Molecular and Cellular Cardiology Program, VA New York Harbor Healthcare System and SUNY Downstate Medical Center, Brooklyn, NY 11209, USA.
J Autoimmun. 2010 Mar;34(2):80-6. doi: 10.1016/j.jaut.2009.06.005. Epub 2009 Jul 28.
Congenital heart block (CHB) is an autoimmune disease associated with autoantibodies against intracellular ribonucleoproteins SSB/La and SSA/Ro. The hallmark of CHB is complete atrioventricular block. We have recently established that anti-SSA/Ro -SSB/La autoantibodies inhibit alpha(1D) L-type Ca current, I(Ca-L), and cross-react with the alpha(1D) Ca channel protein. This study aims at identifying the possible binding sites on alpha(1D) protein for autoantibodies from sera of mothers with CHB children. GST fusion proteins of the extracellular regions between the transmembrane segments (S5-S6) of each of the four alpha(1D) Ca channel protein domains I-IV were prepared and tested for reactivity with sera from mothers with CHB children and controls using ELISA. Sera containing anti-Ro/La autoantibodies from 118 mothers with CHB children and from 15 mothers with anti-Ro/La autoantibodies but have healthy children, and from 28 healthy mothers without anti-Ro/La autoantibodies and healthy children were evaluated. Seventeen of 118 (14.4%) sera from mothers with CHB children reacted with the extracellular loop of domain I S5-S6 region (E1). In contrast, only 2 of 28 (7%) of sera from healthy mothers (-anti-Ro/La) and healthy children reacted with E1 loop and none (0 of 15) of sera from healthy mothers (+anti-Ro/La) and healthy children reacted with the E1 loop. Preincubation of E1 loop with the positive sera decreased the O.D reading establishing the specificity of the response. Electrophysiological characterization of the ELISA positive sera and purified IgG showed inhibition (44.1% and 49.8%, respectively) of the alpha(1D) I(Ca-L) expressed in tsA201 cells. The inhibition was abolished when the sera were pre-incubated with E1 fusion protein. The results identified the extracellular loop of domain I S5-S6 of L-type Ca channel alpha(1D) subunit as a target for autoantibodies from a subset of mothers with CHB children. This novel finding provides insights into the potential development of therapeutic peptides that could bind to the pathogenic antibodies and prevent CHB.
先天性心脏传导阻滞（CHB）是一种与抗细胞内核糖核蛋白 SSB/La 和 SSA/Ro 自身抗体相关的自身免疫性疾病。CHB 的标志是完全性房室传导阻滞。我们最近发现抗 SSA/Ro-SSB/La 自身抗体抑制了α(1D)L 型钙电流，I(Ca-L)，并与α(1D)钙通道蛋白发生交叉反应。本研究旨在确定来自患有 CHB 儿童的母亲的血清中针对α(1D)蛋白的可能结合位点。制备了四个α(1D)钙通道蛋白结构域 I-IV 的跨膜片段（S5-S6）之间的细胞外区域的 GST 融合蛋白，并使用 ELISA 测试了其与来自患有 CHB 儿童的母亲和对照者的血清的反应性。使用 ELISA 评估了来自患有 CHB 儿童的母亲的含有抗 Ro/La 自身抗体的血清 118 份、含有抗 Ro/La 自身抗体但有健康儿童的母亲的血清 15 份和没有抗 Ro/La 自身抗体且健康儿童的母亲的血清 28 份。来自患有 CHB 儿童的母亲的血清 118 份中的 17 份（14.4%）与结构域 I S5-S6 区域的细胞外环（E1）反应。相比之下，来自没有抗 Ro/La 自身抗体和健康儿童的母亲的血清 28 份中只有 2 份（7%）与 E1 环反应，来自有抗 Ro/La 自身抗体和健康儿童的母亲的血清 15 份中没有 1 份（0%）与 E1 环反应。用阳性血清预孵育 E1 环降低了 O.D 读数，确立了反应的特异性。对 ELISA 阳性血清和纯化 IgG 的电生理特性进行了表征，发现它们分别抑制了在 tsA201 细胞中表达的α(1D)I(Ca-L)（44.1%和 49.8%）。当血清与 E1 融合蛋白预孵育时，抑制作用被消除。结果确定 L 型钙通道α(1D)亚基结构域 I S5-S6 的细胞外环是来自患有 CHB 儿童的母亲的自身抗体的一个靶标。这一新发现为开发可能与致病性抗体结合并预防 CHB 的治疗性肽提供了思路。
