Congenital myasthenic syndrome–associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner
Bisei Ohkawara, … , Kinji Ohno, Andrew G. Engel
Bisei Ohkawara, … , Kinji Ohno, Andrew G. Engel
Published April 9, 2020
Citation Information: JCI Insight. 2020;5(7):e132023. https://doi.org/10.1172/jci.insight.132023.
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Research Article Genetics

Congenital myasthenic syndrome–associated agrin variants affect clustering of acetylcholine receptors in a domain-specific manner

Abstract

Congenital myasthenic syndromes (CMS) are caused by mutations in molecules expressed at the neuromuscular junction. We report clinical, structural, ultrastructural, and electrophysiologic features of 4 CMS patients with 6 heteroallelic variants in AGRN, encoding agrin. One was a 7.9-kb deletion involving the N-terminal laminin–binding domain. Another, c.4744G>A — at the last nucleotide of exon 26 — caused skipping of exon 26. Four missense mutations (p.S1180L, p.R1509W, p.G1675S, and p.Y1877D) expressed in conditioned media decreased AChR clusters in C2C12 myotubes. The agrin-enhanced phosphorylation of MuSK was markedly attenuated by p.Y1877D in the LG3 domain and moderately attenuated by p.R1509W in the LG1 domain but not by the other 2 mutations. The p.S1180L mutation in the SEA domain facilitated degradation of secreted agrin. The p.G1675S mutation in the LG2 domain attenuated anchoring of agrin to the sarcolemma by compromising its binding to heparin. Anchoring of agrin with p.R1509W in the LG1 domain was similarly attenuated. Mutations of agrin affect AChR clustering by enhancing agrin degradation or by suppressing MuSK phosphorylation and/or by compromising anchoring of agrin to the sarcolemma of the neuromuscular junction.

Authors

Bisei Ohkawara, XinMing Shen, Duygu Selcen, Mohammad Nazim, Vera Bril, Mark A. Tarnopolsky, Lauren Brady, Sae Fukami, Anthony A. Amato, Uluc Yis, Kinji Ohno, Andrew G. Engel

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Figure 6

Degradation assay of p.S1180L and binding assays of p.G1509W and p.G1675S to a muscle section and to heparan sulfate/heparin.

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Degradation assay of p.S1180L and binding assays of p.G1509W and p.G1675...
(A) Degradation assay of WT and p.S1180L-mutant agrin-mycAP. Purified WT or p.S1180L agrin-mycAP was added to the culture medium of C2C12 myotubes, and the amount of agrin-mycAP was quantified at 0, 2, and 18 hours. The amount of agrin-mycAP was normalized for that of a Coomassie-stained ~200-kDa band and also for the ratio of WT at 0 hours. Mean ± SD (n = 3 independent experiments) are indicated. One-way ANOVA followed by post hoc Tukey test. P < 0.05 is indicated by a, b, c, and d. (B) Overlay of purified WT, p.G1509W, and p.G1675S agrin-mycAP on a section of mouse tibialis anterior muscle. WT agrin was bound to the sarcolemma and especially to the neuromuscular junction (NMJ). p.G1509W and p.G1675S reduced binding to the sarcolemma. p.G1509W also reduced binding to the NMJ. Mean ± SD (n = 3 independent experiments) are indicated in right panels. One-way ANOVA followed by post hoc Tukey test. P < 0.05 is indicated by a and b. (C and D) Plate-binding assay of purified WT, p.G1509W, and p.G1675S agrin-mycAP to heparan sulfate (C) and heparin (D). A total of 80 μL of 50 ng/μL WT and mutant agrin-mycAP in PBS were incubated for 3 hours at room temperature. p.G1509W reduced binding to heparan sulfate and p.G1675S reduced binding to heparin. Mean ± SD (n = 3 wells) are indicated. One-way ANOVA followed by post hoc Tukey test; P < 0.05.