Use ofTorpedo‐mouse hybrid acetylcholine receptors reveals immunodominance of the α subunit in myasthenia gravis antisera

摘要

AbstractThe nicotinic acetylcholine receptor (AChR), a pentameric complex of α2βγδ subunits, is the autoantigen in the human autoimmune disease myasthenia gravis (MG). Anti‐AChR antibodies are found in ∼90 of MG patients and using indirect methods (competitive binding to solubilized AChR), peptides, or synthetic peptides, the majority of these antibodies have been shown to bind to the AChR α subunit. In order to determine directly the AChR subunit specificities of MG antibodies, we employed as antigens a novel set of hybrid AChR composed of species cross‐reacting and non‐cross‐reacting subunits stably expressed in fibroblasts. Sequence similarities of homologous subunits among species can vary widely, with mammalian subunits having 87 ‐96 identity andTorpedo‐mammalian subunits having 54 −80 identity. These findings are reflected in antigenic specificities, with human anti‐AChR antisera frequently recognizing mouse AChR but rarely recognizingTorpedo. By establishing separate cell lines stably expressing all‐Torpedo, all‐mouse, and different combinations ofTorpedoand mouse subunits, we were able to provide the first direct evidence of a predominant anti‐α subunit specificity in MG antisera. Functional hybrid AChR stably expressed in an intact cell membrane provide us, with a system that best mimics thein vivoenvironment of the MG antibody in a binding assay. Such a system allows us to investigate a perplexing observation in the field: a poor correlation between the patient's clinical status and antibody titer. Those antibodies which can interfere with AChR function, such as ones with the ability to cross‐link AChR and induce their accelerated internalization and degradation (antigenic modulation) might represent a subpopulation of MG antibodies important in disease induction or maintenance. In this report, we demonstrate that wild‐type and hybrid AChR expressed in fibroblasts can be antigenically modulated by intermolecular cross‐linking antibodies as AChR are in native muscle cells. Because we can monitor dynamic interactions between AChR and MG antibodies, this system may allow us to define crucial pathogenic epitopes in MG by expressin