Nicolas J P, Lin Y, Lambeau G, Ghomashchi F, Lazdunski M, Gelb M H
Institut de Pharmacologie Moléculaire et Cellulaire, CNRS, 660 Route des Lucioles, Sophia Antipolis, 06560 Valbonne, France.
J Biol Chem. 1997 Mar 14;272(11):7173-81. doi: 10.1074/jbc.272.11.7173.
We have shown previously that neurotoxic venom secretory phospholipases A2 (sPLA2s) have specific receptors in brain membranes called N-type receptors that are likely to play a role in the molecular events leading to neurotoxicity of these proteins. The sPLA2 found in honey bee venom is neurotoxic and binds to this receptor with high affinity. In this paper, we have used a number of mutants of bee venom sPLA2 produced in Escherichia coli to determine the structural elements of this protein that are involved in its binding to N-type receptors. Mutations in the interfacial binding surface, in the Ca2+-binding loop and in the hydrophobic channel lead to a dramatic decrease in binding to N-type receptors, whereas mutations of surface residues localized in other parts of the sPLA2 structure do not significantly modify the binding properties. Neurotoxicity experiments show that mutants with low affinity for N-type receptors are devoid of neurotoxic properties, even though some of them retain high enzymatic activity. These results provide further evidence for the involvement of N-type receptors in neurotoxic processes associated with venom sPLA2s and identify the surface region surrounding the hydrophobic channel of bee venom sPLA2 as the N-type receptor recognition domain.
我们之前已经表明，神经毒性毒液分泌型磷脂酶A2（sPLA2s）在脑膜中有特定的受体，称为N型受体，这些受体可能在导致这些蛋白质神经毒性的分子事件中发挥作用。蜜蜂毒液中的sPLA2具有神经毒性，并以高亲和力与该受体结合。在本文中，我们使用了在大肠杆菌中产生的多种蜜蜂毒液sPLA2突变体，以确定该蛋白质中参与其与N型受体结合的结构元件。界面结合表面、Ca2+结合环和疏水通道中的突变导致与N型受体的结合显著减少，而位于sPLA2结构其他部位的表面残基突变不会显著改变结合特性。神经毒性实验表明，对N型受体亲和力低的突变体没有神经毒性，尽管其中一些保留了高酶活性。这些结果为N型受体参与与毒液sPLA2相关的神经毒性过程提供了进一步证据，并将蜜蜂毒液sPLA2疏水通道周围的表面区域确定为N型受体识别域。
