Functional epitope of muscarinic type 3 receptor which interacts with autoantibodies from Sjogren's syndrome patients

Objectives. Recently, autoantibodies directed against muscarinic type 3 receptor (M3R) have been reported in patients with primary SS. However, the precise epitope(s) of the M3R that interacts with SS autoantibodies remains unclear. The aim of this study was to identify the functional epitope of M3R which interacts with SS immunoglobulin G (IgG). Methods. Purified IgGs were obtained from the sera of seven SS patients (six primary and one secondary SS) and two normal persons. We examined whether SS IgG inhibits M3R function and identified the epitope using six synthetic peptides covering all the extracellular domains of M3R by microspectrofluorimetry and surface plasmon resonance-based optical biosensor system (BIAcore system). Results. A volume of 0.5 mg/ml SS IgG inhibited carbachol (CCh)-induced [Ca2+](i) transient (CICT) in human submandibular gland (HSG) cells. However, co-incubation of SS IgG with the 6th peptide (514-527 amino acid region) corresponding to the third extracellular loop of M3R, recovered CICT. The result was further confirmed by BIAcore analysis. We found that the 6th peptide interacts with IgGs from three primary SS patients in a concentration-dependent manner. The synthetic peptide which consists of amino acids 228-237 corresponding to the COOH-terminus of the second extracellular loop of M3R also bound to SS IgG. However, normal IgGs did not interact with the 6th peptide. Conclusions. The results suggest that the third extracellular loop of M3R represents a functional epitope bound by SS IgG, and thereby partly inhibits M3R function.