Background/Purpose: The messenger RNA (mRNA) processing body (P-body) is a cellular structure that regulates the stability of cytoplasmic mRNA. Approximately 5% of patients with the autoimmune disease primary biliary cirrhosis (PBC) have antibodies directed against this structure and some patients have antibodies that react with several different known P-body components. In this study, we used a proteomic array and serum from a PBC patient to rapidly and efficiently identify a new P-body component.

Methods: Serum from patient 0081 was used to probe a high density protein macroarray which contains approximately 17,000 proteins. The proteins were produced in situ on a polyvinylidene difluoride (PVDF) membrane by Escherichia coli transformed with a prokaryotic expression cDNA library. This library was derived from mRNA prepared from phytohemagglutinin treated human T-lymphocytes. Human auto-antibodies that bound to the protein macroarray were detected using horse-radish peroxidase-conjugated rabbit anti-human IgG antiserum and chemiluminescence. Immunoreactive protein targets of these autoantibodies were determined by their locations on the PVDF membrane. Immunoblot was then used to confirm that serum 0081 contained antibodies that reacted with newly indentified autoantigens. Human epidermoid cancer cells (HEp-2 cells) were transfected with plasmids encoding green fluorescent protein (GFP) fused to the new P-body component candidates, to confirm that the proteins localized to P-bodies.

Results: Serum 0081 reacted with fifty-six proteins on the protein macroarray membrane. One of these new proteins, Limkain B (LMKB), was chosen for further study because it was previously reported to localize to a “subset of peroxisomes”, a staining pattern that might appear similar to the P-body pattern. Antibodies in serum 0081 reacted with glutathione S-transferase (GST)-LMKB, but not GST alone, by immunoblot. A plasmid encoding full-length LMKB fused to GFP was used to express the protein in HEp-2 cells. LMKB localized to cytoplasmic dots and co-localized with Ge-1, a known P-body component.

Conclusion: Our findings demonstrate that the combination of proteomic array technology and human autoantibodies provides a useful tool for identifying new autoantigens. “Autoantigenomics” can be used to rapidly and efficiently discover new proteins in cellular structures. Using this method, we identified LMKB as a novel component of mRNA processing bodies.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/the-use-of-autoantigenomics-to-rapidly-identify-targets-of-human-autoantibodies/