Peptide handling by HLA-B27 subtypes influences their biological behavior, association with ankylosing spondylitis and susceptibility to endoplasmic reticulum aminopeptidase 1 (ERAP1)

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.

Fig. 1.

Mass range (upper panel) and length distribution (lower panel) of 2229 HLA-B27-bound and other peptides. A, The HLA-B27 ligands are listed in supplemental Table S2. Five peptides with MW>1700 Da are not included in the upper panel. B, Mass range (upper panel) and length distribution (lower panel) of HLA-B7 (B*07:02) ligands. C, Mass range (upper panel) and length distribution (lower panel) of HLA-A2 (A*02:01) ligands. Data from panes B and C are based on previously published peptide databases (45) and are included here only for comparison. These series included only peptides up to 1500 Da and 8–11 residues.

Fig. 2.

Residue frequencies (% RF) among subtype-bound HLA-B27 ligands at their main anchor positions P2 and PC. AS-associated (B*27:02, 04, 05, and 07) and non-AS-associated (B*27:06 and 09) subtypes are shown, in that order, with white and black bars, respectively. B*27:03 in shown with a gray bar. For n-fold and statistical significance of relevant differences see supplemental Table S6.

Fig. 3.

Residue frequencies (% RF) among subtype-bound HLA-B27 ligands at their secondary anchor positions P1, P3, and PC-2. B*27:02, 04, 05 and 07 are shown, in that order, with white bars, B*27:06 and 09 are shown with black bars and B*27:03 in shown with a gray bar. For n-fold and statistical significance of relevant differences see supplemental Table S6.

Fig. 4.

MW and length of peptide subsets correlating with AS or molecular stability. A, Mass range distribution of peptides found exclusively in AS-associated (dashed line) or non-AS-associated (solid line) HLA-B27 subtypes. B, Length distribution of peptides found exclusively in AS-associated (white bars) or non-AS-associated (black bars) subtypes. Statistically significant differences in the frequency of 9-mers, 10-mers and 11-mers in both peptide sets (marked with *) were observed (p = 1.6 × 10⁻¹⁶, 5.4 × 10⁻⁸, and 1.7 × 10⁻³, respectively) C, Mass range distribution of peptides found exclusively in high (dashed line) or low (solid line) HLA-B27 thermostability subtypes. D, Length distribution of peptides found exclusively in high (white bars) or low (black bars) thermostability subtypes. Statistically significant differences in the frequency of 9-mers and 10-mers in both peptide sets (marked with *) were observed (p = 1.4 × 10⁻¹¹ and 2.2 × 10⁻⁷ respectively).

Fig. 5.

C-terminal residue usage among peptide subsets correlating with AS or molecular stability. A, Residue frequencies (% RF) among peptides found exclusively in the AS-associated subtypes B*27:02, 04, 05, and 07 (white bars) or the non-AS-associated ones, B*27:06 and 09 (black bars) at the C-terminal position. Statistically significant differences (p < 0.05) are marked with (*) B, Chemical classification of the C-terminal residues from peptides found exclusively in the AS-associated (white bars) or non-AS-associated subtypes (black bars). Statistically significant differences (marked with *) were observed among aliphatic, aromatic and basic residues in both subsets (p = 2.1 × 10⁻⁷⁸, 6.8 × 10⁻⁵¹, and 7.3 × 10⁻⁸, respectively). C, Size-based classification of C-terminal residues from peptides found exclusively in the AS-associated (white bars) or non-AS-associated subtypes (black bars). Statistically significant differences were observed among peptides in the ranges 57–103, 104–115, and 138–186 Da in both subsets (p: 2.5 × 10⁻⁸, 1.0 × 10⁻⁶², and 3.0 × 10⁻⁶⁴, respectively). D, C-terminal residue frequencies among peptides found exclusively in the high (B*27:02, 04, and 05: white bars) or low (B*27:06, 07, and 09: black bars) thermostability subtypes. Statistically significant differences (p < 0.05) are marked with (*). E, Chemical classification of C-terminal residues from peptides found exclusively in the high (white bars) or low thermostability subtypes (black bars). Statistically significant differences (marked with *) were observed among aliphatic, aromatic, and basic residues in both subsets (p = 3.8 × 10⁻¹¹⁰, 6.9 × 10⁻⁶⁷, and 4.6 × 10⁻¹³, respectively). F, Size-based classification of C-terminal residues in peptides found exclusively in the high (white bars) or low thermostability subtypes (black bars). Statistically significant differences were observed among peptides in the ranges 57–103, 104–115, and 138–186 Da in both subsets (p = 1.4 × 10⁻⁹, 1.3 × 10⁻⁸⁰, and 1.7 × 10⁻⁸⁶, respectively).

Fig. 6.

Predicted affinity of HLA-B27 and other ligands and their precursors for TAP. A, TAP binding affinity of the natural HLA-B27 ligands in supplemental Table S2 and their N-terminally extended precursors (Nt) by 1 to 4 residues. Bars indicate the geometrical mean values of each peptide set. Affinity was calculated as previously described (43). The significance of the differences among peptide sets was assessed by the Mann-Whitney test. Natural ligands were better suited to TAP than any of their precursors (p < 0.0001), followed by single-residue extended precursors. B, TAP binding affinity of peptides selectively found among non-AS- or AS-associated subtypes. The latter peptide set was subdivided according to the C-terminal residue as indicated. Peptides from AS-associated subtypes were more adapted to TAP (p < 0.0001). Among these, peptides with C-terminal aromatic residues were most suitable (p < 0.0001), but peptides with C-terminal aliphatic or basic residues were also better TAP binders than non-AS-associated peptides (p < 0.0001). C, TAP binding affinity of HLA-B*07:02 ligands (n = 3214) and their precursors. Conventions are as in panel A. D, TAP binding affinity of HLA-A*02:01 ligands (n = 3502) and their precursors. Conventions are as in panel A. The data in panels C and D are based on previously published sequence databases (45) and are included here only for comparison. Reported sequences assigned only to HLA-B7 or HLA-A2 were included.

Fig. 7.

ERAP1 susceptibility of the flanking and P1 residues of peptides selectively found among HLA-B27 subtypes with differential AS-association or thermostability. A, Joint frequency of highly susceptible residues (score>50) at the P-2, P-1, and P1 positions in the AS-associated (white bars) and the non-AS-associated subtype ligands (black bars). B, Joint frequency of low susceptibility residues (score<10) at the P-2, P-1, and P1 positions in the AS-associated (white bars) and the non-AS-associated subtype ligands (black bars). Statistically significant differences (p = 6.2 × 10⁻³) are indicated (*). (C) Joint frequency of susceptible residues at the P-2, P-1, and P1 positions in the high (white bars) and low thermostability subtype ligands (black bars). D, Joint frequency of low susceptibility residues at the P-2, P-1, and P1 positions in the high (white bars) and low thermostability subtype ligands (black bars). Statistically significant differences are indicated (p = 1.4 × 10⁻⁴). E, Joint frequency of susceptible P1 residues of the peptides selectively found among AS-associated (white bars), non-AS-associated (black bars), high thermostability (light gray bars), or low thermostability (dark gray bars) subtypes. Peptides were classified by length in ≤9-mers and ≥10-mers. Statistically significant differences are indicated (p = 0.016). F, Joint frequency of low susceptibility P1 residues of the same peptide subsets as in panel E. Statistically significant differences (p = 3.5 × 10⁻⁴ and 5.6 × 10⁻⁵, respectively) are indicated.