Soluble form of the MDA5 protein in human sera

Abstract

Viral double-stranded RNA (dsRNA) is sensed by toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), including melanoma differentiation-associated gene 5 (MDA5). MDA5 recognizes the genome of dsRNA viruses and replication intermediates of single-stranded RNA viruses. MDA5 also plays an important role in the development of autoimmune diseases, such as Aicardi-Goutieres syndrome and type I diabetes. Patients with dermatomyositis with serum MDA5 autoantibodies (anti-CADM-140) are known to have a high risk of developing rapidly progressive interstitial lung disease and poor prognosis. However, there have been no reports on the soluble form of MDA5 in human serum. In the present study, we generated in-house monoclonal antibodies (mAbs) against human MDA5. We then performed immunohistochemical analysis and sensitive sandwich immunoassays to detect the MDA5 protein using two different mAbs (clones H27 and H46). As per the immunohistochemical analysis, the MDA5 protein was moderately expressed in the alveolar epithelia of normal lungs and was strongly expressed in the cytoplasm of lymphoid cells in the tonsils and acinar cells of the pancreas. Interestingly, soluble MDA5 protein was detectable in the serum, but not in the urine, of healthy donors. Soluble MDA5 protein was also detectable in the serum of patients with dermatomyositis. Immunoblot analysis showed that human cells expressed a 120 kDa MDA5 protein, while the 60 kDa MDA5 protein increased in the supernatant of peripheral mononuclear cells within 15 min after MDA5 agonist/double-strand RNA stimulation. Hydrogen deuterium exchange mass spectrometry revealed that an anti-MDA5 mAb (clone H46) bound to the epitope (415QILENSLLNL424) derived from the helicase domain of MDA5. These results indicate that a soluble MDA5 protein containing the helicase domain of MDA5 could be rapidly released from the cytoplasm of tissues after RNA stimulation.