Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients
Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor
Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor
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Research Article Immunology

Characterization of pathogenic monoclonal autoantibodies derived from muscle-specific kinase myasthenia gravis patients

Abstract

Myasthenia gravis (MG) is a chronic autoimmune disorder characterized by muscle weakness and caused by pathogenic autoantibodies that bind to membrane proteins at the neuromuscular junction. Most patients have autoantibodies against the acetylcholine receptor (AChR), but a subset of patients have autoantibodies against muscle-specific tyrosine kinase (MuSK) instead. MuSK is an essential component of the pathway responsible for synaptic differentiation, which is activated by nerve-released agrin. Through binding MuSK, serum-derived autoantibodies inhibit agrin-induced MuSK autophosphorylation, impair clustering of AChRs, and block neuromuscular transmission. We sought to establish individual MuSK autoantibody clones so that the autoimmune mechanisms could be better understood. We isolated MuSK autoantibody-expressing B cells from 6 MuSK MG patients using a fluorescently tagged MuSK antigen multimer, then generated a panel of human monoclonal autoantibodies (mAbs) from these cells. Here we focused on 3 highly specific mAbs that bound quantitatively to MuSK in solution, to MuSK-expressing HEK cells, and at mouse neuromuscular junctions, where they colocalized with AChRs. These 3 IgG isotype mAbs (2 IgG4 and 1 IgG3 subclass) recognized the Ig-like domain 2 of MuSK. The mAbs inhibited AChR clustering, but intriguingly, they enhanced rather than inhibited MuSK phosphorylation, which suggests an alternative mechanism for inhibiting AChR clustering.

Authors

Kazushiro Takata, Panos Stathopoulos, Michelangelo Cao, Marina Mané-Damas, Miriam L. Fichtner, Erik S. Benotti, Leslie Jacobson, Patrick Waters, Sarosh R. Irani, Pilar Martinez-Martinez, David Beeson, Mario Losen, Angela Vincent, Richard J. Nowak, Kevin C. O’Connor

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Figure 3

MuSK domain-binding results.

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MuSK domain-binding results. To map the human MuSK mAb epitopes, MuSK co...
To map the human MuSK mAb epitopes, MuSK constructs that had particular domains deleted and full-length MuSK were each expressed in HEK cells and tested with the CBA. (A) The schematic illustrates the mutant forms of MuSK. For example, “ΔIg-1” includes only the Ig-like domains 2 and 3 and the frizzled-like (Fz-like) domain because the Ig-like domain 1 was deleted (shown as “Δ” in the schematic). Similarly, “Ig-1” includes only the Ig-like domain 1 because the Ig-like domains 2 and 3 and Fz-like domain were deleted (shown as “Δ” in the schematic). Binding of mAbs (MuSK1A, MuSK1B, MuSK3-28, and the positive control humanized MuSK mAb 4A3) to these mutant forms of MuSK was tested in our standardized flow cytometry CBA. Results for each (B) mAb or (C) serum specimen are shown. Serum was obtained from the same patients from whom the mAbs were derived. Each data point represents a separate replicate within the same experiment. Bars represent means and error bars SDs.