Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin.
Mapping of a cholinergic binding site by means of synthetic peptides, monoclonal antibodies, and alpha-bungarotoxin.
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通过合成肽、单克隆抗体和 α-银环蛇毒素绘制胆碱能结合位点图谱。
DOI：
10.1021/bi00478a016
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发表时间：
1990
期刊：
影响因子：
2.9
通讯作者：
Maelicke,A
中科院分区：
文献类型：
--
作者：
Conti-Tronconi,BM;Tang,F;Diethelm,BM;Spencer,SR;Reinhardt-Maelicke,S;Maelicke,A
摘要：几个实验室以前的研究已经确定了烟碱乙酰胆碱受体（AChR）a亚基的一个狭窄的序列区域，位于192和193位的半胱氨酰残基两侧，包含胆碱能配体结合位点的主要元素（如果不是全部）。在本研究中，我们使用一组合成肽作为AChR的代表性结构元件，以研究AChR序列的其他片段是否能够结合银环蛇毒素（a-BTX）和几种a-BTX竞争性单克隆抗体（mAb）。使用的mAb（WF 6、WF 5和W2）针对天然电鳐AChR产生，特异性识别α亚基，并以与α-BTX互斥的方式结合AChR。WF 5和W2与电鳐AChR的结合被所有胆碱能配体抑制。WF 6与激动剂竞争，但不与低mol.重量拮抗剂，用于AChR结合。本研究所用的合成肽长约20个残基，相互重叠4-6个残基，与电鳐AChR α亚基的全序列一致。此外，合成了与a166 -203同源的每个电鳐AChR亚基的序列片段对应的重叠肽。α-BTX与含有序列a181 -200的肽结合，并且还与含有序列a55-74的肽结合，尽管程度较低。WF 6结合a181 -200，并在较小程度上结合a55-74和a134 -153。另外两种mAb主要结合α 55 -74，并且在较小程度上结合α 181 -200。肽a181 -200和a55-74均抑制125 I-a-BTX与天然电鳐AChR的结合。与其它亚基序列片段相对应的肽均不与α-BTX或WF 6结合，也不干扰它们的结合。因此，胆碱能结合位点不是一个单一的狭窄的序列区，而是在AChR α亚基的N-末端胞外区内的两个或多个不连续的序列片段，在受体的天然结构中折叠在一起，有助于形成胆碱能结合区。这种结构排列类似于通过抗体-抗原复合物的X射线衍射研究观察到的“不连续表位”[Davies et al.（1988）]。e烟碱乙酰胆碱受体（AChR）1是由同源亚基形成的复杂的跨膜蛋白[在McCarthy et al.（1986）、Maelicke（1988）和Lindstrom et al.（1987）]，其在外周组织如鱼电沉积物和骨骼肌中以化学计量的α 2F 3 Y8组装（Raftery等人，1980; Conti-Tronconi等人，1982年）。AChR亚基在结构上是相关的，表明它们是从共同的祖先进化而来的（Raftery et al.，（1980），并且它们与其它配体门控离子通道形成蛋白质家族，例如甘氨酸和由NSF Grant BNS-8607289（to BMC-T.）NINCDS赠款NS 29919（给BMC-T.），ARO合同DAMD 17-88-C-8120（至BMC-T），Deutsche Forshungsgemeinschaft赠款SFB 168-B2和Ma 599/10-1（to.和化学工业基金会（给AM）。
Revised Manuscript Received January 29, 1990 abstract: Previous studies by several laboratories have identified a narrow sequence region of the nicotinic acetylcholine receptor (AChR) a subunit, flanking the cysteinyl residues at positions 192 and 193, as containing major elements of, if not all, the binding site for cholinergic ligands. In the present study, we used a panel of synthetic peptides as representative structural elements of the AChR to investigate whether additional segments of the AChR sequences are able to binda-bungarotoxin (a-BTX) and several a-BTX-competitive monoclonal antibodies (mAbs). The mAbs used (WF6, WF5, and W2) were raised against native Torpedo AChR, specifically recognize the a subunit, and bind to AChR in a mutually exclusive fashion with a-BTX. The binding of WF5 and W2 to Torpedo AChR is inhibited by all cholinergic ligands. WF6 competes with agonists, but not with low mol. wt. antagonists, for AChR binding. The synthetic peptides used in this study were approximately 20 residue long, overlapped each other by 4-6 residues, and corre-sponded to the complete sequence of Torpedo AChR a subunit. Also, overlapping peptides, corresponding to the sequence segments of each Torpedo AChR subunit homologous to al66-203, were synthesized. a-BTX bound to a peptide containing the sequence al81-200 and also, albeit to a lesser extent, to a peptide containing the sequence a55-74. WF6 bound to al81-200 and to a lesser extent to a55-74 and al34-153. The two other mAbs predominantly bound to a55-74, and to a lesser extent to al81-200. Peptides al81-200 and a55-74 both inhibited binding of 125I-a-BTX to native Torpedo AChR. None of the peptides corresponding to sequence segments from other subunitsbound a-BTX or WF6, or interfered with their binding. Therefore, the cholinergicbinding site is not a single narrow sequence region, but rather two or more discontinuous sequence segments within the N-terminal extracellular region of the AChR a subunit, folded together in the native structure of the receptor, contribute to form a cholinergic binding region. Such a structural arrangement is similar to the “discontinuous epitopes” observed by X-ray diffraction studies of antibody-antigen complexes [reviewed in Davies et al.(1988)]. e nicotinic acetylcholine receptors (AChRs) 1 are complex transmembrane proteins formed by homologous subunits [reviewed in McCarthy et al.(1986), Maelicke (1988), and Lindstrom et al.(1987)] which in peripheral tissues, such as fish electroplax and skeletal muscle, are assembled in a stoichiometry of a2f3y8 (Raftery et al., 1980; Conti-Tronconi et al., 1982). The AChR subunits are structurally related, suggesting that they have evolved from a common ancestor (Raftery et al.,(1980), and they form a family of proteins with other ligand-gated ion channels, such as the glycine and the’Supported by NSF Grant BNS-8607289 (to BMC-T.), NINCDS Grant NS 29919 (to BMC-T.), ARO Contract DAMD 17-88-C-8120 (to BMC-T.), Deutsche Forshungsgemeinschaft Grants SFB 168-B2 and Ma 599/10-1 (to. AM), and a Fonds der Chemischen Industrie Grant (to AM).