Neumann D, Barchan D, Horowitz M, Kochva E, Fuchs S
Department of Chemical Immunology, Weizmann Institute of Science, Rehovot, Israel.
Proc Natl Acad Sci U S A. 1989 Sep;86(18):7255-9. doi: 10.1073/pnas.86.18.7255.
The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.
眼镜蛇科蛇类神经肌肉接头处的乙酰胆碱受体（AcChoR）能结合胆碱能配体，但与其他肌肉型乙酰胆碱受体不同，它不结合α-银环蛇毒素。大量研究表明，乙酰胆碱受体的配体结合位点包括α亚基第192和193位的半胱氨酸残基。我们之前已经表明，对应于电鳗乙酰胆碱受体α亚基第185 - 196位残基的合成十二肽包含配体结合位点的关键元件。为了阐明蛇乙酰胆碱受体精确结合特性的结构基础，我们对蛇乙酰胆碱受体α亚基的一部分进行了测序。首先，用小鼠乙酰胆碱受体α亚基cDNA探针筛选了经大小选择的蛇（棋盘蛇）基因组文库。分离出一个基因组克隆，发现其包含与外显子同源的序列，该外显子包括乙酰胆碱受体α亚基的前两个半胱氨酸（Cys - 128和 - 142）。通过mRNA的逆转录和聚合酶链反应扩增了棋盘蛇和眼镜蛇乙酰胆碱受体α亚基包含四个细胞外半胱氨酸（128、142、192和193）的结构域（氨基酸残基119 - 222），然后进行测序。推导的氨基酸序列表明，蛇α亚基在第192和193位含有两个串联的半胱氨酸，与所有其他乙酰胆碱受体α亚基相似。序列比较显示，蛇α亚基的克隆区域与其他肌肉型乙酰胆碱受体高度同源（75 - 80%），而与同样不结合α-银环蛇毒素的神经元型乙酰胆碱受体不同源。在假定的配体结合位点，在Cys - 192和Cys - 193附近，蛇序列中发生了四个主要替换——在第184位（Trp→Phe）、185位（Lys→Trp）、187位（Trp→Ser）和194位（Pro→Leu）。此外，Asn - 189是一个假定的N - 糖基化位点，仅存在于蛇中。这些变化，或其中一部分，可能解释了蛇乙酰胆碱受体缺乏α-银环蛇毒素结合的原因。
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