Background/Purpose Polymorphisms in endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) are associated with ankylosing spondylitis, Behcet’s disease, and psoriasis/psoriatic arthritis, where HLA class I alleles are also implicated in susceptibility. The mechanism by which ERAP1 influences disease is unknown, although ERAP1 trims peptides that are presented by HLA class I. To better define the functional interaction between ERAP1 and HLA-B27 we examined the effect of knocking down ERAP1 (ERAP1 KD) on the conformation of HLA-B27 in human monocytic U937 cells (U937.B27) that also express HLA-A3/31, HLA-B18/51, HLA-C1/7).

Methods U937.B27 cells stably expressing shRNA for ERAP1 showed a 65% reduction in ERAP1 protein expression. The unfolded and folded forms of HLA class I molecules (including HLA-B27) were assessed by immunoprecipitation with antibodies HC10 and W6/32, respectively, followed by either isoelectric focusing to separate HLA class I heavy chains, or SDS-PAGE under non-reducing or reducing conditions, to assess disulfide-linked (misfolded) complexes of HLA-B27. HLA proteins were visualized by immunoblotting with 3B.10.7. Flow cytometry was employed to measure expression of folded forms of HLA-B27 (HLA.ABC.m3 or ME1), unfolded and folded forms of HLA class I (HC10 and W6/32, respectively), on the cell surface or after permeabilization (cell surface and intracellular). MARB4, which recognizes a subset of HLA-B27 containing longer peptides and also dimers was also used.

Results

The HLA-B27-transfected U937 cells express approximately equal amounts of HLA-B27 and B51 heavy chains, which is greater than the HLA-B18 allele. At steady state, ERAP1 KD resulted in 47% more folded HLA-B27 accumulating on the cell surface (HLA-ABC.m3) and 30% more in total (extra- plus intracellular), whereas there was only an 11% increase in total folded HLA class I (W6/32). When other alleles were examined by focusing, there was no increase in folded forms of HLA-B51 or B18. MARB4-reactive HLA-B27 was increased by 24% on the cell surface in ERAP1 KD cells. ERAP1 KD caused a 53% increase in unfolded HLA class I (B/C) on the cell surface, and led to a 39% increase in the accumulation of high molecular weigh disulfide-linked complexes of HLA-B27 (immunoblots) (P < 0.05 for all comparisons).

When cells were treated with IFNg, ERAP1 KD caused a 25% increase in total unfolded HLA-B27 compared to sc shRNA, but no increase in HLA-B51 or B18. High molecular weigh disulfide-linked complexes of HLA-B27 were also increased.

Conclusion These data demonstrate that reduction of ERAP1 expression significantly increases both folded and unfolded/misfolded forms of HLA-B27. IFNg treatment further increased all forms of HLA-B27 when ERAP1 expression was limited. It is of interest that the effects of ERAP1 KD on HLA-B allele conformation were substantially greater for HLA-B27 than for Behcet’s disease associated HLA- B51.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/effect-of-erap1-knockdown-on-conformation-of-hla-b27-and-other-hla-class-i-molecules-in-human-monocytic-cells/