Identification of delta/notch-like epidermal growth factor-related receptor as the Tr antigen in paraneoplastic cerebellar degeneration
Identification of delta/notch-like epidermal growth factor-related receptor as the Tr antigen in paraneoplastic cerebellar degeneration
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DOI：
10.1002/ana.23550
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发表时间：
2012-06-01
影响因子：
11.2
通讯作者：
Smitt, Peter Sillevis
中科院分区：
文献类型：
--
作者：
de Graaff, Esther;Maat, Peter;Smitt, Peter Sillevis
目的：抗Tr是在副肿瘤性小脑变性（PCD）合并霍奇金淋巴瘤（HL）中描述较好的自身抗体之一;然而，Tr抗原仍未确定。研究方法：我们使用免疫沉淀的总大鼠脑提取物，然后通过质谱法，以确定由抗Trp阳性血清识别的抗原。通过Western印迹和基于细胞的测定，我们测试了总共12个抗Tr阳性血清和246个对照血清，并确定了由抗Tr抗体识别的表位区域。产生缺失和突变构建体以进一步定位抗原区域。结果如下：使用4种不同的抗Tr阳性血清对免疫纯化的大鼠脑提取物进行质谱分析，鉴定出Delta/Notch样表皮生长因子相关受体（DNER）为Tr抗原。在基于HeLa细胞的筛选试验中，246份对照样品中除1份外均为阴性，而12份抗Tr阳性血清中有12份染色了血凝素标记的DNER表达细胞。仅1例HL但无共济失调的对照受试者被发现DNER和Tr均阳性。使用缺失构建体，我们将主要表位精确定位到胞外结构域。在海马和N-糖基化突变中内源性DNER的敲低废除了抗Tr染色，表明DNER的糖基化是被抗Tr抗体识别所必需的。解释：DNER是抗Tr阳性血清检测到的抗原。通过使用基于细胞的筛选试验，现在可以快速可靠地筛选PCD和HL或仅HL患者中抗Tr抗体的存在。ANN NEUROL 2012
Objective: Anti-Tr is among the better described autoantibodies in paraneoplastic cerebellar degeneration (PCD) combined with Hodgkin lymphoma (HL); however, the Tr antigen remains unidentified. Methods: We used immunoprecipitation of total rat brain extract followed by mass spectrometry to identify the antigen recognized by anti-Trpositive sera. By Western blotting and cell-based assays, we tested a total of 12 anti-Trpositive and 246 control sera and determined the region of the epitope recognized by the anti-Tr antibodies. Deletion and mutant constructs were generated to further map the antigenic region. Results: Mass spectrometry analysis of immunopurified rat brain extract using 4 different anti-Trpositive sera led to the identification of Delta/Notch-like epidermal growth factor-related receptor (DNER) as the Tr antigen. All but 1 of 246 control samples were negative in the HeLa cell-based screening assay, whereas 12 of the 12 anti-Trpositive sera stained hemagglutinin-tagged DNER-expressing cells. Only 1 control subject with HL but no ataxia was found to be both DNER and Tr positive. Using deletion constructs, we pinpointed the main epitope to the extracellular domain. Knockdown of endogenous DNER in hippocampal and N-glycosylation mutations abolished the anti-Tr staining, indicating that glycosylation of DNER is required for it to be recognized by anti-Tr antibodies. Interpretation: DNER is the antigen detected by anti-Trpositive sera. Presence of anti-Tr antibodies in patients with PCD and HL or HL only can now be screened quickly and reliably by using a cell-based screening assay. ANN NEUROL 2012