Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 16/10 Miklukho-Maklaya Street, 117997 Moscow, Russia.
J Biol Chem. 2011 Mar 25;286(12):10618-27. doi: 10.1074/jbc.M110.189100. Epub 2011 Jan 20.
Discovery of proteins expressed in the central nervous system sharing the three-finger structure with snake α-neurotoxins provoked much interest to their role in brain functions. Prototoxin LYNX1, having homology both to Ly6 proteins and three-finger neurotoxins, is the first identified member of this family membrane-tethered by a GPI anchor, which considerably complicates in vitro studies. We report for the first time the NMR spatial structure for the water-soluble domain of human LYNX1 lacking a GPI anchor (ws-LYNX1) and its concentration-dependent activity on nicotinic acetylcholine receptors (nAChRs). At 5-30 μM, ws-LYNX1 competed with (125)I-α-bungarotoxin for binding to the acetylcholine-binding proteins (AChBPs) and to Torpedo nAChR. Exposure of Xenopus oocytes expressing α7 nAChRs to 1 μM ws-LYNX1 enhanced the response to acetylcholine, but no effect was detected on α4β2 and α3β2 nAChRs. Increasing ws-LYNX1 concentration to 10 μM caused a modest inhibition of these three nAChR subtypes. A common feature for ws-LYNX1 and LYNX1 is a decrease of nAChR sensitivity to high concentrations of acetylcholine. NMR and functional analysis both demonstrate that ws-LYNX1 is an appropriate model to shed light on the mechanism of LYNX1 action. Computer modeling, based on ws-LYNX1 NMR structure and AChBP x-ray structure, revealed a possible mode of ws-LYNX1 binding.
在中枢神经系统中发现具有与蛇α-神经毒素相同的三指结构的蛋白质，这激发了人们对其在大脑功能中的作用的极大兴趣。原毒素 LYNX1 与 Ly6 蛋白和三指神经毒素都具有同源性，是第一个被鉴定为具有 GPI 锚定的膜结合成员的家族成员，这使得体外研究变得非常复杂。我们首次报道了缺乏 GPI 锚定的人 LYNX1 的水溶性结构域（ws-LYNX1）的 NMR 空间结构及其在烟碱型乙酰胆碱受体（nAChRs）上的浓度依赖性活性。在 5-30 μM 时，ws-LYNX1 与（125）I-α-银环蛇毒素竞争结合乙酰胆碱结合蛋白（AChBPs）和 Torpedo nAChR。将表达α7 nAChR 的非洲爪蟾卵母细胞暴露于 1 μM 的 ws-LYNX1 中，增强了对乙酰胆碱的反应，但对α4β2 和 α3β2 nAChR 没有检测到作用。将 ws-LYNX1 浓度增加到 10 μM 会导致这三种 nAChR 亚型的抑制作用略有增加。ws-LYNX1 和 LYNX1 的一个共同特征是降低了 nAChR 对高浓度乙酰胆碱的敏感性。NMR 和功能分析均表明，ws-LYNX1 是阐明 LYNX1 作用机制的合适模型。基于 ws-LYNX1 NMR 结构和 AChBP X 射线结构的计算机建模揭示了 ws-LYNX1 结合的一种可能模式。
