Functionally distinct ERAP1 allotype combinations distinguish individuals with Ankylosing Spondylitis

Abstract

For more than 40 y, expression of HLA-B27 has been strongly associated with the chronic inflammatory disease Ankylosing Spondylitis (AS); however, the mechanisms underlying this association are still unknown. Single nucleotide polymorphisms within the aminopeptidase endoplasmic reticulum aminopeptidase 1 (ERAP1), which is essential for trimming peptides before they are presented to T cells by major histocompatibility complex (MHC) class I molecules, have been linked with disease. We show that ERAP1 is a highly polymorphic molecule comprising allotypes of single nucleotide polymorphisms. The prevalence of specific ERAP1 allotypes is different between AS cases and controls. Both chromosomal copies of ERAP1 are codominantly expressed, and analysis of allotype pairs provided clear stratification of individuals with AS versus controls. Functional analyses demonstrated that ERAP1 allotype pairs seen in AS cases were poor at generating optimal peptide ligands for binding to murine H-2K(b) and -D(b) and the AS-associated HLA-B*2705. We therefore provide strong evidence that polymorphic ERAP1 alters protein function predisposing an individual to AS via its influence on the antigen processing pathway.

Keywords: Ankylosing Spondylitis; ERAP1; HLA-B27; antigen presentation; antigen processing.

Fig. 1.

Phylogenetic analysis of ERAP1 allotypes. ERAP1 amino acid (A) and nucleotide (B) sequences were used to generate unrooted maximum likelihood phylogenetic trees. The frequency of each allotype in the amino acid tree is indicated. The overall trimming function of each allotype is also indicated; hyperactive trimmers are in red, hypoactive trimmers in blue, intermediate trimmers in green, and efficient trimmers in bold type. Allotypes in italics have not been assessed.

Fig. 2.

ERAP1 allotype pairs isolated from AS cases have impaired trimming capacity. Erap1-deficient cells were transfected with ERAP1 allotypes corresponding to individual allotype pairs identified in cases and controls and X5-SHL8 and assessed for trimming using B3Z. (A and B) Representative line graphs showing trimming of the most common allotype pairs from controls (A) or cases (B) as indicated. The positive (*002) and negative (*002-E320A) control allotypes were also transfected. Data are representative of four experiments. (C and D) The relative maximum B3Z response of observed allotype pairs identified in control or case groups with each symbol representing a single allotype pair transfection (C) or individual allotype pairs compared with *002 allotype pair are shown (D). Data are pooled from four experimental repeats ±SEM (****P < 0.0001; **P < 0.01; *P < 0.05; ns, not significant). (E) The ability of ERAP1 allotype pairs to restore MHC I levels in Erap1-deficient cells was assessed. Erap1-deficient cells were transfected with allotype pairs as above (*002+*011, *010+*011, *002+*003, *005+*013, and *004+*006 not done), and the levels of H-2K^b (filled bars) or H-2D^b (open bars) were assessed and compared with *002. Results show data pooled from three experiments ±SEM (***P < 0.001; **P < 0.01; *P < 0.05; ns, not significant). The dashed line represents 100% restoration of MHC I levels.

Fig. 3.

AS case ERAP1 allotype pairs fail to increase HLA-B*2705 cell surface expression. Flow cytometry analysis of HLA-B*2705 cell surface expression by Erap1-deficient fibroblasts (A–C) or ERAP1 KO 293T cells (D–F) transfected with each of the 15 ERAP1 allotype pairs identified from control and AS case groups compared with *002-E320A. (A and D) Representative histograms showing HLA-B*2705 expression after transfection of Erap1-deficient fibroblasts (A) and ERAP1 KO 293T cells (D) with an example of allotype pairs from control and AS case groups. (B and E) Comparison of HLA-B*2705 cell surface expression in Erap1-deficient fibroblasts (B) or ERAP1 KO 293T cells (E) transfected with allotype pairs from control and AS case groups. Each symbol represents a single allotype pair transfection from three (B) or five (E) independent experiments; ***P < 0.0001. (C and F) The effect of individual ERAP1 allotype pairs from control and AS cases on HLA-B*2705 cell surface expression following transfection into Erap1-deficient fibroblasts (C) or ERAP1 KO 293T cells (F). Data are pooled from three (C) or five (F) independent experiments ±SEM.

Fig. 4.

Model representing the link between the ERAP1 trimming activity of an allotype pair and disease. ERAP1 allotype pairs from individuals have a broad spectrum of trimming activity. Those with trimming activities toward the extreme ends of this spectrum have a greater risk of developing AS. This increased risk is manifested in two different ways: (i) Aberrant ERAP1 activity results in increased misfolded and HLA-B*2705 homodimers in the ER inducing the unfolded protein response. (ii) Aberrant ERAP1 activity generates unstable peptide ligands and results in increased cell surface HLA-B*2705 homodimers activating NK and/or Th17 cells.