Background/Purpose:

The HLA class I allele, HLA-B27, is associated with spondyloarthritis (SpA), an immune-mediated inflammatory disease affecting the axial skeletal, skin, gut and eyes. HLA-B27 has a tendency to misfold, and accumulate in the endoplasmic reticulum (ER) and on the cell surface as disulfide-linked dimers. ER accumulation can generate stress resulting in an unfolded protein response (UPR). The UPR upregulates several pathways that promote folding and secretion and/or ER-associated degradation (ERAD) of accumulated proteins, which involves retrotranslocation and ubiquitination of target proteins in the cytosol where they are degraded by proteasomes. Our recent studies suggest that autophagy as well as ERAD contributes to the disposal of misfolded HLA-B27, yet these pathways are still not sufficient to prevent misfolded HLA-B27 from accumulating. Since these pathways of protein degradation require substrate ubiquitination, we asked whether HLA-B27 heavy chains were ubiquitinated using HLA-B7 for comparison.

Methods: Bone marrow derived macrophages from HLA-B27 and human β₂m (hβ₂m) transgenic rats were examined with and without IFNγ and proteasome or autophagy inhibitors. Immunoprecipitation, western blotting, and immunofluorescence were used to measure HLA-B27 heavy chains and ubiquitination mount. Autophagy was activated using rapamycin, or blocked with bafilomycin. ERAD was inhibited by inhibiting proteasome function with bortezomib. HLA-B7/hβ₂m transgenic rat macrophages were used as controls.

Results: Blocking autophagic flux with bafilomycin resulted in the accumulation of misfolded HLA-B27 dimers and oligomers as well as monomers, comparable to blocking ERAD with the proteasome inhibitor bortezomib. HLA-B7 monomers also accumulated after blocking each degradation pathway. The proportion of ubiquitinated heavy chains was ~3-fold lower for HLA-B27 compared to HLA-B7. Immunoprecipitation experiments using anti-ubiquitin antibody followed by blotting for HLA class I heavy chains, further confirmed that HLA-B7 is significantly more ubiquitinated than HLA-B27. Activation of autophagy with rapamycin rapidly eliminated ~50% of misfolded HLA-B27, while folded HLA-B27 or HLA-B7 monomeric heavy chains were minimally affected.

Conclusion: These results demonstrate for the first time that both autophagy and ERAD play a role in the elimination of excess HLA class I heavy chains expressed in transgenic rats. Our results suggest that impaired ubiquitination of HLA-B27 may play a role in the accumulation of misfolded disulfide-linked dimers, whose elimination can be enhanced by activation of autophagy. Manipulation of the autophagy pathway should be further investigated as a potential therapeutic avenue in experimental spondyloarthritis.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/reduced-ubiquitination-of-misfolded-hla-b27-is-associated-with-inefficient-degradation-by-erad-and-autophagy/