Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russia.
J Biol Chem. 2013 May 31;288(22):15888-99. doi: 10.1074/jbc.M112.436576. Epub 2013 Apr 12.
Human LYNX1, belonging to the Ly6/neurotoxin family of three-finger proteins, is membrane-tethered with a glycosylphosphatidylinositol anchor and modulates the activity of nicotinic acetylcholine receptors (nAChR). Recent preparation of LYNX1 as an individual protein in the form of water-soluble domain lacking glycosylphosphatidylinositol anchor (ws-LYNX1; Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627) revealed the attachment at the agonist-binding site in the acetylcholine-binding protein (AChBP) and muscle nAChR but outside it, in the neuronal nAChRs. Here, we obtained a series of ws-LYNX1 mutants (T35A, P36A, T37A, R38A, K40A, Y54A, Y57A, K59A) and examined by radioligand analysis or patch clamp technique their interaction with the AChBP, Torpedo californica nAChR and chimeric receptor composed of the α7 nAChR extracellular ligand-binding domain and the transmembrane domain of α1 glycine receptor (α7-GlyR). Against AChBP, there was either no change in activity (T35A, T37A), slight decrease (K40A, K59A), and even enhancement for the rest mutants (most pronounced for P36A and R38A). With both receptors, many mutants lost inhibitory activity, but the increased inhibition was observed for P36A at α7-GlyR. Thus, there are subtype-specific and common ws-LYNX1 residues recognizing distinct targets. Because ws-LYNX1 was inactive against glycine receptor, its "non-classical" binding sites on α7 nAChR should be within the extracellular domain. Micromolar affinities and fast washout rates measured for ws-LYNX1 and its mutants are in contrast to nanomolar affinities and irreversibility of binding for α-bungarotoxin and similar snake α-neurotoxins also targeting α7 nAChR. This distinction may underlie their different actions, i.e. nAChRs modulation versus irreversible inhibition, for these two types of three-finger proteins.
人类 LYNX1 属于三指蛋白 Ly6/神经毒素家族，通过糖基磷脂酰肌醇锚定连接在膜上，并调节烟碱型乙酰胆碱受体 (nAChR) 的活性。最近，LYNX1 以缺乏糖基磷脂酰肌醇锚定的水溶性结构域（ws-LYNX1；Lyukmanova, E. N., Shenkarev, Z. O., Shulepko, M. A., Mineev, K. S., D'Hoedt, D., Kasheverov, I. E., Filkin, S. Y., Krivolapova, A. P., Janickova, H., Dolezal, V., Dolgikh, D. A., Arseniev, A. S., Bertrand, D., Tsetlin, V. I., and Kirpichnikov, M. P. (2011) NMR structure and action on nicotinic acetylcholine receptors of water-soluble domain of human LYNX1. J. Biol. Chem. 286, 10618-10627）的形式被单独制备出来，该结构域缺少糖基磷脂酰肌醇锚定，其在乙酰胆碱结合蛋白 (AChBP) 和肌肉型 nAChR 的激动剂结合位点被发现，但在神经元型 nAChR 中，它位于该位点之外。在这里，我们获得了一系列 ws-LYNX1 突变体（T35A、P36A、T37A、R38A、K40A、Y54A、Y57A、K59A），并用放射性配体分析或膜片钳技术研究了它们与 AChBP、加利福尼亚美洲电鳐 nAChR 和由α7 nAChR 细胞外配体结合域和α1 甘氨酸受体跨膜域组成的嵌合受体的相互作用。对于 AChBP，只有 T35A 和 T37A 没有改变活性，K40A 和 K59A 略有降低，其余突变体的活性甚至增强（最明显的是 P36A 和 R38A）。对于两种受体，许多突变体失去了抑制活性，但 P36A 在α7-GlyR 中观察到抑制活性增强。因此，LYNX1 存在识别不同靶标的特定亚基和共同的残基。由于 ws-LYNX1 对甘氨酸受体没有活性，其在α7 nAChR 上的“非经典”结合位点应位于细胞外结构域内。对于 ws-LYNX1 及其突变体，我们测量了微摩尔亲和力和快速冲洗率，与针对α7 nAChR 的α-银环蛇毒素和类似蛇α-神经毒素的纳摩尔亲和力和不可逆结合形成对比。这种差异可能是这两种三指蛋白不同作用的基础，即 nAChR 的调节与不可逆抑制。
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