Laboratory of Neurobiology, National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, P.O. Box 12233, Research Triangle Park, North Carolina 27709, USA.
Biochemistry. 2010 Mar 16;49(10):2279-87. doi: 10.1021/bi902023y.
We identified a homologue of the molluscan acetylcholine-binding protein (AChBP) in the marine polychaete Capitella teleta, from the annelid phylum. The amino acid sequence of C. teleta AChBP (ct-AChBP) is 21-30% identical with those of known molluscan AChBPs. Sequence alignments indicate that ct-AChBP has a shortened Cys loop compared to other Cys loop receptors, and a variation on a conserved Cys loop triad, which is associated with ligand binding in other AChBPs and nicotinic ACh receptor (nAChR) alpha subunits. Within the D loop of ct-AChBP, a conserved aromatic residue (Tyr or Trp) in nAChRs and molluscan AChBPs, which has been implicated directly in ligand binding, is substituted with an isoleucine. Mass spectrometry results indicate that Asn122 and Asn216 of ct-AChBP are glycosylated when expressed using HEK293 cells. Small-angle X-ray scattering data suggest that the overall shape of ct-AChBP in the apo or unliganded state is similar to that of homologues with known pentameric crystal structures. NMR experiments show that acetylcholine, nicotine, and alpha-bungarotoxin bind to ct-AChBP with high affinity, with K(D) values of 28.7 microM, 209 nM, and 110 nM, respectively. Choline bound with a lower affinity (K(D) = 163 microM). Our finding of a functional AChBP in a marine annelid demonstrates that AChBPs may exhibit variations in hallmark motifs such as ligand-binding residues and Cys loop length and shows conclusively that this neurotransmitter binding protein is not limited to the phylum Mollusca.
我们在海洋多毛类环节动物 Cap itella teleta 中鉴定出一种与软体动物乙酰胆碱结合蛋白（AChBP）同源的蛋白。Cap itella teleta AChBP（ct-AChBP）的氨基酸序列与已知的软体动物 AChBPs 有 21-30%的同一性。序列比对表明，ct-AChBP 的 C 环较其他 C 环受体缩短，并且在保守的 C 环三联体上发生了变异，这与其他 AChBPs 和烟碱型乙酰胆碱受体（nAChR）α亚基中的配体结合有关。在 ct-AChBP 的 D 环中，nAChRs 和软体动物 AChBPs 中的一个保守芳香族残基（Tyr 或 Trp）直接参与配体结合，被异亮氨酸取代。质谱结果表明，当在 HEK293 细胞中表达时，ct-AChBP 的 Asn122 和 Asn216 发生糖基化。小角度 X 射线散射数据表明，apo 或未结合配体状态下的 ct-AChBP 的整体形状与具有已知五聚体晶体结构的同源物相似。NMR 实验表明，乙酰胆碱、尼古丁和α-银环蛇毒素以高亲和力结合 ct-AChBP，其 K(D) 值分别为 28.7μM、209nM 和 110nM。胆碱的结合亲和力较低（K(D) = 163μM）。我们在海洋环节动物中发现了一种功能性 AChBP，这表明 AChBPs 可能在标志性基序（如配体结合残基和 C 环长度）上发生变异，并确凿地表明这种神经递质结合蛋白不仅限于软体动物门。
