Congenital heart block: identification of autoantibody binding site on the extracellular loop (domain I, S5-S6) of alpha(1D) L-type Ca channel

Abstract

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.

Published by Elsevier Ltd.

Figures

Figure 1

Panel A: Schematic structure of L-type Ca channel α1D subunit. α1 subunit is organized in four domains, I-IV, each consisting of transmembrane segments S1–S6 connected with a small stretch of amino acids. The extracellular loop between transmembrane segments S5–S6, E-Loop, and is the ion conductance pore and selectivity filter. The 4 extracellular loops in each domain that were prepared and tested in this study are shown. Panel B: Coomassie stain of total extract from BL21 E-coli following induction with 0.1 mM Isopropyl β-D-1-thiogalactopyranoside for 2 hours, and the successful expression of the extracellular loops E1-E4 of representative domains I-IV S5–S6 segments. Panel C: Immunoblot of the extracellular proteins E1-E4 on 15% SDS-PAGE after pulldown using 50% slurry of Glutathione Sepharose 4B and probed with monoclonal anti-GST antibody at 1:1000 dilution.

Figure 2

ELISA results of 161 serum samples against the GST fusion proteins of the E1 loop of the α1D L-type Ca channel. Seventeen of 118 (14.4%) samples from the CHB group were ELISA positive and above the O.D. cutoff point of 0.1 which represents the average for the healthy sera plus 2 standard deviation. In contrast, sera (with anti-SSA/Ro and SSB/La antibodies) from mothers with healthy children did not react (0/15) with the E1 loop, while in the sera (without anti-SSA/Ro and SSB/La antibodies) from mothers with healthy children, only 2 of 18 (7%) were ELISA positive.

Figure 3

Competitive ELISA assay using 4 healthy controls sera and 8 CHB samples with the highest O.D. out of the 17 ELISA positive sera in Figure 2. Sera were incubated with 10 μg of E1 loop at 37°C for 3 hours, centrifuged and used as the probe in the ELISA experiment. The O.D. of each of the 8 CHB sera decreased by more than half to a value below the cutoff of 0.1 nm.

Figure 4

Cross reactivity of the ELISA positive sera with the E1 protein by Western blot. Five micrograms of the E1 protein was resolved on a 15% SDS-PAGE and probed using a healthy control serum and 3 ELISA positive sera with the highest O.D. from the congenial heart block (CHB).

Figure 5

A. Effect of ELISA positive sera on the α1D ICa-L expressed in tsA201 cells. α1D ICa-L was recorded using whole cell mode of the patch clamp technique with 2 mmol/L Ca as a charge carrier. Panel A shows the current-voltage relationships for the α1D ICa-L densities during control and in the presence of ELISA positive serum 1. Panel B shows the current-voltage relationships for the α1D ICa-L densities during control and purified IgG from serum 1. Panel C shows the current-voltage relationships of the α1D ICa-L densities before and after the application of preincubated serum 1 with E1 fusion protein. Panel D shows the current voltage relationships for the α1D ICa-L densities before and after application of the healthy control serum.