Epitomic profiling and functional characteristics of pemphigus vulgaris autoantibody binding to keratinocyte M3 muscarinic acetylcholine receptor

Patients with pemphigus vulgaris (PV) develop IgG autoantibodies (AuAbs) binding to keratinocyte desmogleins (Dsg), acetylcholine (ACh) receptors, mitochondrial proteins, and some other self-antigens. In this study, we identified linear and discontinuous peptide tetrameric epitope segments (ES) of M3 muscarinic ACh receptor (M3AR) targeted by different anti-M3AR AuAbs. As positive controls, we identified Dsg1 and Dsg3 ES targeted by PV sera. Healthy individuals also possessed natural antibodies targeting M3AR, Dsg1 and Dsg3 epitopes that were different from those targeted by AuAbs produced by patients with PV. The two targeted M3AR pentameric ES encompass the 10 amino acids-long epitope LSEPTITFGT included the tetramer TFGT containing Thr235 which is a part of the ACh-binding pocket. Previously, it has been demonstrated that the anti-M3AR AuAb produces an agonist-like effect on downstream signaling, but its long-term effect is receptor desensitization. In this study, we compared the functional consequences of binding anti-M3AR AuAbs that target the ACh-binding pocket with that of AuAbs that target M3AR outside of its ACh-binding pocket. While the former AuAbs induced a very high elevation of phospholipase C, inositol triphosphate and diacylglycerol, which represents an agonist-like effect, the latter AuAbs produced a much weaker signaling response. These results indicate that patients with PV develop two types of anti-M3AR AuAbs. One type attaches to orthosteric, i.e., ACh-binding, site and elicits a strong signaling response comparable to that induced by a full pharmacologic agonist, whereas another type binds to an allosteric site and elicits submaximal signaling response comparable to that induced by a partial (allosteric) agonist.