Division of Rheumatology, Department of Medicine Solna, Karolinska Institutet, Stockholm, Sweden.
Center for Molecular Medicine, Karolinska Institutet, Stockholm, Sweden.
Rheumatology (Oxford). 2024 May 2;63(5):1466-1473. doi: 10.1093/rheumatology/kead400.
Clinical observations in patients with dermatomyositis (DM) and autoantibodies against the melanoma differentiation-associated protein 5 (MDA5) suggest that the autoantibodies contribute to the pathogenesis of MDA5(+) DM. To gain insight into the role of the anti-MDA5 autoantibodies, we aimed to identify their binding sites on the different domains of the MDA5 protein.
We developed an in-house ELISA to assess the reactivity against the MDA5 domains (conformational epitopes) in plasma (n = 8) and serum (n = 24) samples from MDA5(+) patients with varying clinical manifestations and disease outcomes. The reactivities were also assessed using western blot (linearized epitopes). An ELISA-based depletion assay was developed to assess cross-reactivity among the different MDA5 domains.
All eight plasma samples consistently showed reactivity towards conformational and linearized epitopes on the helicase domains of the MDA5 protein. The ELISA-based depletion assay suggests that anti-MDA5 autoantibodies specifically target each of the three helicase domains. Twenty-two of the 24 serum samples showed reactivity in the in-house ELISA and all 22 displayed reactivity towards the helicase domains of the MDA5 protein.
Our data revealed that the main immunogenic targets of anti-MDA5 autoantibodies from MDA5(+) patients are the helicase domains. Considering that the helicase domains are responsible for the enzymatic activity and subsequent triggering of an inflammatory response, our findings suggest that binding of anti-MDA5 autoantibodies could alter the canonical activity of the MDA5 protein and potentially affect the downstream induction of a pro-inflammatory cascade.
对皮肌炎（DM）患者和抗黑色素瘤分化相关蛋白 5（MDA5）自身抗体的临床观察表明，这些自身抗体有助于 MDA5（+）DM 的发病机制。为了深入了解抗 MDA5 自身抗体的作用，我们旨在确定其在 MDA5 蛋白不同结构域上的结合位点。
我们开发了一种内部 ELISA 来评估来自 MDA5（+）患者的不同临床表现和疾病结局的血浆（n=8）和血清（n=24）样本中对 MDA5 结构域（构象表位）的反应性。还使用 Western blot（线性表位）评估了反应性。开发了基于 ELISA 的耗竭测定法来评估不同 MDA5 结构域之间的交叉反应性。
所有 8 份血浆样本均一致显示对 MDA5 蛋白解旋酶结构域的构象和线性表位的反应性。基于 ELISA 的耗竭测定法表明，抗 MDA5 自身抗体特异性针对三个解旋酶结构域中的每一个。24 份血清样本中的 22 份在内部 ELISA 中显示反应性，并且所有 22 份均显示对 MDA5 蛋白的解旋酶结构域的反应性。
我们的数据表明，MDA5（+）患者的抗 MDA5 自身抗体的主要免疫原性靶标是解旋酶结构域。考虑到解旋酶结构域负责酶活性和随后引发炎症反应，我们的发现表明，抗 MDA5 自身抗体的结合可能改变 MDA5 蛋白的经典活性，并可能影响下游促炎级联的诱导。
