Anti-MuSK autoantibodies block binding of collagen Q to MuSK

Abstract

Objective: Muscle-specific receptor tyrosine kinase (MuSK) antibody-positive myasthenia gravis (MG) accounts for 5%-15% of autoimmune MG. MuSK mediates the agrin-signaling pathway and also anchors the collagenic tail subunit (ColQ) of acetylcholinesterase (AChE). The exact molecular target of MuSK-immunoglobulin G (IgG), however, remains elusive. As acetylcholine receptor (AChR) deficiency is typically mild and as cholinesterase inhibitors are generally ineffective, we asked if MuSK-IgG interferes with binding of ColQ to MuSK.

Methods: We used 3 assays: in vitro overlay of the human ColQ-tailed AChE to muscle sections of Colq-/- mice; in vitro plate-binding assay to quantitate binding of MuSK to ColQ and to LRP4; and passive transfer of MuSK-IgG to mice.

Results: The in vitro overlay assay revealed that MuSK-IgG blocks binding of ColQ to the neuromuscular junction. The in vitro plate-binding assay showed that MuSK-IgG exerts a dose-dependent block of MuSK binding to ColQ by but not to LRP4. Passive transfer of MuSK-IgG to mice reduced the size and density of ColQ to ∼10% of controls and had a lesser effect on the size and density of AChR and MuSK.

Conclusions: As lack of ColQ compromises agrin-mediated AChR clustering in Colq-/- mice, a similar mechanism may lead to AChR deficiency in MuSK-MG patients. Our experiments also predict partial AChE deficiency in MuSK-MG patients, but AChE is not reduced in biopsied NMJs. In humans, binding of ColQ to MuSK may be dispensable for clustering ColQ, but is required for facilitating AChR clustering. Further studies will be required to elucidate the basis of this paradox.

Figure 1. Muscle-specific receptor tyrosine kinase (MuSK)–immunoglobulin G (IgG) recognizes the neuromuscular junction (NMJ) of Colq−/− mice

Nonreducing (A) and reducing (B) sodium dodecyl sulfate–polyacrylamide gel electrophoresis of serum proteins and purified IgG of patient 1. Gels are stained with Coomassie brilliant blue. M = molecular weight markers; 1 = serum before purification; 2 = purified IgG. Arrowheads point to IgG of 160 kD (A), as well as the heavy (50 kD) and light (25 kD) chains of IgG (B). (C) In vitro overlay of purified IgG on a 10-μm skeletal muscle section of Colq−/− mice. IgG is visualized with FITC-labeled antihuman IgG and acetylcholine receptor with Alexa594-labeled α-bungarotoxin. Scale bar = 50 μm.

Figure 2. In vitro overlay assays

Purified recombinant collagen Q (ColQ)-tailed acetylcholinesterase (AChE) was overlaid on a 10-μm quadriceps muscle section of Colq−/− mice in the presence of the indicated purified muscle-specific receptor tyrosine kinase–immunoglobulin G. ColQ is stained with anti-ColQ antibody and acetylcholine receptor (AChR) with Alexa594-labeled α-bungarotoxin. Scale bar = 50 μm.

Figure 3. In vitro plate-binding assays

(A) Increasing amounts of muscle-specific receptor tyrosine kinase (MuSK)–immunoglobulin G (IgG) block binding of the purified recombinant collagen Q (ColQ)-tailed acetylcholinesterase (AChE) to the extracellular domain of human MuSK that is coated on a 96-well plate. Bound ColQ-tailed AChE is quantified by AChE activity. AChE activities are normalized for that at 1 pg IgG of each sample. Mean and SEM of 3 experiments are plotted. *p < 0.01 between controls and patients. (B) MuSK-IgG does not block binding of the purified FLAG-tagged extracellular domain of human LRP4 (LRP4N-FLAG) to MuSK that is coated on a 96-well plate. Bound LRP4N-FLAG is quantified with anti-FLAG-HRP. HRP activities are normalized for that at 1 pg IgG of each sample. Mean and SEM of 3 experiments are plotted.

Figure 4. Passive transfer of muscle-specific receptor tyrosine kinase (MuSK)–immunoglobulin G (IgG) of control 2 and patient 2 to C57BL/6J mice

(A, B) Quadriceps muscle sections of mice injected with IgG of control 2 or patient 2 are stained for acetylcholine receptor (AChR) by Alexa594-labeled α-bungarotoxin, collagen Q (ColQ) and MuSK by immunostaining, acetylcholinesterase (AChE) by cytochemical staining. Scale bar = 40 μm. Signal areas (C), intensities (D), and densities (intensity/area) (E) of the indicted molecules per neuromuscular junction (NMJ) are shown in mean and SEM. (F) Densities of ColQ and MuSK are normalized for the density of AChR to estimate the number of ColQ and MuSK per AChR. For AChR, ColQ, and MuSK, we analyzed 44 NMJs of control 2 and 23 NMJs of patient 2. For MuSK, we analyzed 82 NMJs of control 2 and 42 NMJs of patient 2. Areas and intensities are quantified by the BZ-9000 microscope (Keyence). Open and closed bars represent control 2 and patient 2, respectively. *p < 0.05, ***p < 0.001. NS = not significant.