Congenital heart block: Identification of autoantibody binding site on the extracellular loop (domain I, S5–S6) of α_1D L-type Ca channel

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• 作者：Eddy Karnabi ; Yongxia Qu ; Raj Wadgaonkar ; Salvatore Mancarella ; Yuankun Yue ; Mohamed Chahine ; Robert M. Clancy ; Jill P. Buyon ; Mohamed Boutjdir
• 关键词：Autoantibodies ; Calcium ; Heart block ; Ion channels ; Sinoatrial node
• 刊名：Journal of Autoimmunity
• 出版年：2010
• 出版时间：March 2010
• 年：2010
• 卷：34
• 期：2
• 页码：80-86
• 全文大小：767 K

文摘

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 α_1D L-type Ca current, I_Ca-L, and cross-react with the α_1D Ca channel protein. This study aims at identifying the possible binding sites on α_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 α_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 α_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 α_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.