Background/Purpose: Polymorphisms in endoplasmic reticulum (ER) aminopeptidase 1 (ERAP1) are associated with susceptibility to ankylosing spondylitis in HLA-B27 (B27) positive individuals, implying a functional interaction between the gene products. A principle function of ERAP1 is to trim peptides that bind to HLA class I molecules such as B27. Since the quantity and quality of peptides is critical for the efficient folding and assembly of class I molecules, we developed a system to examine how reduced ERAP1 activity affects properties of HLA-B27 in cells expressing other HLA class I molecules. Our immediate objective was to determine how ERAP1 knockdown affects HLA class I and specifically B27 expression in U937 cells (U937.B27).

Methods:

Lentiviral shRNA was used to knock down (KD) ERAP1 expression in U937.B27 cells (65% reduction), which were compared to U937.B27 cells expressing scrambled shRNA as a control. Total class I heavy chain expression was examined by immunoblotting whole cell lysates. Total folded and unfolded forms of HLA class I were assessed by immunoprecipation with conformation-specific monoclonal antibodies (mAb) W6/32 (anti-HLA-A,B,C) and HC10 (anti-HLA-B,C), respectively, followed by quantitative immunoblotting. Cell surface expression of folded and unfolded forms of class I were assessed by flow cytometry, and HLA-ABC.m3 was used to measure expression of folded B27.

Results:

ERAP1 KD resulted in a ~20% increase in total HLA class I heavy chain in whole cell lysates by immunoblotting. Similarly, total folded and unfolded forms of immunoprecipitable HLA class I heavy chain were slightly increased, with folded forms predominating. In contrast, total folded and unfolded HLA class I heavy chain on the cell surface detected by flow was unchanged by ERAP1 KD. For HLA-B27, folded forms on the cell surface were increased by ~40%. Interestingly, treatment with IFNg resulted in more total folded heavy chain in ERAP1 KD cells, whereas the increase in total unfolded heavy chain was limited. Although IFNg induces ERAP1, the ERAP1 KD cells maintained a 50% reduction in ERAP1 expression compared to scrambled shRNA controls. Surprisingly, ERAP1 KD limited the increase in cell surface folded B27 by approximately ~15%. ERAP1 KD also resulted in greater accumulation of disulfide-linked heavy chain oligomers both without and with IFNg treatment.

Conclusion:

These data demonstrate that folded and unfolded forms of HLA class I are clearly affected by reduced ERAP1 expression. Loss of ERAP1 function through KD results in a small increase in the accumulation of total cellular HLA class I heavy chain, with more folded compared to unfolded forms, but this difference is not readily apparent on the cell surface, suggesting that it may be intracellular. Differences are further increased with IFNg. Interestingly, while diminished ERAP1 function results in more folded B27 on the cell surface at steady state, upregulation of folded forms can be limited by ERAP1 KD. Our studies highlight the importance of ERAP1 in modulating folded and misfolded forms of B27, and provide a system to probe additional biological effects of these two gene products.

---

« Back to 2013 ACR/ARHP Annual Meeting

ACR Meeting Abstracts - https://acrabstracts.org/abstract/effects-of-erap1-knockdown-on-hla-class-i-and-hla-b27-expression-in-human-monocytic-u937-cells/