Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers
Synthetic analogues of glycosylphosphatidylinositol-anchored proteins and their behavior in supported lipid bilayers
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DOI：
10.1021/ja073271j
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发表时间：
2007-09-19
影响因子：
15
通讯作者：
Bertozzi, Carolyn R.
中科院分区：
文献类型：
--
作者：
Paulick, Margot G.;Wise, Amber R.;Bertozzi, Carolyn R.
糖基化磷脂酰肌醇（GPI）锚位于许多真核蛋白质的C-末端，是一种翻译后修饰，将修饰的蛋白质锚定在质膜的外小叶中。GPI锚定蛋白在信号转导、脊椎动物免疫应答和锥虫的病理生物学中起着重要作用。虽然许多GPI锚定蛋白已被表征，但GPI锚的生物学功能尚未在分子水平上阐明。我们合成了一系列带有修饰的锚结构的GPI-蛋白类似物，这些结构被设计用于剖析各种聚糖组分对GPI-蛋白的膜行为的贡献。这些锚类似物在长度上与天然GPI锚相似，并且包括天然结构的三个结构域的模拟物。使用表达的蛋白质连接和天然化学连接的组合将这些类似物连接到绿色荧光蛋白（GFP）。将这些修饰的GFP掺入到支持的脂质双层中，并使用荧光相关光谱法分析它们的迁移率。来自这些实验的数据表明GPI锚不仅仅是一个简单的膜锚定装置;它还可以防止附着的蛋白质和下面的脂质双层之间的瞬时相互作用，从而允许在双层中快速扩散。产生GPI锚定蛋白的化学定义的类似物的能力是阐明这种有趣的翻译后修饰的分子功能的重要一步。
Positioned at the C-terminus of many eukaryotic proteins, the glycosylphosphaticlylinositol (GPI) anchor is a posttranslational modification that anchors the modified proteins in the outer leaflet of the plasma membrane. GPI-anchored proteins play vital roles in signal transcluction, the vertebrate immune response, and the pathobiology of trypanosomal parasites. While many GPI-anchored proteins have been characterized, the biological functions of the GPI anchor have yet to be elucidated at a molecular level. We synthesized a series of GPI-protein analogues bearing modified anchor structures that were designed to dissect the contribution of various glycan components to the GPI-protein's membrane behavior. These anchor analogues were similar in length to native GPI anchors and included mimics of the native structure's three domains. A combination of expressed protein ligation and native chemical ligation was used to attach these analogues to the green fluorescent protein (GFP). These modified GFPs were incorporated in supported lipid bilayers, and their mobilities were analyzed using fluorescence correlation spectroscopy. The data from these experiments suggest that the GPI anchor is more than a simple membrane-anchoring device; it also may prevent transient interactions between the attached protein and the underlying lipid bilayer, thereby permitting rapid diffusion in the bilayer. The ability to generate chemically defined analogues of GPI-anchored proteins is an important step toward elucidating the molecular functions of this interesting post-translational modification.