Structural and functional characteristics of binding of pemphigus vulgaris antibody to keratinocyte M3 muscarinic acetylcholine receptor

Summary: Abstract Body: Patients with pemphigus vulgaris (PV) develop IgG autoantibodies (AuAbs) binding to keratinocyte desmogleins (Dsg), acetylcholine (ACh) receptors, mitochondrial proteins, and some other autoantigens. In this study, we used epitomic approach to immunoselect random sequences from a phage display library and determine the sequence patterns that are common to PV AuAbs. We wanted to identify linear and discontinuous peptide epitope segments of Dsg1, Dsg3 and M3 muscarinic ACh receptor (M3AR) targeted by different AuAbs on the same protein target. PV sera specifically targeted 278 Dsg1, 242 Dsg3, and 122 M3AR tetramers. Since the M3AR protein shares only 2 tetramers with Dsg1 and 4 with Dsg3, targeting of M3AR by cross-reacting anti-Dsg1/3 PV was ruled out. The targeted M3AR pentamers encompass the 10 amino acids-long epitope LSEPTITFGT (amino acids 226-235) located on the border of the second extracellular loop and the fifth transmembrane helix, including 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, because of which ACh regulation of keratinocytes via M3AR is lost. In this study, we compared the functional consequences of binding anti-M3AR AuAbs that targeted the ACh-binding pocket with that of AuAbs that targeted M3AR outside of its ACh-binding pocket. While the former AuAbs induced a very high elevation of phospholipase C, which is consistent with an agonist-like effect, the latter AuAbs produced a much weaker response. These results indicate that PV patients develop two types of anti-M3AR AuAbs. One type attaches to orthosteric, ie, ACh-binding, site and elicits a very strong signaling response comparable to a full pharmacologic agonist, whereas another type binds to an allosteric site and elicits submaximal signaling response comparable to a partial (allosteric) pharmacologic agonist. Jorge Mauricio Reyes-Ruiz¹, Alexander Chernyavsky¹, Charles Glabe¹, Sergei Grando¹ 1. University of California Irvine, Irvine, CA, United States. Adaptive and Auto-Immunity