In the present study, we reported higher frequencies and titres of anti-M3R antibodies against all extracellular domains in SS patients than the control. SS and controls for 12 5-Iodo-A-85380 2HCl h. After loading with Fluo-3, HSG cells were stimulated with cevimeline hydrochloride, and intracellular Ca2+concentrations [(Ca2+)i] were measured. Antibodies to the N-terminal, first, second and third loops were detected in 429% (18 of 42), 476% (20 of 42), 548% (23 of 42) and 452% (19 of 5-Iodo-A-85380 2HCl 42) of SS, while in 48% (two of 42), 71% (three of 42), 24% (one of 42) and 24% (one of 42) of controls, respectively. Antibodies to the second loop positive SS-IgG inhibited the increase of (Ca2+)i induced by cevimeline hydrochloride. Antibodies to the N-terminal positive SS-IgG and antibodies to the first loop positive SS-IgG enhanced it, while antibodies to the third loop positive SS-IgG showed no effect on (Ca2+)i as well as anti-M3R antibody-negative SS-IgG. Our results indicated the presence of several B cell epitopes on M3R in SS. The influence of anti-M3R antibodies on salivary secretion might differ based on these epitopes. Keywords:autoantibodies, epitopes, function, M3 muscarinic acetylcholine receptor, Sjgren’s syndrome == Introduction == Sjgren’s syndrome (SS) is an autoimmune disease that affects exocrine glands, including salivary and lacrimal glands. It is characterized by lymphocytic infiltration into exocrine glands, leading to dry mouth and eyes. A number of autoantibodies, such as anti-SS-A and SS-B antibodies, are detected in patients with SS. However, 5-Iodo-A-85380 2HCl no SS-specific pathological autoantibodies have yet been found in this condition [1]. Data from recent studies have suggested that some patients with SS carry inhibitory autoantibodies directed against muscarinic acetylcholine receptors, especially M3 muscarinic acetylcholine receptor (M3R) [1]. To date, five subtypes of muscarinic acetylcholine receptors (M1RM5R) have been identified, and M3R is expressed in MDNCF exocrine glands and plays crucial roles in exocrine secretion. Acetylcholine binds to and activates M3R on salivary gland cells, causing a rise in intracellular Ca2+via inositol 1, 4, 5-trisphosphate (IP3) and IP3 receptors. Consequently, the rise in intracellular Ca2+activates apical membrane Clchannels and induces salivary secretion [1]. Activation of M3R also induces trafficking of aquaporin 5 (AQP5) to the apical membrane from the cytoplasm, which causes rapid transport of water across the cell membrane [2]. M3R has four extracellular domains: the N-terminal region and the first, second and third extracellular loops. Among these domains, the second extracellular loop is critical for receptor activation by agonists [3]. Therefore, the second extracellular loop of M3R has been the focus of our interest, and we report a subgroup of SS patients who had anti-M3R antibodies that recognized the second extracellular loop of M3R [4,5]. Although these data indicate that the second extracellular loop is the target antigen, the precise epitopes are currently unknown. A recent study reported that the third extracellular loop represents a functional epitope bound by IgG derived from SS patients [6]. The present study was designed to clarify the precise B cell epitopes of M3R and the function of anti-M3R antibodies. For this purpose, we screened sera of SS patients for anti-M3R autoantibodies against all four extracellular domains of M3R by enzyme-linked immunosorbent assay (ELISA) using synthetic peptide antigens and performed functional assays of these antibodies using human salivary gland (HSG) cells. We assessed the correlation between epitopes and function and various clinical features. == Materials and methods == == Study population == Serum samples were collected from 42 Japanese patients with SS (15 with primary SS and 27 with secondary SS) who had been followed-up at the Division of Rheumatology, University of Tsukuba Hospital, Ibaraki, Japan. All patients with SS satisfied the Japanese Ministry of Health criteria for the diagnosis of SS. These criteria included four clinicopathological findings: lymphocytic infiltration of the salivary or lacrimal glands, dysfunction of salivary secretion, keratoconjunctivitis sicca and presence of anti-SS-A or SS-B antibodies. The diagnosis of SS was based on the presence of two or more of the above items. We also recruited 42 healthy controls (HC). Approval for this study was obtained from the local ethics committee and signed informed consent was obtained from each subject. == Synthesis of peptide antigens == We synthesized different peptides encoding the extracellular domains of human-M3R. The N-terminal of human-M3R has a 66-mer amino acid sequence, and accordingly we divided this domain into three segments. The sequences were: MTLHNNSTTSPLFPNISSSWIHSPSDAGLP for N-terminal 1, IHSPSDAGLPPGTVTHFGSYNVSRAAGNFS for N-terminal 2 and NVSRAAGNFSSPDGTTDDPLGGHTVWQV for N-terminal 3 (Sigma-Aldrich Japan, Ishikari,.
