Pinal-Fernandez Iago, Musai Jon, Casal-Dominguez Maria, Pak Katherine, Kaplan Mariana, Warner Blake M, Rider Lisa G, Aggarwal Rohit, Oddis Chester V, Moghadam-Kia Siamak, Garrabou Gloria, Selva-O'Callaghan Albert, Milisenda Jose C, Chiorini John A, Mammen Andrew L, Burbelo Peter D
Muscle Disease Section, National Institute of Arthritis and Musculoskeletal and Skin Disease, National Institutes of Health, Bethesda, MD, USA.
Johns Hopkins University School of Medicine, Baltimore, MD, USA.
medRxiv. 2025 Mar 5:2025.03.04.25323364. doi: 10.1101/2025.03.04.25323364.
Plant homeodomain (PHD) fingers are present in many chromatin-binding proteins. We recently discovered that anti-Mi2 autoantibodies recognize PHD fingers in Mi2 and AIRE. The purpose of this study was to characterize anti-Mi2 autoantibody recognition of PHD fingers in SP140L and TIF1γ as well as to explore recognition of TIF1γ by both anti-TIF1γ and anti-Mi2 autoantibodies.
Luciferase immunoprecipitation system (LIPS) assays were performed to detect autoantibodies against full-length and protein fragments of SP140L and TIF1γ in serum samples from myositis patients, disease controls, and healthy controls.
Anti-Mi2 autoantibodies recognized SP140L. When a 49 amino acid fragment of the PHD finger of SP140L was used as the target, the specificity for selectively detecting anti-Mi2 autoantibodies increased. Additionally, anti-Mi2 autoantibodies weakly bound TIF1γ compared to anti-TIF1γ autoantibodies. Excluding the TIF1γ PHD finger from the TIF1γ target autoantigen eliminated cross-reactivity with anti-Mi2 autoantibodies, confirming that anti-Mi2 autoantibodies specifically target the PHD finger of TIF1γ. Switching two amino acids in the TIF1γ PHD finger to resemble those in AIRE markedly enhanced anti-Mi2 autoantibody immunoreactivity. Anti-TIF1γ autoantibodies primarily recognized the N-terminal fragment outside of the PHD finger, indicating this region contains the immunodominant epitopes.
Anti-Mi2 autoantibodies recognize the PHD fingers of SP140L and TIF1γ. TIF1γ is recognized by two different myositis-specific autoantibodies: anti-Mi2 autoantibodies bind the C-terminal PHD domain and anti-TIF1γ autoantibodies predominantly bind the N-terminal region. Removing the PHD finger from the anti-TIF1γ target autoantigen can improve the specificity of anti-TIF1γ autoantibody assays by reducing cross-reactivity with anti-Mi2 autoantibodies.
植物同源结构域（PHD）指存在于许多染色质结合蛋白中。我们最近发现抗Mi2自身抗体可识别Mi2和AIRE中的PHD指。本研究的目的是表征抗Mi2自身抗体对SP140L和TIF1γ中PHD指的识别，并探索抗TIF1γ和抗Mi2自身抗体对TIF1γ的识别。
进行荧光素酶免疫沉淀系统（LIPS）分析，以检测来自肌炎患者、疾病对照和健康对照的血清样本中针对SP140L和TIF1γ全长及蛋白片段的自身抗体。
抗Mi2自身抗体可识别SP140L。当使用SP140L的PHD指的49个氨基酸片段作为靶点时，选择性检测抗Mi2自身抗体的特异性增加。此外，与抗TIF1γ自身抗体相比，抗Mi2自身抗体与TIF1γ的结合较弱。从TIF1γ靶自身抗原中去除TIF1γ PHD指可消除与抗Mi2自身抗体的交叉反应，证实抗Mi2自身抗体特异性靶向TIF1γ的PHD指。将TIF1γ PHD指中的两个氨基酸替换为与AIRE中的氨基酸相似的氨基酸，可显著增强抗Mi2自身抗体的免疫反应性。抗TIF1γ自身抗体主要识别PHD指以外的N端片段，表明该区域包含免疫显性表位。
抗Mi2自身抗体可识别SP140L和TIF1γ的PHD指。TIF1γ可被两种不同的肌炎特异性自身抗体识别：抗Mi2自身抗体结合C端PHD结构域，抗TIF1γ自身抗体主要结合N端区域。从抗TIF1γ靶自身抗原中去除PHD指可通过减少与抗Mi2自身抗体的交叉反应来提高抗TIF1γ自身抗体检测的特异性。
