Background/Purpose

Endoplasmic reticulum aminopeptidase 1 (ERAP1) is a multifunctional enzyme involved in the processing of peptide cargo for major histocompatibility complex (MHC) class I complexes, and can have significant effects on peptide repertoire, and cell surface expression and stability of MHC class I molecules. ERAP1 variants are associated several MHC class I-associated inflammatory diseases, such as ankylosing spondylitis (AS), Behcet’s disease, and psoriasis/psoriatic arthritis, with evidence for epistasis with MHC class I susceptibility alleles. Since peptide supply is a critical determinant of MHC class I folding and assembly, we asked whether ERAP1 knockdown would affect HLA-B27 misfolding and endoplasmic reticulum (ER) stress in HLA-B27 transgenic (Tg) rats.

Methods Bone marrow derived macrophages from HLA-B27 Tg, HLA-B7 Tg and wild type (WT) rats were transduced with lentiviral ERAP1 shRNA or scrambled shRNA as a control. Protein expression including folded, unfolded, and misfolded forms of HLA-B27 was evaluated using immunoblotting of whole cell lysates and immunoprecipitates. To evaluate the effects of cytokines, macrophages were treated without or with IFNg (100 ng/ml) or IFNg and TNFa (30 ng/ml) for 24-48 hr. ER stress was assessed using real time PCR and XBP-1 splicing.

Results

ERAP1 protein expression was reduced 55-77% by ERAP1 shRNA as measured by immunoblotting blotting in several experiments, before and after treatment with cytokines. ERAP1 knockdown led to increased accumulation of aberrant disulfide-liked HLA-B27 complexes in whole cell lysates and HC10 immunoprecipitates. Interestingly, HLA-B7 heavy chains, which do not misfold under normal conditions, could be detected forming dimers with prolonged exposure in ERAP1 KD cells, although quantitatively much less than for HLA-B27. Expression of Bip and CHOP mRNA and XBP1 splicing were elevated in ERAP1 KD cells compared to sc shRNA. With cytokine stimulation, the there was increased UPR target gene expression and XBP1 splicing consistent with accumulation of aberrant HLA-B27 complexes.

Conclusion

In summary, these results suggest that ERAP1 loss-of-function impacts HLA-B27 misfolding and may affect the pathogenesis of AS via the aberrant biology of HLA-B27 and ER stress.

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« Back to 2014 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/erap1-knockdown-affects-hla-b27-misfolding-and-endoplasmic-reticulum-stress-in-hla-b27-transgenic-rat-macrophages/