchor positions of the peptide specificity of HLA–B27 subtypes differentially associated with ankylosing spondylitis

OBJECTIVE To investigate the rules governing peptide binding to HLA-B*2705, and to B*2704 and B*2706, which are 2 subtypes differentially associated with ankylosing spondylitis. METHODS Poly-Ala analogs carrying the HLA-B27 motif Arg-2, and substitutions at anchor positions P1, P3, or Pomega, were used to determine a binding score for each residue at each position. Binding was assessed in a quantitative epitope stabilization assay, where the cell surface expression of HLA-B27 was measured by flow cytometry as a function of peptide concentration. RESULTS Peptide anchor residues contributed additively to B*2705 binding. About 15% of the natural B*2705 ligands used a deficient P3 or Pomega anchor, but never both, indicating that detrimental anchoring at one of these positions is always compensated by a good anchor at the other one. About 50% of the B*2705 ligands used suboptimal P1 residues. However, this was compensated with optimal P3 and/or Pomega anchoring. Peptides that were longer than decamers used good anchor residues at the 3 positions, suggesting more stringent binding requirements. B*2704 and B*2706 differed in their residue specificity at P1, P3, and Pomega. The rules derived for B*2705 also applied to the known ligands of these 2 subtypes. CONCLUSION The B*2705, B*2704, and B*2706 peptide repertoires are limited by the allowed residue combinations described in this study. The differential association of B*2704 and B*2706 with spondylarthropathy correlates with differences in their peptide specificity at multiple anchor positions. However, it is now possible to predict the peptide features that determine this differential binding to both subtypes.

Limited Diversity of Peptides Related to an Alloreactive T Cell Epitope in the HLA-B27-Bound Peptide Repertoire Results from Restrictions at Multiple Steps Along the Processing-Loading Pathway

The influence of various factors along the processing-loading pathway in limiting the diversity of HLA-B27-bound peptides around a core protein sequence was analyzed. The C5 proteasome subunit-derived RRFFPYYV and RRFFPYYVY peptides are natural B*2705 ligands. The octamer is an allospecific CTL epitope. Digestion of a 27-mer fragment of C5 revealed that both ligands are generated from this precursor substrate with the 20S proteasome in vitro in a ratio comparable to that in the B*2705-bound peptide pool. The C5 sequence allowed to derive a nested set of six additional peptides with 8–11 residues containing the core octamer sequence and the Arg2 motif of HLA-B27, none of which was found in the B27-bound pool. Together, low proteasomal yield, disfavored TAP-binding motifs, and low affinity for B*2705 accounted for the absence of four of the six peptides. The two remaining differed from the natural octamer or nonamer ligands only by an additional N-terminal Ser residue. Their stability in complex with B*2705 was lower than the respective natural ligands, raising the possibility that N-terminal trimming might have favored a shift toward the more stable peptides. The results suggest that the B*2705-bound peptide repertoire has a highly restricted diversity around a core alloantigenic sequence. This is not explained by a single bottleneck feature, but by multiple factors, including proteasomal generation, TAP-binding motifs, MHC-binding efficiency, and perhaps optimized stability through N-terminal trimming. Tapasin-dependent restrictions, although not excluded, were not required to explain the absence in vivo of the particular peptide set in this study.

Structure of the HLA-A*0204 antigen, found in South American Indians. Spatial clustering of HLA-A2 subtype polymorphism

The primary structure of the HLA-A2 subtype A*0204 (isoelectric focusing variant A2.A) has been determined. cDNA encoding this subtype was amplified by the polymerase chain reaction. Four independent full-lenght cDNA clones encoding A*0204 were analyzed to obtain a consensus sequence for this subtype. A*0204 differs from A*0201 by a single nucleotide change of G to T through the coding regions, resulting in an Arg to Met change at position 97. This substitution accounts for the isoelectric focusing pattern of the subtype. The same change occurs in other HLA-A specificities in association with other changes in its vicinity. The absence of additional substitutions in A*0204 suggests that it could have arisen from A*0201 by point mutation, and that recurrent mutations may take place during HLA diversification. The spatial location of this change implies that A*0204 must be a functional variant. Comparison of its sequence with other HLA-A2 subtypes reveals that much of the HLA-A2 subtype polymorphism is generated by variations in four neighboring positions, including position 97, which are located in two adjacent β-strands on the floor of the peptide binding site of the molecule.