New anti-huntingtin monoclonal antibodies: Implications for huntingtin conformation and its binding proteins
New anti-huntingtin monoclonal antibodies: Implications for huntingtin conformation and its binding proteins
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DOI：
10.1016/s0361-9230(01)00599-8
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发表时间：
2001-10-01
影响因子：
3.8
通讯作者：
Patterson, PH
中科院分区：
文献类型：
--
作者：
Ko, J;Ou, S;Patterson, PH
我们制备了八种抗亨廷顿蛋白(Htt)的单抗，其中几种具有新的结合模式。多肽阵列表位图谱显示，单抗MW1-6特异性结合Htt外显子1的多Q结构域。在突变型Htt敲入小鼠脑提取液和亨廷顿病淋巴母细胞瘤细胞株的Western blotting中，MW1-5都强烈地与Htt的扩展多Q重复形式结合，与正常Htt没有检测到结合。这些结果表明，多聚Q结构域可以呈现不同的构象，这些构象可以通过mAbs来区分。这一观点得到了野生型(WT)和突变型Htt转基因小鼠(R6)脑免疫组织化学的支持。尽管在Western blotts上有相同的表位和结合偏好，但MW1-5显示出不同的染色模式。MW1呈点状胞浆和神经束染色，MW2-5呈神经元高尔基复合体强染。相比之下，MW6染色神经元胞体和神经纤维层。此外，尽管它们在印迹图上倾向于突变的Htt，但这些单抗中没有一株在R6脑中显示出比WT更强的染色，并且在R6脑中没有显示含有Htt的核包涵体的结合。这表明，在其不同的亚细胞位置，Htt的多Q结构域要么具有不同的构象，要么被Htt结合蛋白差异封闭。与MW1-6相比，MW7和8可以通过染色R6/2脑中的核包涵体来区分转基因和WT小鼠；MW8在WT脑中没有检测到染色，而在R6/2脑中只染色到包涵体。表位图谱显示，MW7和MW8分别与息肉结构域和83-90氨基酸特异性结合。与MW1-6一样，MW7和MW8的表位在发现Htt的不同亚细胞室中是不同的。(C)2001年爱思唯尔科学公司。
We produced eight anti-huntingtin (Htt) monoclonal antibodies (mAbs), several of which have novel binding patterns. Peptide array epitope mapping shows that mAbs MW1-6 specifically bind the polyQ domain of Htt exon 1. On Western blots of extracts from mutant Htt knock-in mouse brain and Huntington's disease lymphoblastoma cell lines, MW1-5 all strongly prefer to bind to the expanded polyQ repeat form of Htt, displaying no detectable binding to normal Htt. These results suggest that the polyQ domain can assume different conformations that are distinguishable by mAbs. This idea is supported by immunohistochemistry with wild type (WT) and mutant Htt transgenic mouse (R6) brains. Despite sharing the same epitope and binding preferences on Western blots, MW1-5 display distinct staining patterns. MW1 shows punctate cytoplasmic and neuropil staining, while MW2-5 strongly stain the neuronal Golgi complex. MW6, in contrast, stains neuronal somas and neuropil. In addition, despite their preference for mutant Htt on blots, none of these mAbs show enhanced staining of R6 brains over WT, and show no binding of the Htt-containing nuclear inclusions in R6 brains. This suggests that in its various subcellular locations, the polyQ domain of Htt either takes on different conformations and/or is differentially occluded by Htt binding proteins. In contrast to MW1-6, MW7, and 8 can differentiate transgenic from WT mice by staining nuclear inclusions in R6/2 brain; MW8 displays no detectable staining in WT brain and stains only inclusions in R6/2 brain. Epitope mapping reveals that MW7 and 8 specifically bind the polyp domains and amino acids 83-90, respectively. As with MW1-6, the epitopes for MW7 and 8 are differentially available in the various subcellular compartments where Htt is found. (C) 2001 Elsevier Science Inc.