Anti-Sez6l2 antibody, detected in a patient with immune-mediated cerebellar ataxia, inhibits complex formation of GluR1 and Sez6l2
Anti-Sez6l2 antibody, detected in a patient with immune-mediated cerebellar ataxia, inhibits complex formation of GluR1 and Sez6l2
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在免疫介导的小脑性共济失调患者中检测到的抗 Sez6l2 抗体可抑制 GluR1 和 Sez6l2 复合物的形成
DOI：
10.1007/s00415-018-8785-z
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发表时间：
2018
期刊：
影响因子：
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通讯作者：
S.
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作者：
Yaguchi;H.;Yabe;I.;Takahashi;H.;Watanabe;M.;Nomura;T.;Kano;T.;Watanabe;M. and Hatakeyama;S.
自身抗体对某些小脑共济失调很重要[2,4]。我们之前在小脑性共济失调患者[7]中检测到一种新的抗神经元抗体，抗癫痫相关6同源物2 （Sez6l2）抗体。Sez6l2在海马和小脑皮层高度表达，是1型膜蛋白之一[3,5]。我们之前发现Sez6l2蛋白可以结合内收蛋白（ADD）和谷氨酸受体1 (GluR1)，并且Sez6l2可能通过调节ADD磷酸化[8]来调节AMPA受体的功能。我们之前的研究结果表明，Sez6l2是AMPA受体的辅助亚基之一，并作为连接GluR1和ADD[8]的支架蛋白。GluR1是构建AMPA受体的重要蛋白，AMPA受体是自身免疫介导的神经系统疾病[1]的靶受体之一。此外，我们之前发现Sez6l2和GluR1通过它们的细胞外结构域直接结合，并且患者的血清能够识别Sez6l2[8]的细胞外结构域。此外，用转染了FLAG-Sez6l2胞外结构域[FLAG-Sez6l2 (EX)]和HA-GluR1胞外结构域[HA-GluR1 (EX)]的HeLa细胞进行免疫荧光分析，发现Sez6l2 （EX）和GluR1 （EX）共定位（补充图1A）。用转染了全长FLAG-Sez6l2 [FLAG-Sez6l2 (FL)]和全长GluR1 [GluR1 (FL)]的HeLa细胞进行免疫荧光分析，发现FLAG-Sez6l2 （FL）和GluR1 （FL）部分共定位（补充图1B）。因此，确定患者的抗Sez6l2抗体是否直接阻止Sez6l2与GluR1结合是很重要的。因此，在本研究中，我们通过生化实验来验证抗sez6l2抗体的发病机制。实验方法与之前报道的一样[6-8]。为了明确抗Sez6l2抗体对患者的影响，我们使用纯化的FLAG-Sez6l2 （EX）和HA-GluR1 (EX)[8]重组蛋白，在有或没有患者血清的情况下，对Sez6l2的细胞外结构域[Sez6l2 (EX)]和GluR1的细胞外结构域[GluR1 (EX)]进行了体外结合实验（图1）。将FLAG-Sez6l2 （EX）蛋白与患者血清一起或不一起旋转。PBS洗涤后，加入或不加入HA-GluR1 （EX）蛋白。用抗flag抗体进行免疫沉淀。最后，用抗ha抗体对免疫沉淀物进行免疫印迹分析（图1）。患者血清可识别FLAG-Sez6l2 （EX）蛋白（图2a）。我们发现，与没有患者血清的样品相比，与患者血清旋转的样品中FLAG-Sez6l2 （EX）蛋白和HA-GluR1 （EX）蛋白之间的结合减少（图2b）。这意味着患者血清中纯化的每一种重组蛋白都抑制了Sez6l2 （EX）和GluR1 （EX）之间的直接相互作用。此外，我们比较了患者的血清和健康对照血清（补充图2）。我们发现FLAG-Sez6l2 （EX）蛋白之间的结合减少
Autoantibodies are important for some cerebellar ataxias [2, 4]. We previously detected a new anti-neuronal antibody, anti-seizure-related 6 homolog like 2 (Sez6l2) antibody, in a cerebellar ataxia patient [7]. Sez6l2 is highly expressed in the hippocampus and cerebellar cortex and is one of the type 1 membrane proteins [3, 5]. We previously showed that Sez6l2 protein binds to both adducin (ADD) and glutamate receptor 1 (GluR1) and that Sez6l2 may be a modulator of AMPA receptor function via the regulation of ADD phosphorylation [8]. Our previous results indicate that Sez6l2 is one of the auxiliary subunits of the AMPA receptor and acts as a scaffolding protein to link GluR1 to ADD [8]. GluR1 is an important protein constructing the AMPA receptor, which is one of the target receptors for autoimmune-mediated neurological diseases [1]. Furthermore, we previously showed that Sez6l2 and GluR1 directly bind using their extracellular domains and that the patient’s serum recognized the extracellular domain of Sez6l2 [8]. In addition, immunofluorescence analysis using HeLa cells transfected with the extracellular domain of FLAG-Sez6l2 [FLAG-Sez6l2 (EX)] and the extracellular domain of HA-GluR1 [HA-GluR1 (EX)] showed co-localization of Sez6l2 (EX) and GluR1 (EX)(supplemental Fig. 1A). Immunofluorescence analysis using HeLa cells transfected with the full length of FLAG-Sez6l2 [FLAG-Sez6l2 (FL)] and the full length of GluR1 [GluR1 (FL)] showed partial co-localization of FLAG-Sez6l2 (FL) and GluR1 (FL)(supplemental Fig. 1B). It is therefore important to determine whether anti-Sez6l2 antibody from the patient directly prevents binding between Sez6l2 and GluR1. Hence, in this study, we conducted a biochemistry experiment to verify the pathogenesis of anti-Sez6l2 antibodies.Experimental methods were performed as reported previously [6–8]. To clarify the influence of anti-Sez6l2 antibodies in the patient, we performed in vitro binding assays between the extracellular domain of Sez6l2 [Sez6l2 (EX)] and the extracellular domain of GluR1 [GluR1 (EX)] using purified recombinant proteins of FLAG-Sez6l2 (EX) and HA-GluR1 (EX)[8] with or without the patient’s serum (Fig. 1). FLAG-Sez6l2 (EX) proteins with or without the patient’s serum were rotated. After washing with PBS, HA-GluR1 (EX) proteins were added or not. Then immunoprecipitation with anti-FLAG antibody was performed. Finally, immunoprecipitates were subjected to immunoblot analysis with anti-HA antibody (Fig. 1). The patient’s serum recognized FLAG-Sez6l2 (EX) protein (Fig. 2 a). We found a decrease of binding between FLAG-Sez6l2 (EX) protein and HA-GluR1 (EX) protein in the sample rotated with the patient’s serum compared to that in the sample without the patient’s serum (Fig. 2 b). This means that there was the inhibition of direct interactions between Sez6l2 (EX) and GluR1 (EX) using each purified recombinant protein by the patient’s serum. Moreover, we compared the patient’s serum and healthy control serum (supplemental Fig. 2). We found a decrease of binding between FLAG-Sez6l2 (EX) protein