Cai Bin, Zheng Qi, Teng Xin-Chen, Chen Dong, Wang Yang, Wang Ke-Qiang, Zhou Guo-Ming, Xie Yi, Zhang Ming-Jie, Sun Hong-Zhe, Huang Zhong-Xian
Chemical Biology Laboratory, Department of Chemistry, Fudan University, 200433 Shanghai, China.
J Biol Inorg Chem. 2006 Jun;11(4):476-82. doi: 10.1007/s00775-006-0097-6. Epub 2006 Apr 7.
GIF, a member of the metallothionein (MT) family (assigned as MT3), is a neuron growth inhibitory factor that inhibits neuron outgrowth in Alzheimer's disease. The conserved Thr5 is one of the main differences between GIF and other members in the MT family. However, natural sheep GIF has an unusual Ala5, casting doubt on the role of common Thr5. We constructed a series of human GIF mutants at site 5, and characterized their biochemical properties by UV spectroscopy, circular dichroism spectroscopy, EDTA reaction, 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) reaction, and pH titration. Their inhibitory activity toward neuron survival and neurite extension was also examined. Interestingly, the T5A mutant exhibited distinct metal thiolate activity in the EDTA and DTNB reactions, and also lost its bioactivity. Meanwhile, the T5S mutant had similar biochemical properties and biological activity as wild-type human GIF, indicating the hydroxyl group on the Thr5 was critical to the bioactivity of human GIF. We suggest the hydroxyl group in human GIF may help stabilize the biologically active conformation. On the other hand, lack of the hydroxyl group in sheep GIF may be partially compensated by its abnormal structure.
生长抑制因子（GIF）是金属硫蛋白（MT）家族的成员之一（被指定为MT3），是一种在阿尔茨海默病中抑制神经元生长的神经元生长抑制因子。保守的苏氨酸5（Thr5）是GIF与MT家族其他成员的主要差异之一。然而，天然绵羊GIF具有不寻常的丙氨酸5（Ala5），这让人对常见的Thr5的作用产生怀疑。我们构建了一系列第5位点的人GIF突变体，并通过紫外光谱、圆二色光谱、乙二胺四乙酸（EDTA）反应、5,5'-二硫代双（2-硝基苯甲酸）（DTNB）反应和pH滴定来表征它们的生化特性。还检测了它们对神经元存活和神经突延伸的抑制活性。有趣的是，T5A突变体在EDTA和DTNB反应中表现出独特的金属硫醇盐活性，并且也失去了其生物活性。同时，T5S突变体具有与野生型人GIF相似的生化特性和生物活性，表明Thr5上的羟基对人GIF的生物活性至关重要。我们认为人GIF中的羟基可能有助于稳定其生物活性构象。另一方面，绵羊GIF中缺乏羟基可能部分由其异常结构所补偿。
