New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjogren's syndrome
New epitopes and function of anti-M3 muscarinic acetylcholine receptor antibodies in patients with Sjogren's syndrome
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DOI：
10.1111/j.1365-2249.2010.04188.x
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发表时间：
2010-10-01
影响因子：
4.6
通讯作者：
Sumida, T.
中科院分区：
文献类型：
--
作者：
Tsuboi, H.;Matsumoto, I.;Sumida, T.
毒蕈碱型乙酰胆碱受体（M3 R）在唾液腺分泌唾液的过程中起着重要作用。据报道，一些干燥综合征（SS）患者携带M3 R抑制性自身抗体。本研究的目的是阐明SS中抗M3 R抗体的表位和功能。我们合成了编码人-M3 R胞外结构域的肽，包括N-末端区域和第一、第二和第三胞外环。对这些地区的抗体进行了检查酶联免疫吸附试验在42 SS和42名健康对照血清。为了进行功能分析，将人唾液腺（HSG）细胞与从抗M3 R抗体阳性SS、阴性SS和对照血清中分离的免疫球蛋白G（IgG）预孵育12 h。在用Fluo-3加载后，用盐酸西维美林刺激HSG细胞，并测量细胞内Ca 2+浓度[（Ca 2+）i]。在42个中心点中检测到N-末端、第一、第二和第三环的抗体，（42个中的18个），47个中心点6%（42个中的20个），54个中心点8%（42个中的23个）和45个中心点2%（42例中的19例），而在4个中心点中，8%（42个中的2个）、7个中心点1%（42个中的3个）、2个中心点4%（42个中的1个）和2个中心点4%（42个中的1个）。抗第二环阳性SS-IgG抗体可抑制盐酸西维美林引起的（Ca ~（2+））i升高。抗N-末端阳性SS-IgG抗体和抗第一环阳性SS-IgG抗体增强了（Ca ~（2+））_i，而抗第三环阳性SS-IgG抗体对（Ca ~（2+））_i和抗M_3R抗体阴性SS-IgG无影响。我们的研究结果表明SS中M3 R上存在几个B细胞表位。抗M3 R抗体对唾液分泌的影响可能基于这些表位而不同。
P>M3 muscarinic acetylcholine receptor (M3R) plays a crucial role in the secretion of saliva from salivary glands. It is reported that some patients with Sjogren's syndrome (SS) carried inhibitory autoantibodies against M3R. The purpose of this study is to clarify the epitopes and function of anti-M3R antibodies in SS. We synthesized peptides encoding the extracellular domains of human-M3R including the N-terminal region and the first, second and third extracellular loops. Antibodies against these regions were examined by enzyme-linked immunosorbent assay in sera from 42 SS and 42 healthy controls. For functional analysis, human salivary gland (HSG) cells were preincubated with immunoglobulin G (IgG) separated from sera of anti-M3R antibody-positive SS, -negative SS and controls for 12 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 42 center dot 9% (18 of 42), 47 center dot 6% (20 of 42), 54 center dot 8% (23 of 42) and 45 center dot 2% (19 of 42) of SS, while in 4 center dot 8% (two of 42), 7 center dot 1% (three of 42), 2 center dot 4% (one of 42) and 2 center dot 4% (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.