The very strong association of human leukocyte antigen (HLA)-B27 with spondyloarthritis might be related to its peptide-presenting properties. The natural polymorphism of this molecule influences both peptide specificity and disease susceptibility. In this study, we present a comprehensive compilation of known natural ligands of HLA-B27 arising from endogenous proteins of human cells, together with a statistical assessment of residue usage among constitutive peptide repertoires of multiple HLA-B27 subtypes. This analysis provides evidence that every peptide position, including "non-anchor" ones, may be subjected to selection on the basis of its contribution to HLA-B27 binding and also allows a quantization of residue preferences at known anchor positions. The present registry is intended as a basis on which to build up reliable criteria to assess the effect of HLA-B27 polymorphism on peptide presentation, for T-cell epitope predictions, and for molecular mimicry studies.

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.

Four inflammatory diseases are strongly associated with Major Histocompatibility Complex class I (MHC-I) molecules: birdshot chorioretinopathy (HLA-A*29:02), ankylosing spondylitis (HLA-B*27), Behçet's disease (HLA-B*51), and psoriasis (HLA-C*06:02). The endoplasmic reticulum aminopeptidases (ERAP) 1 and 2 are also risk factors for these diseases. Since both enzymes are involved in the final processing steps of MHC-I ligands it is reasonable to assume that MHC-I-bound peptides play a significant pathogenetic role. This review will mainly focus on recent studies concerning the effects of ERAP1 and ERAP2 polymorphism and expression on shaping the peptidome of disease-associated MHC-I molecules in live cells. These studies will be discussed in the context of the distinct mechanisms and substrate preferences of both enzymes, their different patterns of genetic association with various diseases, the role of polymorphisms determining changes in enzymatic activity or expression levels, and the distinct peptidomes of disease-associated MHC-I allotypes. ERAP1 and ERAP2 polymorphism and expression induce significant changes in multiple MHC-I-bound peptidomes. These changes are MHC allotype-specific and, without excluding a degree of functional inter-dependence between both enzymes, reflect largely separate roles in their processing of MHC-I ligands. The studies reviewed here provide a molecular basis for the distinct patterns of genetic association of ERAP1 and ERAP2 with disease and for the pathogenetic role of peptides. The allotype-dependent alterations induced on distinct peptidomes may explain that the joint association of both enzymes and unrelated MHC-I alleles influence different pathological outcomes.

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 Cterminal 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 ␤ 2 -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.

The emergence of proteomics has placed great interest in the understanding of the mechanisms of MS/MS fragmentation of peptides under low-energy collision-induced dissociation. In this work, we describe the presence of anomalous fragments, which correspond to neutral loss elimination of internal amino acids from ions of the b series in quadrupole ion trap MS/MS spectra from naturally occurring peptides. Internal amino acid elimination occurred preferentially with aliphatic amino acids. The phenomenon was more apparent when doubly charged precursors were fragmented and was inhibited when peptides were N-acetylated at the N-terminus. Fragmentation of isomeric peptides where some internal amino acids were relocated in N-terminal position produced MSn spectra indistinguishable from those of the original peptides, indicating that some b ions underwent a structural rearrangement process. Formation of anomalous fragments required a minimum activation time. Our data are consistent with a nucleophile attack of the N-terminal nitrogen over the electrophilic carbonyl carbon at one peptide bond, forming a cyclic b ion intermediate that, by reopening at preferential sites, exposes internal amino acids to the C-terminal side.

The association of HLA-B27 with ankylosing spondylitis and other spondyloarthropathies ranks among the strongest between any HLA antigen and a human disease. Yet, in spite of intense research and advanced knowledge of the biochemistry and biology of major histocompatibility complex molecules, the mechanism of this association remains unknown. This review attempts a critical assessment of current pathogenetic hypotheses from evidence concerning the epidemiology of HLA-B27 association with disease, its peptide-binding specificity, and other aspects of the molecular biology and immunology of this molecule.

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.

No abstract